WO2010040571A2 - Method for a genome wide identification of expression regulatory sequences and use of genes and molecules derived thereof for the diagnosis and therapy of metabolic and/or tumorous diseases - Google Patents

Method for a genome wide identification of expression regulatory sequences and use of genes and molecules derived thereof for the diagnosis and therapy of metabolic and/or tumorous diseases Download PDF

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WO2010040571A2
WO2010040571A2 PCT/EP2009/007431 EP2009007431W WO2010040571A2 WO 2010040571 A2 WO2010040571 A2 WO 2010040571A2 EP 2009007431 W EP2009007431 W EP 2009007431W WO 2010040571 A2 WO2010040571 A2 WO 2010040571A2
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diseases
genes
hnf4α
gene
metabolic
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WO2010040571A3 (en
WO2010040571A4 (en
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Jürgen BORLAK
Fridjof Weltmeier
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Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • the invention is directed to the use of particular human genes, nucleic acids hybridizing to said genes, and gene products encoded thereby in the context of the diagnosis and/or therapy of metabolic and/or cancerous diseases, preferably of diabetes mellitus and/or colorectal cancer.
  • the invention further relates to a method for a genomewide identification of functional binding sites at specifically targeted DNA sequences with high resolution. Areas of application are the life sciences: biology, biochemistry, biotechnology, medicine and medical technology.
  • Hepatic nuclear factor (HNF)-4 ⁇ is a member of the nuclear rece?ptor superfamily and known to be expressed in the liver, intestine, and pancreas (for review see Sladek et al. f 2001 ; Schrem, 2002). Many reports have highlighted the importance of HNF4 ⁇ in the regulation of developmental processes in determining the hepatic phenotype, as well as the regulation of diverse metabolic pathways (e.g., glucose, cholesterol, and fatty acid metabolism) (Sladek et al., 1990; Jiang et al., 1995; Yamagata et al., 1996; Hadzopoulou-Cladaras et al., 1997).
  • HNF4 ⁇ is considered as a hepatic master regulatory protein, ln ⁇ contrast to other members of the nuclear receptor superfamily, HNF4 ⁇ binds to its cognate DNA binding site as a homodimer (Jiang 1997; Sladek 1990). HNF4 ⁇ is one of the best characterized transcription factors, and in the past some dozent direct binding sites were reported. The employment of ChlP-chip technologies demonstrated however that these are only the smallest fraction of the actual HNF4 ⁇ binding sites. Notably, Rada-lglesias et al. (2005) used custom made arrays with a low resolution encompasing the ENCODE regions, i. e.
  • the aim of the invention is thus to provide a method allowing a genome-wide high resolution map of binding sites relevant for the transcription regulation induced by HNF4 ⁇ , and the identification of human chromosomal genes having at least one specific regulatory sequence in their natural environment, and the use of said genes or gene products encoded thereby or of RNA hybridizing to said genes for the diagnosis and therapy of diseases, preferably of adenocarcinomas of the colon, caused by an deregulation of HNF4 ⁇
  • the invention is directed to the use of a human gene, in particular the coding region thereof, or of a gene product encoded thereby or of an antibody directed against said gene product, or of DNA or RNA sequences hybridizing to said gene and coding for a polypeptide having the function of said gene product for the therapy and/or diagnosis of metabolic and/or cancerous diseases and/or to screen for and to identify drugs against metabolic and/or cancerous diseases, such as diabetes mellitus and/or colorectal cancer may be, wherein the gene is selected from the group of the human chromosomal genes having at least one expression regulatory sequence according to matrix 1 ("de novo" HNF4 ⁇ matrix) in the range of 100000 nucleotides upstream or downstream of their transcription start site in the human genome, and wherein the at least one expression regulatory sequence according to matrix 1 is located within the chromosomal position specified by the start and end sites according to Tables 9-32 (chromosomes 1-22, X-, Y-chromosome, wherein Table 9 refers
  • the term "gene” according to the invention is directed to both the template strand, which refers to the sequence of the DNA that is copied during the synthesis of mRNA, and to the coding strand corresponding to the codons that are translated into a protein.
  • the genes according to the invention and gene products encoded thereby can be easily derived from the common databases, as such are known to the person skilled in the art, wherein the UCSC Genome Database is particularly preferred: Karolchik D, Kuhn, RM, Baertsch R, Barber GP, Clawson H, Diekhans M, Giardine B, Harte RA, Hinrichs AS 1 Hsu F, Miller W, Pedersen JS, Pohl A, Raney BJ, Rhead B, Rosenbloom KR 1 Smith KE, Stanke M, Thakkapallayil A, Trumbower H, Wang T 1 Zweig AS, Haussler D, Kent WJ.
  • coding region is directed to the portion of DNA or RNA that is transcribed into the mRNA, which then is translated into a protein. This does not include gene regions such as a recognition site, initiator sequence, or termination sequence.
  • DNA sequences hybridizing to said gene or “DNA sequences, which hybridize to said gene”, respectively, according to the invention preferably relates to DNA molecules hybridizing with the coding strand of said gene, in particular with the coding region thereof.
  • hybridizing refers to conventional hybridization conditions, preferably to hybridization conditions under which the Tm value is between 37°C to 70 0 C, preferably between 41 0 C to 66 0 C. [fl] An example for low stringency conditions is e.g.
  • hybridisation under the conditions 42°C, 2*SSC, 0.1% SDS and an example for high stringency conditions is e.g. hybridization under the conditions: 65°C, 2*SSC, 0.1% SDS, wherein in the case that washing is necessary for equilibrium, the hybridization solution is used for the washings.
  • hybridization refers to stringent hybridization conditions.
  • transcription start site refers to the start codon or initiation codon in eukaryotes, in particular to the respective DNA sequence ATG of the coding strand coding for methionin.
  • colonal cancer within the context of the invention is in particular directed to adenocarcinoma of the colon, in particular to colorectal adenocarcinoma associated with an upregulation or hyperactivity of HNF4 ⁇ .
  • matrix or “matrices” according to the invention is directed to position weight matrix/matrices (PWM(s)), which are known to the person skilled in the art.
  • PWM position weight matrix/matrices
  • the first column specifies the position
  • the second column provides the number of occurrences of A at that position
  • the third column provides the number of occurrences of C at that position
  • the fourth column provides the number of occurrences of G at that position
  • the fifth column provides the number of occurrences of T at that position, such that the matrix contains log odds weights for computing a match score and provides a likelihood ratio to specify whether an input sequence matches the motif or not.
  • the cut-offs for the matrices according to the invention are preferably set to minFN to maximize the sensitivity of the site prediction (false negative rate of 10%).
  • the gene is selected from the group of genes, wherein genes having an expression regulatory sequence according to matrix 2 (CTCF matrix) between the transcription start site and the at least one expression regulatory sequence (matrix 1 ("de novo" HNF4 ⁇ matrix)) in the human genome are excluded from the group of genes.
  • CTCF matrix matrix 2
  • matrix 1 matrix 1
  • the gene is selected from the group of genes further having at least one expression regulatory sequence selected from the group matrices 3- 6 (AP1 , GATA2, ER, HNF1 matrices), preferably according to matrix 3, in the range of 20-60 nucleotides upstream or downstream of the at least one expression regulatory sequence according to matrix 1 ("de novo" HNF4 ⁇ matrix) in the human genome.
  • group matrices 3- 6 AP1 , GATA2, ER, HNF1 matrices
  • the gene is selected from the group of genes, wherein genes having an expression regulatory sequence according to matrix 7 (CART1 matrix) in the range of 20-60 nucleotides upstream or downstream of the at least one expression regulatory sequence according to matrix 1 ("de novo" HNF4 ⁇ matrix)) in the human genome are excluded from the group of genes.
  • CART1 matrix genes having an expression regulatory sequence according to matrix 7 (CART1 matrix) in the range of 20-60 nucleotides upstream or downstream of the at least one expression regulatory sequence according to matrix 1 ("de novo" HNF4 ⁇ matrix)) in the human genome are excluded from the group of genes.
  • the gene is selected from the group of genes located on the human chromosome 6 or the human chromosome 10, wherein genes located on chromosome 10 are particularly preferred.
  • the gene is selected from the group of genes coded at least partially in the genomic regions according to table 33, which represent high density clusters of identified HNF4 ⁇ binding sites.
  • the gene is selected from the group of the genes according to Table 34.
  • Table 34 list of human chromosomal genes: 7A5, A2M, A4GALT, AAA1 , AADAC, AAK1 , AARS, AASDH, ABAT, ABC1, ABCA1, ABCA10, ABCA3, ABCA8, ABCA9, ABCB6, ABCC10, ABCC11, ABCC4, ABCD2, ABCD3, ABCG2, ABCG5, ABHD12, ABHD2, ABHD3, ABHD4, ABHD5, ABHD6, ABHD7, ABM, ABI3BP, ABL1, ABL2, ABLIM1, ABLIM3, ABP1, ABR, ABRA, ABTB2, ACACA, ACADM, ACADS, ACAT2, ACBD3, ACBD5, ACBD6, ACCN1, ACE2, ACHE, ACIN1, ACOI.ACOTI.ACOTH, ACOT12, ACOT2, ACOT6, ACOT7, ACOX2, ACP1, ACPL2, ACPP, ACPT, ACRC, ACSL1, ACSL3, ACSL4, ACSL5, ACSL6,
  • GSTA2 GSTA5, GSTCD, GSTK1, GSTO1, GSTP1, GTDC1, GTF2A1, GTF2H5, GTF2IRD1, GTPBP4, GUCA1B, GUCA1C, GUCA2B, GUCY2C, GUCY2D, GULP1, GYPA, H1F0, H2AFB1, H2AFY, H2AFZ, H2-ALPHA, HABP2, HACE1, HACL1, HADH, HAK, HAO2, HAPLN 1, HAVCR1, HBLD1, HCCS, HCN3, HCN4, HCP1, HCRTR2, HDAC11, HDAC7A, HDAC9, HDC, HDDC2, HDGF, HDGFL1, HDHD3, HEBP2, HECA, HECTD1, HECW1, HECW2, HELZ 1 HEMK1, HEMK2, HEPH, HERC1, HERPUD1, HERPUD2, HERV- FRD, HES1, HES3, HEXB,
  • PRTC7 PPTC7, PPYR1, PQLC1, PQLC2, PRDM1, PRDM10, PRDM8, PRDX1, PRDX4, PRDX6, PREP, PRF1, PRG1, PRG2, PRH1, PRICKLE1, PRICKLE2, PRKAA1, PRKAA2, PRKAG2, PRKAR1A, PRKAR2B, PRKCA, PRKCBP1, PRKCD, PRKCI, PRKG2, PRKRIR, PRL, PRLHR, PRM1, PRMT1, PROC, PR0CA1, PRODH, PR0M1, PR0S1, PR0X1, PRPF38B, PRPF39, PRPSAP1, PRPSAP2, PRR13, PRR15, PRR17, PRR3, PRR6, PRR8, PRRG4, PRRT1, PRSS12, PRSS16, PRSS23, PRSS3, PRSS35, PRSS8, PRTFDC1, PRTG, PRUNE, PSCD4, PSCDBP, PSD3, PSD4, PSEN1, PSKH2, PSMAL, PSMB3, PSMB4, PS
  • the gene is selected from the group of genes regulated on the transcriptional level by the HNF4 ⁇ inducing agent Aroclor 1254, wherein the gene is preferably selected from the group of genes according to Table 35.
  • Table 35 list of genes regulated on the transcriptional level by the HNF4 ⁇ inducing agent Aroclor 1254: ABCA1, ABCC3, ABP1, ACE2, ACOX1, ACSL5, ACY3, ACYP2, ADH4, AFF1, AFP, AGR2, AGT, AGXT2, AHSG, AIG1, AKAP7, ALB, ALDH6A1, ALDOB, AMICA1, AMMECR1, ANKRD9, ANKZF1, ANTXR2, ANXA4, AP1S3, APOA1, APOA4, APOB, APOBEC1, APOC3, APOM, AQP3, ARHGAP18, ARL4C, ASGR1, ATAD4, ATXN1, AXIN2, BCL2L14, BCMP11.
  • the first aspect of the invention is, in one example, directed to the use, in particular the in vitro use, of
  • (C) the template strand of a (A) or (B), wherein (C) is preferably a recombinant DNA molecule, or
  • (D) the coding strand of (A) or (B), wherein (D) is preferably a recombinant DNA molecule, or
  • the invention is thus also directed to the use of an antibody directed against a gene product encoded by a human gene selected from one of the groups of genes described herein, in particular of the group described in claim 1 , for the diagnosis, prognosis and/or treatment monitoring of metabolic and tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer, preferably colorectal adenocarcinoma, or said antibody is used for the preparation of a diagnostic agent for for the diagnosis, prognosis and/or treatment monitoring of metabolic and tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer, preferably colorectal adenocarcinoma.
  • antibodies are understood to include monoclonal antibodies and polyclonal antibodies and antibody fragments (e.g., Fab, and F(ab') 2 ) specific for one of said polypeptides.
  • Polyclonal antibodies against selected antigens may be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, goats, rabbits, mice, rats, chicken or preferably of eggs derived from immunized chicken.
  • Monoclonal antibodies may be generated using conventional techniques (see Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are incorporated herein by reference).
  • the invention is thus further directed to the use of primer sequences, preferably primer pairs, directed against the mRNA of a human gene selected from one of the groups of genes described herein, in particular of the group described in claim 1 , for the diagnosis, prognosis and/or treatment monitoring of metabolic and tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer, preferably colorectal adenocarcinoma, or said primer sequences are used for the preparation of a diagnostic agent for the diagnosis, prognosis and/or treatment monitoring of metabolic and tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer, preferably colorectal adenocarcinoma,
  • a primer or of primers such as a primer pair may be, selected from Table M1 is particularly preferred.
  • the first aspect of the invention is accordingly directed to the use of a human gene, in particular the coding region thereof, or of a gene product encoded thereby or of an antibody directed against said gene product or of RNA or DNA sequences, which hybridize to said gene and which code for a polypeptide having the function of said gene product, as a biomarker in the diagnosis, prognosis and/or treatment monitoring of metabolic and tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer, wherein the gene is selected from one of the groups described herein, in particular to the group described in claim 1 , and the use is preferably performed for monitoring the therapeutic treatment of a patient suffering from a metabolic and/or tumorous disease, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuro
  • a method for diagnosing, prognosing and/or staging metabolic and tumorous diseases in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer and/or monitoring the treatment of at least one of said diseases, is provided by
  • biomarker is a gene selected from one of the groups of genes described herein, in particular of the group described in claim 1 in a patient or in a sample of a patient suffering from or being susceptible to a metabolic and/or tumorous disease, and
  • the first aspect of the invention may also be used in a method of qualifying the HNF4 ⁇ activity in a patient suffering or being susceptible to metabolic and/or tumorous disease, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer or for classifying a patient suffering from or being susceptible to at least one said diseases, comprising determining in a sample of a subject suffering from or being susceptible to one of said diseases the level of at least one biomarker, wherein the biomarker is the gene product encoded by a gene selected from one of the groups of genes described herein, in particular of the group described in claim 1 , and/or the biomarker is the mRNA sequence encoding the gene product of said selected gene, and wherein the sample level of the at least one biomarker being significantly higher or lower than the level of said biomarker(s) in the sample of a subject without a disease associated
  • Such method of qualifying the HNF4 ⁇ activity in a patient suffering from metabolic and/or tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer to a method of treating such diseases preferably comprises administering a drug identified by the first aspect of the invention, or by the second aspect of the invention as described hereinafter, or administering a HNF4 ⁇ activity modulator, wherein the level of the at least one biomarker being significantly higher or lower than the level of said biomarker(s) in a subject without cancer associated with increased activity of HNF4 ⁇ is indicative that the subject will respond therapeutically to a method of treating cancer comprising administering said drug or administering a HNF4 ⁇ activity modulator.
  • Such methods of qualifying the HNF4 ⁇ activity may be also used for monitoring the therapeutically response of a patient suffering from metabolic and/or tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer to a method of treating at least one of said diseases comprising administering a drug identified by the first aspect of the invention, or by the second aspect of the invention as described hereinafter, or administering a HNF4 ⁇ activity modulator, wherein the level of the at least one biomarker before and after the treatment is determined, and a significant decrease or increase of said level(s), preferably a decrease or increase to the normal level(s), of the at least one biomarker after the treatment is indicative that the subject therapeutically responds to the administration said drug or to the administration of the HNF4 ⁇ activity modulator.
  • metabolic and/or tumorous diseases in particular type 1 and/or type 2 diabetes mellit
  • RT-PCR real time
  • the invention is also directed to a composition for qualifying the HNF4 ⁇ activity in a patient suffering or being susceptible to a metabolic and/or tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer or for classifying a patient suffering from or being susceptible to at least one of said diseases, wherein the composition comprises an effective amount of at least one biomarker, and wherein the biomarker is a gene selected from one of the groups of genes described herein, in particular of the group described in claim 1 , and/or the biomarker is the gene product encoded by said gene, and/or wherein the composition comprises an effective amount of an antibody directed against said gene product.
  • a metabolic and/or tumorous diseases in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases
  • the gene according to the invention may be part of a recombinant DNA molecule for use in cloning a DNA sequence in bacteria, yeasts or animal cells.
  • the gene according to the invention, or the DNA sequences hybridizing to said gene and encoding a polypeptide having the function of the gene product of said gene may be part by a vector.
  • the invention is thus also directed to the use of a vector for the therapy and/or diagnosis of metabolic and/or cancerous diseases and/or to screen for and to identify drugs against metabolic and/or cancerous diseases, such as diabetes mellitus and/or colorectal cancer may be, wherein the vector comprises a gene selected from one of the goups of genes described herein, in particular the group according to claim 1 , or the vector comprises DNA sequences hybridizing to said gene and encoding a polypeptide having the function of the gene product of said gene.
  • composition for qualifying the HNF4 ⁇ activity is used for the production of a diagnostic agent, in particular of a diagnostic standard.
  • this composition is used for the production of a diagnostic agent for qualifying the HNF4 ⁇ activity in a patient suffering or being susceptible to a metabolic and/or tumorous disease, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer or for classifying a patient suffering from or being susceptible to at least one of said diseases.
  • a diagnostic agent for qualifying the HNF4 ⁇ activity in a patient suffering or being susceptible to a metabolic and/or tumorous disease, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer or for classifying a patient suffering from or being susceptible to at least one of said diseases.
  • this compositon is used for the production of a diagnostic agent for predicting or monitoring the response of a patient suffering from a metabolic and/or tumorous disease, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer to a method of treating at least one of said diseases with a drug, in particular a drug identified according to the first aspect of the invention , or according to the second aspect of the invention as described hereinafter, and/or with a HNF4 ⁇ activity modulator.
  • a diagnostic agent for predicting or monitoring the response of a patient suffering from a metabolic and/or tumorous disease, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer to a method of treating at least one of
  • the first aspect of the invention is, in another example, directed to the use, in particular the in vitro use, of
  • (C) the template strand of a (A) or (B), wherein (C) is preferably a recombinant DNA molecule, or
  • (D) the coding strand of (A) or (B), wherein (D) is preferably a recombinant DNA molecule, or
  • the first aspect of the invention is, in a further example, directed to the use, in particular the in vitro use, of
  • (C) the template strand of a (A) or (B) 1 wherein (C) is preferably a recombinant DNA molecule, or
  • (D) the coding strand of (A) or (B), wherein (D) is preferably a recombinant DNA molecule, or
  • the gene is preferably selected from the group of genes identified by the method according to the second aspect of the invention.
  • an agent selected from the group consisting of: the agonists, antagonists, drugs, agents, antiestrogens, and compounds according to Tables 36-54, sulfonlyurea derivates, and Aroclor 1254, is used and/or tested and/or screened as the drug and/or as the HNF4 ⁇ activity modulator.
  • Aroclor 1254 decreases the activity of pyruvate carboxylase (PC), phosphoenolpyrutvate carboxykinase (PEPCK) and glucose-6-phosphatase in diabetic liver.
  • PC pyruvate carboxylase
  • PEPCK phosphoenolpyrutvate carboxykinase
  • glucose-6-phosphatase glucose-6-phosphatase
  • Arocolor 1254 is thus a, at least putative, drug against diabetes mellitus.
  • the administration of insulin to diabetic rats brings the increased amount of pyruvate carboxylase back to the normal level, as disclosed by Wallace & Jitrapakdee Biochemical Journal 340 1-16 (1999).
  • Aroclor 1254 can inhibit tumor growth in rats, as described, for example, by the article "Inhibition of tumor growth in rats by feeding a polychlorinated biphenyl, Aroclor 1254" by Kervliet & Kimeldorf in Bull Environ Contam 1977 Aug , 18 (2) :243-6.
  • Aroclor 1254 is thus a, at least putative, drug against cancerous diseases.
  • Aroclor 1254 is thus a, at least putative, drug against metabolic diseases, in particular against diabetes mellitus, and/or against cancerous diseases.
  • the second aspect of the invention provides a method for a genomewide identification of functional binding sites at targeted DNA sequences bound to DNA complexes in cells, healthy and diseased tissues and/or organs, wherein the method comprises, or preferably consists of, the steps of a. chromatin immunoprecipitation and b. DNA-DNA hybridisation for the c. de novo identification of gene targets.
  • the method according to invention is used for the identification of the functional binding sites of a protein of interest, preferably of a transcription factor, and/or the method is characterized in that a. the chromatin immunoprecipitation comprises or consists of two rounds of sequential chromatin immunoprecipitation, b. a genome wide tiling array is used for the DNA-DNA hybridisation, and/or c. the de novo identification of gene targets comprises reducing the number of false enhancer-gene associations.
  • the method according to invention is used for determining a region of ChIP enrichment in the immunoprecipitated sample (enrichment site), with respect to a control or to genomic DNA, preferably a HNF4 ⁇ binding site, and/or the method is characterized in that a. an anti HNF4 ⁇ antibody is used for the immunoprecipiation b. a human or murine array with a genome-wide 20-50 bp resolution is used, and/or c. all genes, which are separated from their associated enhancer by a CTCF-binding site, are removed from the group of the de novo identified genes (target list).
  • the method according to invention is used for mapping the in vivo enrichment sites ' of a specific protein of interest, and/or the method is characterized in that a. Caco-2 cells are used, b. human tiling arrays with a genome-wide 35 bp resolution are used, and/or c. all genes, which are separated from their associated enhancer by the CTCF-binding site according to matrix 2, are removed from the target list.
  • the method according to the invention further comprises the use of DNA-sequences and/or genes identified by the second aspect of the invention to screen for and to identify novel drug targets for the treatment of metabolic and cancerous diseases, preferably comprising the use according to the first aspect of the invention.
  • novel identified sequences and genes encoding DNA are used.
  • polypeptides encoded by novel identified sequences and genes are used.
  • ES detection is further improved based on the frequency of motives of the protein of interest, in particular of HNF4 ⁇ -motives, within the enriched regions, which is determined by application of the MATCH algorithm,
  • ChIP regions are analyzed for overrepresented motifs, in particular using motif analysis programs, such as from Biobase and Genomatix, as well as the tool CEAS -transcription factors cross-talking with the protein of interest, in particular with HNF4 ⁇ , are determinded
  • the genomic position of the highest scoring motif of the protein of interest (highest likelihood ratio) , in particular of the HNF4 ⁇ motif (highest likelihood ratio), within the ChIP regions is retrieved and extended for 500 nucleotides to both flanks, and within these sequences, other enriched motifs are detected and preferably the distance to the DNA- binding motif of the protein of interest, in particular to the HNF4 ⁇ motif, is calculated
  • AP1 an GATA motifs have their highest enrichment in a distance of 20 to 60 nucleotides to the DNA-binding motifs of the protein of interest, in particular to the HNF4 ⁇ motifs,
  • RefSeq genes with a TSS separated by less than 100000 nucleotides from these binding sites are selected for associating the binding sites of the protein of interest, in particular the HNF4 ⁇ binding sites, identified by the ChlP-chip approach with target genes,
  • the ChIP regions are scanned for transcription factor motifs using position-specific score matrices (PSSM) from TRANSFAC, in particular 533 well-defined PSSM,
  • PSSM position-specific score matrices
  • Ontology categorization is performed with GOFFA and DAVID, -de novo identified genes associated with the DNA binding sites of the protein of interest , in particular with the HNF4 ⁇ binding sites, are grouped by Ontology terms, in particular in genes in metabolic and cancerous disease,
  • the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to develop new medications for the treatment of disease as a result of HNF4 ⁇ dysfunction.
  • the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to develop new drug candidates for treatment of metabolic and tumorous diseases by interfering with the activity of polypeptides, as described herein, encoded by novel identified sequences and genes are used for the purpose of normalizing its activity and to restore a healthy condition.
  • the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to optimize novel chemical entities for the purpose of drug development.
  • the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to identify drugs targeting chromatin and its regulation by interfering with protein-DNA, protein-protein and multiprotein- DNA complexes for the purpose of normalizing gene activities in metabolic and cancerous diseases.
  • the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to identify drugs targeting carbohydrate metabolism in metabolic and tumorous diseases for the purpose of its normalization, wherein a gene, in particular the coding region thereof, selected from the group consisting of
  • the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to identify drugs targeting lipid metabolism in metabolic and tumorous disease for the purpose of its normalization, wherein a gene, in particular the coding region thereof, selected from the group consisting of A4GALT, ABCA1 , ABCD2, ABCG1 , ABHD4, ACAA2, ACACA, ACADM, ACADS, ACAT2, ACBD3, ACLY, ACOT1 , ACOT11 , ACOT12, ACOT2, ACOT7, ACOX1 , ACOX2, ACSL1 , ACSL3, ACSL4, ACSL5, ACSL6, ACSS2, ADIPOR2, ADM, AGPAT1 , AGPAT2, AGPAT3, AKR1 B10, AKR1C1 , AKR1C3, AKR1C4, AKR1 D1 , ALDH3A2, ALOX5AP, ALOXE3, ANGPTL3, AOAH, APOA1 ,
  • the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to identify drugs targeting intracellular signaling in metabolic and tumorous disease for the purpose of its normalization, wherein a gene, in particular the coding region thereof, selected from the group consisting of ABL1, ABL2, ABR, ABRA 1 ACOT11, ACR, ADCY5, ADCY6, ADCY8, ADCY9, ADIPOR2, ADORA2A, ADORA2B, ADORA3, ADRA1B, ADRA1D, ADRB1, AGTR1, AKAP13, AKAP7, AMBP, ANP32A, APBB2, ARF1, ARF6, ARFGEF2, ARHGAP29, ARHGAP5, ARHGAP6, ARHGEF1, ARHGEF10L, ARHGEF11, ARHGEF12, ARHGEF17, ARHGEF18, ARHGEF3, ARHGEF5, ARHGEF7, ARID1A, ARL1, ARL4A, ARL4C, ARL4D, ARL5
  • the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to identify drugs targeting cell cycle and cell proliferation in metabolic and tumorous disease for the purpose of its normalization , wherein a gene, in particular the coding region thereof, selected from the group consisting of
  • the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to identify drugs targeting programmed cell death in metabolic and tumorous disease for the purpose of its normalization, wherein a gene, in particular the coding region thereof, selected from the group consisting of ABL1, ACIN1, ACTN1, ACTN4, ADORA2A, AHR, ALB, AMID, AMIG02, ANXA4, ANXA5, APOE, APP 1 ASAH2, ATG5, AXIN1, BAD, BAG2, BAG3, BBC3, BCAR1, BCL10, BCL2, BCL2A1, BCL2L1, BCL2L10, BCL2L11, BCL2L14, BCLAF1, BID, BIRC2, BIRC3, BIRC4, BMF, BRAF, BRCA1, BRE, BTG1, CARD10, CARD4, CASP10, CASP3, CASP6, CBX4, CD28, CD3E, CD74, CDC2, CDK5R1,
  • the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to identify drugs targeting cell morphogenesis and cell / organ development in metabolic and tumorous disease for the purpose of its normalization , wherein a gene, in particular the coding region thereof, selected from the group consisting of
  • RNA sequences which hybridize to said gene and which code for a polypeptide having the function of said gene product, are used.
  • the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to select drug candidates of an antisense molecule, ribozyme, triple helix molecule or other new chemical entities targeting the chromatin.
  • the invention further comprises a kit for qualifying the HNF4 ⁇ activity in a patient suffering or being susceptible to metabolic and/or tumorous disease, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer or for classifying a patient suffering from or being susceptible to at least one of said diseases, in particular for predicting or monitoring the response of a patient to suffering from at least one of said diseases by a method of treating metabolic and/or tumorous diseases comprising administering a HNF4 ⁇ activity modulator, comprising at least one standard indicative of the level of a biomarker selected from the groups of genes described herein, preferably the group according to claim 1 , or from the group of gene products encoded by said groups of genes in normal individuals or individuals having metabolic and/or tumorous disease associated with increased HNF4 ⁇ activity, and instructions for the use of the kit, and, preferably, wherein the
  • the kit according to the invention further comprises at least one primer or primer pair specifically hybridizing with the mRNA of a biomarker selected from one of the groups of genes and/or at least one antibody specific for a biomarker selected from the group of gene products encoded by said genes, and reagents effective to detect said biomarker(s) in a serum sample.
  • a medicament for the treatment of metabolic and tumorous diseases in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer
  • the medicament comprises a composition that decreases the expression or activity of a HNF4 ⁇ modulated gene selected from one of the groups of genes described herein, preferably from the group specified in claim 1.
  • siRNA composition wherein the siRNA composition reduces the expression of a de novo identified HNF4 ⁇ modulated gene selected from one of the groups of genes described herein, preferably from the group specified in claim
  • the present invention thus employs SiRNA oligonucleotides directed to said genes specifically hybridizing with nucleic acids encoding the gene products of said genes and interfering with gene expression of said genes.
  • the siRNA composition comprises siRNA (double stranded RNA) that corresponds to the nucleic acid ORF sequence of the gene product coded by one of said human genes or a subsequence thereof; wherein the subsequence is 19, 20, 21 , 22, 23, 24, or 25 contiguous RNA nucleotides in length and contains sequences that are complementary and non-complementary to at least a portion of the mRNA coding sequence.
  • nucleotide sequences and siRNA according to the invention may be prepared by any standard method for producing a nucleotide sequence or siRNA, such as by recombinant methods, in particular synthetic nucleotide sequences and siRNA is preferred.
  • an antisense composition comprising a nucleotide sequence complementary to a coding sequence of a HNF4 ⁇ modulated gene selected from one of the groups of genes described herein, preferably from the group specified in claim 1.
  • coding sequence is directed to the portion of an mRNA which actually codes for a protein.
  • nucleotide sequence complementary to a coding sequence in particular is directed to an oligonucleotide compound, preferably RNA or DNA, more preferably DNA, which is complementary to a portion of an mRNA, and which hybridizes to and prevents translation of the mRNA.
  • the antisense DNA is complementary to the 5' regulatory sequence or the 5' portion of the coding sequence of said mRNA.
  • the antisense composition comprises a nucleotide sequence containing between 10-40 nucleotides , preferably 12 to 25 nucleotides, and having a base sequence effective to hybridize to a region of processed or preprocessed human mRNA.
  • the composition comprises a nucleotide sequence effective to form a base-paired heteroduplex structure composed of human RNA transcript and the oligonucleotide compound, whereby this structure is characterized by a Tm of dissociation of at least 45 0 C.
  • the siRNA composition and/or the antisense composition is/are used for the preparation of a medicament, in particular for the preparation of a medicament for preventing, treating, or ameliorating metabolic and tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer.
  • metabolic and tumorous diseases in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer.
  • siRNA composition and/or said antisense composition a composition is used.
  • a nucleic acid, in particular DNA, hybridizing with the expression regulatory sequence of matrix 1 is used.
  • This cell line differentiates into enterocytes upon confluence (Soutoglou et al., 2002). Furthermore, in differentiated Caco-2 cells HNF4 ⁇ protein expression is comparable to its expression in liver (Niehof and Borlak, 2008). Therefore, Caco-2 cells are an interesting system to study the HNF4 ⁇ gene regulatory network.
  • the study according to the invention is the first genome-wide approach to identify nearby 6600 RefSeq genes targeted by HNF4 ⁇ .
  • the importance of promoter-distal regions in HNF4 ⁇ mediated transcriptional control is highlighted, and a basis for elucidation of transcriptional networks formed by cooperating transcription factors acting in concert with HNF4 ⁇ is offered.
  • good agreement between de novo identified genes and their expression in Caco-2 cells is demonstrated.
  • HNF4 ⁇ binding sites described in literature and present in the Biobase database, including /WT (R00114), GCC (R08885), PCK (R12074), APOB (R01612), CYP2C9 (R15905), AKR1C4 (R13037), ACADM (R15923) or CYP27A1 (R15917).
  • SHBG SHBG
  • ALDH2 ALDH2
  • HNF4 ⁇ binds predominantly to enhancer elements
  • ES overall distribution of ES is very similar to that observed in genome-wide ChlP-chip for another member of the nuclear receptor family, i. e. the estrogen receptor (Carroll et al., 2006).
  • a significant lack of preference for binding to 5' promoter-proximal regions has also been reported for other transcription factors, e.g. p53, cMyc or p63 (Table 7; Cawley et al., 2004; Wei et al., 2006; Yang et al., 2006).
  • transcription factors like E2F1 show a clear preference for binding to 5' promoter- proximal regions (Bieda et al., 2006), but accumulating evidence is highly suggestive for promoter-proximal regions to constitute only a small fraction of mammalian gene regulatory sequences. Indeed, members of the nuclear receptor family clearly display higher activity at enhancer rather than promoter binding sites, as evidenced by the invention and other investigators (e.g. Bolton et al., 2007). Consequently, studies based on promoter sequence containing arrays can be misleading.
  • the HNF4 ⁇ motif is highly enriched within the ChIP regions:
  • HNF4 ⁇ binding motifs are highly abundant within the ChIP regions. Using stringent criteria to minimize false positives, an up to > 14-fold enrichment was detected for different HNF4 ⁇ matrices compared to the genomic background (Table 3). This enrichment allowed an easy 'de novo' identification of the HNF4 ⁇ motif: Among 5 motifs identified by a Gibbs motif sampler, 2 motifs represented HNF4 ⁇ binding sites (Fig. 4).
  • regions with a low enrichment show a higher percentage of promoter- proximal binding sites, and a lower number of HNF4 ⁇ motifs, than regions with higher enrichment (Figure S3b). Therefore, it was demonstrated that often HNF4 ⁇ binding to promoter- proximal regions is indirect (due to interaction with other transcription factors), and therefore weaker. However, as HNF4 ⁇ motifs are still enriched in promoter-proximal regions it seems likely that HNF4 ⁇ can act as a promoter binding as well as an enhancer binding transcription factor.
  • Enhancer elements defined by HNF4 ⁇ display high conservation
  • the ChIP regions for overrepresented motifs were analyzed, using motif analysis programs from Biobase and Genomatix, as well as the tool CEAS.
  • motifs with the highest fold enrichment are matrices similar to the HNF4 ⁇ binding motif, e. g. the binding motifs for COUP-TF, PPAR or LEF1 (Table 3; Fig. 6).
  • These transcription factors are known to compete with HNF4 ⁇ for common binding sites (Galson et al., 1995; Hertz et al., 1995; Hertz et al., 1996; Dongol et al., 2007; For motif similarity see also Kielbasa et al., 2005).
  • HNF4 ⁇ many motifs dissimilar to HNF4 ⁇ , e.g. the binding motifs for HNF1 , AP1 , GATA transcription factors or CREB, where also enriched within the ChIP regions (Table 3; Supplementary tables S1 , S2 and S3; Fig. 7). Significant overrepresentation of these motifs has been further confirmed with different sets of ChIP regions defined by high- and low stringency cut-off criteria (data not shown). The corresponding transcription factors can therefore be expected to form composite modules with HNF4 ⁇ . If these factors act combinatorial with HNF4 ⁇ , it could be further expected that the frequency of their motifs increases with decreasing distance to the HNF4 ⁇ binding sites. Therefore, the frequency of their motifs relative to the HNF4 ⁇ binding sites was analyzed identified in this study.
  • HNF4 ⁇ and estrogen receptor (ER) binding motifs at flanking sequences were observed (Fig. 9). Enrichment of ER motifs, and possibly other motifs as well, could therefore be coincidental for enrichment of HNF4 ⁇ .
  • the genomic position of the highest scoring HNF4 ⁇ motif (highest likelihood ratio) within the ChIP regions was retrieved and extended for 500 nucleotides to both flanks. Within these sequences, other enriched motifs were detected and the distance to the HNF4 ⁇ motif (i.e.
  • Binding sites for ER and HNF4 ⁇ were compared, and a considerable overlap was found (Fig. 1 1). Using the low stringency set of HNF4 ⁇ binding sites binding, 15% of the ER binding sites were also targeted by HNF4 ⁇ , supporting the idea of cooperation between HNF4 ⁇ and the ER nuclear receptor.
  • ChlP-chip experiments were employed by Rada-lglesias et al (2005) to identify HNF4 ⁇ binding sites within the ENCODE regions in HepG2 hepatoma cells.
  • a ChlP-chip protocol was used by Odom et al. (2004) to identify binding sites within promoter regions in human hepatocytes and pancreatic islets.
  • the genomewide approach according to the invention was compared with the aforementioned studies to identify regions which overlap amongst these platforms, and the data according to the invention was compared to the binding sites reported in these publications (Fig. 12; Table 7).
  • enhancers appear to be promiscuous and can regulate multiple genes (West et al., 2005). Additionally, the genes with the TSS closest to the enhancer are not necessarily the ones regulated by this enhancer (Blackwood, 1998). Enhancer action can thus take place over hundreds of kilobases (Dekker et al.2008), and even cases of inter-chromosomal regulation by enhancers are known (Spilianakis et al., 2005). Nonetheless, most known enhancer elements are within 100000 nt of their respective TSS.
  • Enhancer-blocking insulators Activity of an enhancer is defined by enhancer-blocking insulators. If an active insulator is placed between an enhancer and a promoter, no communication between them, and therefore no activation of transcription by the enhancer, is possible. In vertebrates, binding of the protein CTCF to an insulator element is required for insulator function. Recently, genome-wide data of insulator regions became available. Kim et al. (2007) identified 13.804 CTCF binding sites. Furthermore, they compared insulator activity between different cell types, and found that CTCF localization is largely invariant.
  • HNF4 ⁇ is well known to have an important role in development (Sladek et al., 1990; Sladek et al, 1993), and is an essential driver for epithelial differentiation (Watt et al., 2003). Needless to say, HNF4 ⁇ ko mice die around E 8_5 due to deficient organ development.
  • HNF4 ⁇ can be seen as a regulator of an epithelial phenotype, and several lines of evidence suggest HNF4 ⁇ to play an essential role in activation of the expression of genes encoding cell junction molecules (Spa ' th and Weiss, 1998; Chiba et al., 2003; Parviz et al., 2003; Satohisa et al., 2005; Battle et al., 2006).
  • HNF4 ⁇ insulin receptor signaling.
  • HNF4 ⁇ is well known to control the insulin secretory pathway (Miura et al., 2006), and was linked to rare monogenic disorder, maturity-onset diabetes of the young (MODY), confirming its role in insulin signaling (Yamagata et al., 1996).
  • MODY maturity-onset diabetes of the young
  • the overrepresentation of GO categories representing well known functions of HNF4 ⁇ supports the general biological significance of the association of binding sites according to the invention with target genes.
  • HNF4 ⁇ gene targets Caco-2 cell cultures were treated with Aroclor 1254, a known HNF4 ⁇ inducer (Borlak et al., 2001). Indeed, after Aroclor 1254 treatment of Caco-2 cells, binding of the HNF4 ⁇ protein to the HNF1 promoter was increased (Niehof and Borlak, 2008). Caco-2 cells were treated with Aroclor 1254 for 48 and 72 hours. Strong induction of HNF4 ⁇ was confirmed by western blot analysis (Fig. 12) and increased binding activity by EMSA, and genome-wide expression profiling by microarray analyses was performed to search for regulated genes.
  • HNF4 ⁇ targets based on its overexpression in mammalian cell lines were compared. Essentially, a high overlap, ranging from 65 to 94 %, of the genes identified by expression profiling experiments was found (Table 6). Interestingly, the highest overlap was found with the targets identified by Sumi et al. (2007).
  • the approach used in this study differs from the others in the point that targets were not only identified by overexpression of HNF4 ⁇ , but also by knock down by si ' RNA, and only genes increased by overexpression and decreased by siRNA were reported as target genes. Therefore, targets reported here might be considered more reliable.
  • ChlP-chip assays genome-wide scans for transcription factor binding sites becomes feasible.
  • HNF4 ⁇ the transcription factor
  • HNF4 ⁇ binding sites achieved by the use of high resolution tiling array.
  • the regions surrounding the HNF4 ⁇ binding sites were analyzed for overrepresentation of transcription factor binding motifs from different databases with different algorithms, and it was shown for several motifs to be significantly overrepresented in these regions. Moreover, there are also motifs which are significantly underrepresented, such as CART. Based on a thorough and detailed analysis, it was possible to evidence a close relationship between HNF4 ⁇ and AP1 , GATA, ER or HNF1 binding sites. The close association of these motifs enabled to predict composite models formed by the corresponding transcription factors on bound enhancer elements.
  • HNF4 ⁇ While single cases of synergistic action of HNF4 ⁇ with HNF1 (Fourel et al.,1996), ER (Harnish et al., 1996) or GATA transcription factors (Sumi et al., 2007; Alrefai et al., 2007) are already known, to the best of our knowledge the data according to the invention is the first report to suggest a collaborative action of HNF4 ⁇ and AP1. While over 95% of the identified binding sites are promoter distal sequences identified as enhancer elements, promoter proximal sites are highly enriched compared to random controls. This suggests that HNF4 ⁇ can also interact directly with the basal transcriptional machinery. The actual number of promoter-proximal binding sites will even be higher, as transcription start sites only of RefSeq annotated genes were included.
  • enhancers could hardly be identified by available methods; their genome-wide mapping is only possible through the advent of ChlP-chip technology.
  • One of the most promising aspects of future research will therefore be the integration of all the transcription factor binding sites into a genome-wide map of enhancer elements, like it is currently done within the ENCODE regions (See The ENCODE Project Consortium, 2007).
  • Current methods for association of enhancers and their target genes are mostly restricted to CCC or related methods, which allow only the analysis of single enhancer elements, and are technically challenging (Dekker, 2006).
  • association of promoter-distal transcription factor binding sites with their target genes is mostly based on the search for the closest transcription start site or the closest gene.
  • insulator elements in determining enhancer activity has just been unravelled; Their position can be analyzed genome-wide (Dorman et al., 2007; Kim et al., 2007). Therefore, available data on insulator elements was used to narrow down the number of genes associated with the HNF4 ⁇ binding sites identified in the study according to the invention. Based on such analysis ⁇ 6000 RefSeq annotated gene targets were determined.
  • HNF4 ⁇ targeted genes will help to identify novel drug targets for the treatment of cancerous and metabolic disease. This principle has been demonstrated by the invention and other investigators based on identification of novel HNF4 ⁇ gene targets in liver cancer and metabolic diseases (Niehof and Borlak, 2005; Niehof and Borlak, 2005; Niehof and Borlak, 2008)
  • Caco-2 cells were obtained from and cultivated as recommended by Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), seeded with a density of 4 X 10 6 cells/75 cm 2 flask, and harvested after 11 days. Aroclor 1254 treatment was performed as described in Borlak et al. (2001).
  • Chromatin immunoprecipitation (ChIP)
  • Chromatin immunoprecipitation (ChIP) procedures were carried out as described by Weinmann et al. (2001) with some modifications. The samples were sonicated on ice until cross-linked chromatin was fragmented to approximately 0.2 to 1.6 kilobase pairs. Protein A Sepharose CLB4 (Amersham Biosciences, Freiburg, Germany) was blocked with 1 mg/ml bovine serum albumin and washed extensively before use. Chromatin preparations were precleared by incubation with "blocked" Protein A-Sepharose for 1 h at 4°C. Precleared chromatin from 2.5 X 10 7 cells was incubated with 1 ⁇ g of HNF4 ⁇ antibody and rotated at 4°C overnight.
  • ChIP chromatin immunoprecipitation
  • the GeneChlP WT Double-Stranded DNA Terminal Labeling Kit (P/N 900812) from Affymetrix was used. Fragmentation success was confirmed with the Agilent Bioanalyzer. The labeled samples were hybridized to Affymetrix Human tiling 2.0R arrays with a 35 base pair resolution.
  • Raw data (CEL-files generated by GCOS after scanning) were analyzed for enriched regions by three independent algorithms, TAS (Affymetrix/Cawley et al., 2004), MAT (Johnson et al., 2006) and Tilemap (Ji et al., 2005).
  • Initial cut off criteria were determined based on the detection of a weakly enriched positive control (OTC).
  • OTC weakly enriched positive control
  • ES Parameters for enrichment site (ES) detection were further improved based on the rate of HNF4 ⁇ -motives within the enriched regions, which was determined by application of the MATCH algorithm (KeI et al., 2003).
  • ChIP was performed as previously described (Niehof et al., 2005). ChIP-DNA from three independent experiments (122, 124 and f3) was used for enrichment validation.
  • Realtime PCR was performed on the Light Cycler (Roche Diagnostics, Mannheim, Germany) with the following conditions denaturation at 94°C for 120 s, extension at 72 0 C for different times, and fluorescence at different temperatures. Primer sequences, annealing times and temperatures, extension times and fluorescence temperatures are summarized in Table M1. The reaction was stopped after a total of 45 cycles, and at the end of each extension phase, fluorescence was observed and used for quantification within the linear range of amplification. ⁇ ct-values were calculated versus diluted total input, and normalization, i. e. calculation of ⁇ ct-values was performed using a ⁇ -actin negative control, HNF1 upstream. No enrichment of on negative control against the other was observed. Sequence conservation analysis
  • Enrichment site centers left or Peak positions detected by MAT (right) were extend to 200 bp in both directions, and analyzed with CEAS (Ji et al., 2006) for conservation and motif content.
  • CEAS extends the 200 bp genomic regions to 3000 bp, and calculates for each nucleotide the average conservation score, based on the high-quality phast- Cons (Siepel et al., 2005) information from the UCSC GoldenPath genome Resource. The average conservation scores were plotted against the nucleotides position.
  • Total RNA was isolated using QIAGEN's RNeasy total RNA isolation kit according to the manufacturer's recommendations. 10 ⁇ g of total RNA were prepared for hybridization using the respective Affymetrix kits according to the manufacturer's recommendations. Samples were hybridized to Affymetrix U133Plus2.0 genechip arrays. GCOS 1.4 was used to calculate the level of differential expression at each time point relative to 0 h.
  • HNF4 ⁇ gene targets Caco-2 cell cultures were treated with Aroclor 1254, a known HNF4 ⁇ inducer36. After Aroclor 1254 treatment of Caco-2 cells, binding of the HNF4 ⁇ protein to the HNF1 promoter was increased7. Caco-2 cells were treated with Aroclor 1254 for 48 and 72 hours, respectively. Strong induction of HNF4 ⁇ was confirmed by western blot analysis and increased binding activity was observed by EMSA (Fig. 7). A genome-wide expression profiling was performed by microarray analyses to search for regulated genes. Using stringent criteria, 536 RefSeq-annotated genes were identified to be regulated (Supplementary Table 4).
  • HNF4 ⁇ ChIP was performed and enrichment of novel HNF4 ⁇ binding sites detected by ChlP-chip was confirmed by real time PCR.
  • ChIP-DNA from three independent experiments was used. Normalization was performed using a ⁇ -actin negative control, and the values are shown as fold enrichment versus total input.
  • HNF1 upstream is a second negative control, located upstream of the known HNF4 ⁇ binding site in the HNF1 promoter, and is used to confirm the ⁇ -actin negative control.
  • Fig. 2 Distribution of ES relative to RefSeq loci. a) Location of HNF4 ⁇ binding sites relative to the closest transcription start sites (TSS) of RefSeq genes, compared to random distribution. The Y-axis gives the number of binding sites, and the X-axis the distance to closest TSS in nucleotides, b) Overrepresentation of HNF4 ⁇ ES in first, second an third introns of RefSeq annotated genes, relative to a random control regions.
  • TSS transcription start sites
  • Fig. 3 A) Each chromosome was divided into 150 'bins', and within each bin the number of ES was counted. In the grey line chart, the number of HNF4 ⁇ binding sites within each bin is represented as a single data point. Below each chromosome the minimum and maximum number of binding sites located in a single bin is given. B) The distribution of HNF4 ⁇ binding sites (grey line chart, upper half) is compared to the distribution of known genes on chromosome 10. Green arrows mark two gene-sparse regions, in which also hardly ES are found. The red arrows mark two region with a high number of HNF4 ⁇ binding sites and a low number of genes. Analyses where performed using the Ensembl tool Karyoview ( http://www.ensembl.org/Homo_sapiens/karyoview ).
  • HNF4 ⁇ ChlP-chip enrichment sites allow easy 'de novo' prediction of the HNF4 ⁇ binding motif.
  • the HNF4 ⁇ -motif as described by the TRANSFAC matrices M01031or M00134, was actually detected two times, with the second motif presenting only a half site.
  • Fig. 5 a) Conservation of all HNF4 ⁇ binding sites (blue line). Enrichment site centers (blue) or Peak positions (red) were extend to 1000 bp in both directions, and for each nucleotide the average conservation score, based on the high-quality phast-Cons (Siepel et al., 2005) information from the UCSC GoldenPath genome Resource, was calculated. The average conservation scores were plotted against the nucleotides position. Analyses where performed with CEAS (Ji et al., 2006). b) HNF4 ⁇ motifs in the area of 1000 bp surrounding enrichment site peak or center positions were detected with MATCH, using cut offs to minimize false positives.
  • the distance of the center of detected motifs to the peak or center position of the enrichment sites was calculated.
  • a Histogram was created using bins of 50 nucleotides around the center or peak positions.
  • the blue line shows the deviation of HNF4 ⁇ motifs relative to the enrichment site center, the red line shows the deviation relative to the peak position.
  • Fig. 6 Computational screens were performed for motifs which are enriched within the detected HNF4 ⁇ ChlP-chip enriched regions. Binding sites were defined as the area of 300 surround the peak positions. Besides the expected overrepresentation of HNF4 ⁇ -motifs, an enrichment of HNF4 ⁇ -similiar motifs like COUP-TF, PPAR or LEF was found. Similarity of the motifs is visualized by using Weblogo depiction (http://weblogo.berkeley.edu/). A complete list of enriched motifs can be found in Table 3.
  • Fig. 7 As described in Fig. 6, HNF4 ⁇ -dissimilar motifs are displayed. Dissimilarity is visiualized by using Weblogo.
  • Fig. 8 Distribution of AP1 , CART, ER, GATA2, HNF1 and SREBP motifs within the regions enriched by HNF4 ⁇ -ChlP, relative to the peak position (represented as 0). Enrichment site centers were extend to 500 nt in both directions, and Motifs were detected by use of the MATCH algorithm using cutoff criteria to minimize the sum of false positives and false negatives. Regions were segmented into bins of 25 nt, and the number of occurrences of the different motifs within each bin was counted.
  • Fig. 9 Display of the overlap between the binding motifs of HNF4 ⁇ and the estrogen receptor by use of Weblogo illustrations. Both motifs show an partial overlap.
  • Fig. 10 Plot of the relative distance of HNF4 ⁇ motifs to other motifs enriched in the ChIP region. Within ChIP regions the most conserved HNF4 ⁇ motifs where identified. The sequences of the 500 nucleotides surrounding these most conserved HNF4 ⁇ motifs where retrieved and analyzed for those motifs of other TF that were also enriched in the ChIP regions. Then, the distance between these motifs and the HNF4 ⁇ motif was calculated using Cisgenome for motif detection, and plotted as histogram, using bins of 20 nt. The HNF4 ⁇ motif is found at the center, reaching from nt -6 to nt +6.
  • Fig. 11 Overlap between estrogen receptor (ER) binding sites and HNF4 ⁇ binding sites.
  • Fig. 12 For Rada Iglesias, the Random Control group (600nt) was used. For Odom et al, a number of promoter regions from the Huk13 array used in their study equal to the number of promoters they detected as binding sites was selected by random. This was necessary, because Odom et al. tested only promoter regions, and these regions are highly enriched within binding sites identified in this study. Therefore, comparison to the Random Control group (600nt) would have a high negative bias.
  • Fig. S1 Venn diagram of overlap between ES identified by variable algorithms.
  • ES were identified by use of three different algorithms. Although different parameter settings (e. g. band width of 200, 300 and 400 nucleotides) and different algorithms were used, the overlap was surprisingly high.
  • the Venn diagram was calculated using the intersect function of Galaxy (Giardine et al., 2005).
  • FIG. S2 HNF4 ⁇ induction in Caco-2 cells.
  • Promoter-proximal ES might be indirect HNF4 ⁇ binding sites
  • 100 promoter-proximal ES (-138 to -2 relative to the TSS) were compared to 100 promoter-distal ES (-24972 to -23489 relative to the TSS) by the bootstrapping analysis tool POBO (Kankainen and Holm, 2004).
  • Promoter-proximal ES show a significantly lower number of HNF4 ⁇ motifs
  • b) ES (300bp surround the peak position) were sorted by their Pvalue (as calculated by the MAT algorithm) and divided into bins of 1000 ES. For each bin the number of HNF4 ⁇ motif occurrences and the percentage of promoter-proximal ES was calculated. As can be clearly seen, ES with a high Pvalue (weak ES) are more likely to be located promoter-proximal, and to contain no HNF4 ⁇ binding motif.

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Abstract

The invention is directed to the use of particular human genes, nucleic acids hybridizing to said genes, and gene products encoded thereby in the context of the diagnosis and/or therapy of metabolic and/or cancerous diseases, preferably of diabetes mellitus and/or colorectal cancer, wherein the gene is selected from the group of the human chromosomal genes having at least one expression regulatory sequence according to matrix 1 ("de novo" HNF4α matrix) in the range of 100000 nucleotides upstream or downstream of their transcription start site in the human genome, and wherein the at least one expression regulatory sequence according to matrix 1 is located within the chromosomal position specified by particular start and end sites. The invention further relates to a method for a genomewide identification of functional binding sites at specifically targeted DNA sequences with high resolution, wherein the method comprises, or preferably consists of, the steps of: a) chromatin immunoprecipitation and b) DNA-DNA hybridisation for the c) de novo identification of gene targets.

Description

Method for a genome wide identification of expression regulatory sequences and use of genes and molecules derived thereof for the diagnosis and therapy of metabolic and/or tumorous diseases
The invention is directed to the use of particular human genes, nucleic acids hybridizing to said genes, and gene products encoded thereby in the context of the diagnosis and/or therapy of metabolic and/or cancerous diseases, preferably of diabetes mellitus and/or colorectal cancer. The invention further relates to a method for a genomewide identification of functional binding sites at specifically targeted DNA sequences with high resolution. Areas of application are the life sciences: biology, biochemistry, biotechnology, medicine and medical technology.
Hepatic nuclear factor (HNF)-4α is a member of the nuclear rece?ptor superfamily and known to be expressed in the liver, intestine, and pancreas (for review see Sladek et al.f 2001 ; Schrem, 2002). Many reports have highlighted the importance of HNF4α in the regulation of developmental processes in determining the hepatic phenotype, as well as the regulation of diverse metabolic pathways (e.g., glucose, cholesterol, and fatty acid metabolism) (Sladek et al., 1990; Jiang et al., 1995; Yamagata et al., 1996; Hadzopoulou-Cladaras et al., 1997). Therefore, HNF4α is considered as a hepatic master regulatory protein, ln^contrast to other members of the nuclear receptor superfamily, HNF4α binds to its cognate DNA binding site as a homodimer (Jiang 1997; Sladek 1990). HNF4α is one of the best characterized transcription factors, and in the past some dozent direct binding sites were reported. The employment of ChlP-chip technologies demonstrated however that these are only the smallest fraction of the actual HNF4α binding sites. Notably, Rada-lglesias et al. (2005) used custom made arrays with a low resolution encompasing the ENCODE regions, i. e. 1 % of the genome, for ChlP-chip, and thus mapped 194 HNF4α binding sites in the human hepatoma cell line HepG2. In another study binding sites in hepatocytes and pancreatic islets were mapped, but the approach focused on promoter regions only (Odom et al, 2004; Odom et al, 2006). Based on the findings published by Rada-lglesias et al. (2005) only a small fraction of HNF4α binding sites are located in proximal promoter regions.
As of today, a genome-wide footprinting of binding sites targeted by HNF4α has not been reported.
However, such a system would allow to identify the common rationale underlying the genome wide regulation processes induced by protein-DNA binding within the context of diseases caused by HNF4α upregulation, such as adenocarcinomas of the colon may be, namely to identify specific regulatory sequences in the chromosomal genome which (a) bind to HNF4α and (b) bind to proteins other than HNF4α, wherein said proteins bind to HNF4α.
The aim of the invention is thus to provide a method allowing a genome-wide high resolution map of binding sites relevant for the transcription regulation induced by HNF4α, and the identification of human chromosomal genes having at least one specific regulatory sequence in their natural environment, and the use of said genes or gene products encoded thereby or of RNA hybridizing to said genes for the diagnosis and therapy of diseases, preferably of adenocarcinomas of the colon, caused by an deregulation of HNF4α
To this end, the implementation of the actions and embodiments as described in the claims provides appropriate means to fulfill these demands in a satisfying manner.
Thus, the invention in its different aspects and embodiments is implemented according to the claims.
In the first aspect, the invention is directed to the use of a human gene, in particular the coding region thereof, or of a gene product encoded thereby or of an antibody directed against said gene product, or of DNA or RNA sequences hybridizing to said gene and coding for a polypeptide having the function of said gene product for the therapy and/or diagnosis of metabolic and/or cancerous diseases and/or to screen for and to identify drugs against metabolic and/or cancerous diseases, such as diabetes mellitus and/or colorectal cancer may be, wherein the gene is selected from the group of the human chromosomal genes having at least one expression regulatory sequence according to matrix 1 ("de novo" HNF4α matrix) in the range of 100000 nucleotides upstream or downstream of their transcription start site in the human genome, and wherein the at least one expression regulatory sequence according to matrix 1 is located within the chromosomal position specified by the start and end sites according to Tables 9-32 (chromosomes 1-22, X-, Y-chromosome, wherein Table 9 refers to the human chromosome 1 , Table 10 refers to the human chromosome 2, etc., Table 30 refers to the human chromosome 22, Table 31 refers to the human X-chromosome, and Table 32 refers to the human Y-chromosome).
The term "gene" according to the invention is directed to both the template strand, which refers to the sequence of the DNA that is copied during the synthesis of mRNA, and to the coding strand corresponding to the codons that are translated into a protein. The genes according to the invention and gene products encoded thereby can be easily derived from the common databases, as such are known to the person skilled in the art, wherein the UCSC Genome Database is particularly preferred: Karolchik D, Kuhn, RM, Baertsch R, Barber GP, Clawson H, Diekhans M, Giardine B, Harte RA, Hinrichs AS1 Hsu F, Miller W, Pedersen JS, Pohl A, Raney BJ, Rhead B, Rosenbloom KR1 Smith KE, Stanke M, Thakkapallayil A, Trumbower H, Wang T1 Zweig AS, Haussler D, Kent WJ. The UCSC Genome Browser Database: 2008 update. Nucleic Acids Res. 2008 Jan;36:D773, which is incorporated herein by reference. The term "coding region" according to the invention is directed to the portion of DNA or RNA that is transcribed into the mRNA, which then is translated into a protein. This does not include gene regions such as a recognition site, initiator sequence, or termination sequence.
The term "RNA sequences hybridizing to said gene" or "RNA sequences, which hybridize to said gene", respectively, according to the invention relates to RNA molecules hybridizing with the template strand of said gene, in particular with the coding region. The term "DNA sequences hybridizing to said gene" or "DNA sequences, which hybridize to said gene", respectively, according to the invention preferably relates to DNA molecules hybridizing with the coding strand of said gene, in particular with the coding region thereof. The term "hybridizing" as used herein refers to conventional hybridization conditions, preferably to hybridization conditions under which the Tm value is between 37°C to 700C, preferably between 410C to 660C. [fl] An example for low stringency conditions is e.g. hybridisation under the conditions 42°C, 2*SSC, 0.1% SDS and an example for high stringency conditions is e.g. hybridization under the conditions: 65°C, 2*SSC, 0.1% SDS, wherein in the case that washing is necessary for equilibrium, the hybridization solution is used for the washings. Most preferably, the term hybridization refers to stringent hybridization conditions.
The term "function of said gene product" is directed to biological activities of the polypeptide encoded by the selected gene, wherein the biological activities may easily be derived from common gene or protein databases, such as the ncbi or the SWISS-Prot databases may be and the biological activities can be determined according to the literature provided therein. Within the context of the invention the term "transcription start site" refers to the start codon or initiation codon in eukaryotes, in particular to the respective DNA sequence ATG of the coding strand coding for methionin.
The term "colorectal cancer" within the context of the invention is in particular directed to adenocarcinoma of the colon, in particular to colorectal adenocarcinoma associated with an upregulation or hyperactivity of HNF4α.
The term "matrix" or "matrices" according to the invention is directed to position weight matrix/matrices (PWM(s)), which are known to the person skilled in the art.
In the matrix/matrices according to the invention, the first column specifies the position, the second column provides the number of occurrences of A at that position, the third column provides the number of occurrences of C at that position, the fourth column provides the number of occurrences of G at that position, the fifth column provides the number of occurrences of T at that position, such that the matrix contains log odds weights for computing a match score and provides a likelihood ratio to specify whether an input sequence matches the motif or not.The cut-offs for the matrices according to the invention are preferably set to minFN to maximize the sensitivity of the site prediction (false negative rate of 10%).
Figure imgf000005_0001
In one embodiment of the invention the gene is selected from the group of genes, wherein genes having an expression regulatory sequence according to matrix 2 (CTCF matrix) between the transcription start site and the at least one expression regulatory sequence (matrix 1 ("de novo" HNF4α matrix)) in the human genome are excluded from the group of genes.
Figure imgf000005_0002
Figure imgf000006_0001
In another preferred embodiment of the invention the gene is selected from the group of genes further having at least one expression regulatory sequence selected from the group matrices 3- 6 (AP1 , GATA2, ER, HNF1 matrices), preferably according to matrix 3, in the range of 20-60 nucleotides upstream or downstream of the at least one expression regulatory sequence according to matrix 1 ("de novo" HNF4α matrix) in the human genome.
Figure imgf000006_0002
Figure imgf000007_0001
In a further preferred embodiment of the invention the gene is selected from the group of genes, wherein genes having an expression regulatory sequence according to matrix 7 (CART1 matrix) in the range of 20-60 nucleotides upstream or downstream of the at least one expression regulatory sequence according to matrix 1 ("de novo" HNF4α matrix)) in the human genome are excluded from the group of genes.
Figure imgf000008_0001
In another preferred embodiment of the invention, the gene is selected from the group of genes located on the human chromosome 6 or the human chromosome 10, wherein genes located on chromosome 10 are particularly preferred.
In yet another preferred embodiment of the invention, the gene is selected from the group of genes coded at least partially in the genomic regions according to table 33, which represent high density clusters of identified HNF4α binding sites.
Figure imgf000008_0002
Figure imgf000009_0001
In a particular preferred embodiment of the invention the gene is selected from the group of the genes according to Table 34.
Table 34 - list of human chromosomal genes: 7A5, A2M, A4GALT, AAA1 , AADAC, AAK1 , AARS, AASDH, ABAT, ABC1, ABCA1, ABCA10, ABCA3, ABCA8, ABCA9, ABCB6, ABCC10, ABCC11, ABCC4, ABCD2, ABCD3, ABCG2, ABCG5, ABHD12, ABHD2, ABHD3, ABHD4, ABHD5, ABHD6, ABHD7, ABM, ABI3BP, ABL1, ABL2, ABLIM1, ABLIM3, ABP1, ABR, ABRA, ABTB2, ACACA, ACADM, ACADS, ACAT2, ACBD3, ACBD5, ACBD6, ACCN1, ACE2, ACHE, ACIN1, ACOI.ACOTI.ACOTH, ACOT12, ACOT2, ACOT6, ACOT7, ACOX2, ACP1, ACPL2, ACPP, ACPT, ACRC, ACSL1, ACSL3, ACSL4, ACSL5, ACSL6, ACSM2, ACSS1, ACSS2, ACTG1, ACTL6A, ACTL8, ACTN4, ACTR10, ACTR2, ACTR3B, ACTR6, ACVR1B, ACVR2A, ACY3, ACYP2, ADA, ADAM10, ADAM22, ADAM7, ADAMDECI.ADAMTSIO, ADAMTS12, ADAMTS14, ADAMTS15, ADAMTS16, ADAMTS19, ADAMTS6, ADAMTSL3, ADAMTSL4, ADAR1 ADARB1, ADCK5, ADCY5, ADCY6, ADCY8, ADCY9, ADD1, ADD3, ADFP, ADH1A, ADH4, ADH7, ADM, ADIPOQ1 ADIPOR2, ADK1 ADM, ADORA2A, ADORA2B, ADPRHL1, ADRA1B, ADRA1D, ADRB1, AFAP, AFF1, AFF3, AFF4, AFG3L2, AFM, AFMID, AFP1 AGBL2, AGGF1, AGPAT2, AGPAT3, AGR2, AGTR1, AGTRAP, AHCTF1, AHCY1 AHM1 AHNAK1 AHR, AHSA1, AIF1, AIFM2, AIG1, AIM1, AIM1L, AK3L1, AK3L2, AKAP1, AKAP2, AKAP4, AKAP6, AKAP7, AKR1A1, AKR1B10, AKR1C1, AKR1CL2, AKR1D1, ALB, ALDH18A1, ALDH1L1, ALDH3A2, ALDH6A1, ALDOB, ALG14, ALG8, ALG9, ALKBH5, ALKBH7, ALMS1, ALOX5AP, ALOXE3, ALS2CR7, AMAC 1, AMAC 1L2, AMD1, AMDHD1, AMDHD2, AMELX, AMFR, AMICA1, AMIGO1, AMIGO2, AMMECR1, AMOTL1, AMPD2, AMZ2, ANAPC4, ANGEL2, ANGPTL2, ANGPTL3, ANK2, ANK3, ANKFN1 , ANKFY1 , ANKH, ANKRD1 , ANKRD11 , ANKRD13A, ANKRD15, ANKRD30A, ANKRD38, ANKRD39, ANKRD49, ANKRD55, ANKRD56, ANKRD57, ANKRD9, ANKS1A, ANKS4B, ANKZF1, ANP32A, ANTXR2, ANXA11 , ANXA2, ANXA3, ANXA4, ANXA8, AOAH, AOF2, AP1B1, AP1S3, AP2A2, AP3S1, AP3S2, AP4S1, APBB1, APC1 APCDD1, APCDD1L, APEX2, APH1B, APLP2, APOBEC1, APOBEC2, APOBEC3A, APOBEC4, APOE, APOF, APOLD1, APOM, AQP11, AQP4, AQP8, AR, ARCN1, AREG, ARF1, ARF6, ARFGAP3, ARFGEF2, ARHGAP10, ARHGAP12, ARHGAP17, ARHGAP18, ARHGAP19. ARHGAP21, ARHGAP24, ARHGAP25, ARHGAP27, ARHGAP29, ARHGAP5, ARHGAP6, ARHGAP8, ARHGEF1, ARHGEF10L, ARHGEF11, ARHGEF12, ARHGEF18, ARHGEF3, ARHGEF5, ARID1A, ARID1B, ARID3A, ARID3B, ARID4A, ARID5B, ARL14, ARL15, ARL4A, ARL4C, ARL5A, ARL5B, ARL6IP2, ARL8B, ARMC1 , ARMC2, ARMC4, ARNTL, ARPC5L, ARPM1, ARPP-19, ARRB1, ARRB2, ARRDC2, ARRDC4, ARSB, ARSD, ARSH, ARSJ, ARTS-1, ARX, AS3MT, ASAH1, ASAH2, ASAHL, ASAM, ASB1, ASB13, ASB14, ASB18, ASB2, ASB4, ASB9, ASCC3, ASCL1, ASF1A, ASH1L, ASH2L, ASIP, ASL, ASPA, ASPH, ASS1, ASTE1, ASTN2, ASXL1, ATAD4, ATBF1, ATF3, ATF7IP, ATG16L2, ATG3, ATG5, ATIC, ATOH1, ATOX1, ATP10A, ATP11A, ATP13A2, ATP13A5, ATP1A1, ATP1A2, ATP1B1, ATP2A2, ATP2B1, ATP2B2, ATP2B4, ATP2C2, ATP5E, ATP5G1, ATP5H, ATP5I, ATP6V0A4, ATP6V0D2, ATP6V1A, ATP6V1B2, ATP6V1E1, ATP6V1E2, ATP6V1G1, ATP6V1G3, ATP8A1, ATP8B1, ATP8B4, ATP9A, ATP9B, ATPAF1, ATPIF1, ATRN1 ATXN1, ATXN10, ATXN2, ATXN7, ATXN7L4, AUH, AVPR1A, AXIN1, AXIN2, AYTL2, B3GALNT1, B3GALNT2, B3GALT1, B3GALT5, B3GALTL, B3GNT2, B3GNT3, B3GNT5, B4GALNT2, B4GALNT3, B4GALT1, B4GALT4, B4GALT5, B4GALT6, BAAT1 BACH1, BAG2, BAG3, BAHD1, BAIAP2L1, BAIAP2L2, BAMBI, BANK1, BARX2, BATF, BAZ2A, BBC3, BBOX1, BCAR3, BCAS1, BCAS3, BCKDHB, BCL10, BCL2, BCL2A1, BCL2L10, BCL2L11, BCL2L14, BCL9, BCL9L, BCLAF1, BCMO1, BCMP11, BCOR, BDH1, BDKRB1, BDKRB2, BDNF, BEST3, BID, BIN1 , BIRC2, BLCAP, BLM, BLNK, BLOC1S2, BLVRA, BMF, BMP2, BMP2K, BMP3, BMP4, BMP7, BMP8A, BMP8B, BMPR1A, BMS1 L, BOLA1 , BPNT1 , BRAF, BRD2, BRD8, BRE BRI3, BRI3BP, BRP44, BRP44L, BRPF3, BRWD1 , BRWD2, BRWD3, BTAF1 , BTBD1 , BTBD11 , BTBD16, BTBD3, BTBD5, BTBD9, BTC, BTF3L4, BTG2, BTN 1A1 , BTN3A2, BTNL2, BTNL3, BTNL8, BUB1 , BUD13, BVES, BZW1 , BZW2, C10orf107, C10orf108, C10orf1 1 , C10orf1 14, C10orf1 18, C10orf12, C10orf125, C10orf132, C10orf22, C10orf25, C10orf28, C10orf30, C10orf32, C10orf4, C10orf46, C10orf47, C10orf53, C10orf54, C10orf56, C10orf57, C10orf58, C10orf6, C10orf67, C10orf79, C10orf81 , C10orf82, C10orf85, C10orf88, C10orf9, C10orf92, C10orf96, C11orf11 , C11orf37, C11orf44, C11orf47, C11orf49, C11orf52, C1 1orf54, C11orf56, C11orf69, C11orf75, C11orf77, C12orf28, C12orf33, C12orf34, C12orf35, C12orf39, C12orf4, C12orf42, C12orf43, C12orf45, C12orf49, C12orf59, C12orf62, C13orf1 , C13orf16, C13orf18, C13orf21 , C13orf23, C13orf3, C13orf7, C14orf100, C14orf101 , C14orf102, C14orf103, C14orf104, C14orf105, C14orf108, C14orf11 , C14orf1 12, C14orf115, C14orf1 18, C14orf135, C14orf140, C14orf147, C14orf155, C14orf156, C14orf159, C14orf161 , C14orf165, C14orf166B, C14orf169, C14orf174, C14orf177, C14orf179, C14orf29, C14orf32, C14orf4, C14orf43, C14orf46, C14orf94, C15orf21 , C15orf28, C15orf34, C15orf38, C15orf39, C15orf41 , C15orf48, C16orf5, C16orf51 , C16orf69, C16orf70, C17orf27, C17orf46, C17orf50, C17orf54, C17orf57, C17orf58, C17orf59, C17orf61 , C17orf76, C17orf78, C17orf79, C17orf85, C18orf17, C18orf19, C18orf37, C18orf43, C18orf45, C18orf56, C19orf12, C19orf21 , C19orf33, C19orf43, C19orf54, C1GALT1, C1orf100, C1orf103, C1orf104, C1orf105, C1orf106, C1orf107, C1orf108, C1 orf110, C1orf113, C1orf114, C1orf115, C1orf116, C1orf121 , C1orf123, C1orf125, C1orf129, C1 orf130, C1orf131 , C1orf137, C1orf14, C1orf141 , C1orf144, C1 orf146, C1orf151 , C1orf156, C1orf161, C1orf163, C1orf164, C1orf168, C1orf174, C1orf175, C1orf180, C1orf181 , C1orf19, C1orf191 , C1orf210, C1orf215, C1orf24, C1orf42, C1orf45, C1orf50, C1orf55, C1orf61 , C1orf62, C1orf63, C1orf71 , C1 orf74, C1orf82, C1orf83, C1orf85,i C1orf87, C1orf9, C1orf96, C1QL3, C1QTNF1 , C1QTNF3, C1QTNF9, C20orf112, C20orf118, C20orf121 , C20orf175, C20orf179, C20orf19, C20orf196, C20orf197, C20orf23, C20orf24, C20orf38, C20orf39, C20orf42, C20orf46, C20orf52, C20orf67, C20orf74, C20orf75, C21orf125, C21orf129, C21orf24, C21orf25, C21orf34, C21orf7, C21orf88, C21orf91 , C22orf13, C22orf29, C22orf31 , C22orf9, C2orf25, C2orf27, C2orf28, C2orf29, C2orf38, C2orf39, C2orf40, C2orf43, C2orf46, C2orf47, C2orf49, C3orf15, C3orf19, C3orf20, C3orf25, C3orf26, C3orf32, C3orf33, C3orf34, C3orf52, C3orf59, C3orf62, C4BPB, C4orf11 , C4orf16, C4orf18, C4orf19, C4orf24, C4orf31 , C4orf32, C4orf33, C4orf34, C4orf36, C5AR1 , C5orf15, C5orf16, C5orf21 , C5orf28, C5orf32, C5orf33, C5orf35, C5orf4, C6, C6orf10, C6orf105, C6orf106, C6orf107, C6orf120, C6orf122, C6orf128, C6orf130, C6orf141 , C6orf142, C6orf15, C6orf151 , C6orf153, C6orf166, C6orf170, C6orf182, C6orf195, C6orf213, C6orf218, C6orf48, C6orf57, C6orf60, C6orf70, C6orf72, C6orf75, C6orf81 , C6orf85, C6orf96, C6orf97, C7orf23, C7orf30, C7orf36, C8A, C8orf13, C8orf22, C8orf38, C8orf40, C8orf41 , C8orf42, C8orf54, C8orf70, C8orf78, C8ORFK23, C8ORFK32, C9orf102, C9orf103, C9orf11 , C9orf123, C9orf126, C9orf127, C9orf152, C9orf18, C9orf24, C9orf3, C9orf30, C9orf32, C9orf4, C9orf41 , C9orf52, C9orf64, C9orf66, C9orf72, C9orf80, C9orf91 , C9orf97, CA1 , CA13, CA2, CA4, CA7, CA8, CA9, CAB39, CABLES1 , CACHD1 , CACNA1 D, CACNA2D4, CACNB2, CACNB3, CACNG3, CACNG4, CADPS, CALCOCO2, CALCR, CALCRL, CALD1 , CALM1 , CALM2, CALM3, CALML3, CALML4, CALML5, CAMK1 , CAMK1 D, CAMK2G, CAMK2N1 , CAMTA1 , CAND1 , CANX, CAP1 , CAPG, CAPN10, CAPN13, CAPN2, CAPN3, CAPN7, CAPN9, CAPZA3, CAPZB, CARD10, CARHSP1 , CARKL, CART1 , CARTPT, CASC3, CASC4, CASC5, CASD1 , CASK, CASP10, CASP3, CASQ2, CAST, CASZ1 , CAT, CATSPER2, CATSPER3, CAV2, CBLB, CBR1 , CBWD1 , CBWD2, CBWD3, CBWD5, CBX2, CBX4, CCBP2, CCDC100, CCDC103, CCDC107, CCDC108, CCDC112, CCDC113, CCDC12, CCDC125, CCDC129, CCDC13, CCDC130, CCDC25, CCDC28A, CCDC3, CCDC34, CCDC41 , CCDC44, CCDC50, CCDC54, CCDC58, CCDC6, CCDC64, CCDC65, CCDC68, CCDC71 , CCDC90A, CCDC90B, CCDC91 , CCDC92, CCDC97, CCDC99, CCKAR, CCL14, CCL15, CCL16, CCL20, CCL4, CCM2, CCNA2, CCND1 , CCND2, CCNE2, CCNJ, CCNJL, CCNL1 , CCNT2, CCR1 , CCR4, CCR6, CCRK, CCRL1 , CCRN4L, CCT3, CD160, CD164, CD180, CD200, CD200R2, CD244, CD247, CD28, CD2AP, CD34, CD36, CD3E, CD44, CD46, CD55, CD58, CD74, CD86, CD8A, CD9, CD99, CDC14B, CDC2, CDC25C, CDC26, CDC2L6, CDC40, CDC42BPA, CDC42EP4, CDC42EP5, CDC42SE2, CDC91 L1 , CDCA2, CDCA3, CDCA7L, CDCP1 , CDGAP, CDH1 , CDH11 , CDH12, CDH13, CDH17, CDH26, CDH6, CDH7, CDH9, CDK10, CDK3, CDK4, CDK5R1, CDK5RAP2, CDK7, CDKAL1, CDKL1, CDKL2, CDKN1A, CDKN2B, CDKN3, CDON, CDR2, CDRT1, CDRT4, CDS1, CDS2, CDT1, CDV3, CDX2, CDYL, CDYL2, CEACAM3, CEACAM6, CEBPD, CEBPG, CECR5, CEL1 CENPF, CENTD1, CENTD2, CENTD3, CENTG2, CEP135, CEP152, CEP192, CEP250, CEP350, CEP68, CEP70, CERK, CERKL1 CES1, CFB, CFC1, CFLAR, CFTR1 CG018, CGM 15, CHAC1, CHAF1A, CHCHD3, CHCHD4, CHCHD5, CHCHD6, CHCHD8, CHD1, CHD2, CHD6, CHDH, CHES1, CHGB, ChGn, CHKA, CHML, CHMP1B, CHMP2B, CHMP4B, CHN2, CHORDC1, CHPT1, CHRDL2, CHRM1, CHRM2, CHRM4, CHRNA4, CHRNA9, CHRNB3, CHRND1 CHRNE, CHST12, CHST13, CHST3, CHST9, CIAS1, CLASP1, CLCA4, CLCN5, CLDN1, CLDN12, CLDN14, CLDN16, CLDN23, CLDN4, CLDN7, CLDN9, CLEC3A, CLIC1, CLIC2, CLIC4, CLIC5, CLINT1, CLLU1OS, CLMN, CLSTN1, CMAS, CMIP, CMPK1 CMTM4, CMTM6, CMTM7, CMTM8, CNBP, CNDP1, CNDP2, CNFN, CNIH, CNIH4, CNKSR3, CNN3, CNNM2, CNNM3, CNOT1, CNP, CNTF, CNTN2, CNTN4, CNTNAP4, CNTNAP5, COBL1 COCH, COG1, COG3, COG5, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL22A1, COL28A1, COL2A1, COL4A1, COL5A2, COL6A1, COL9A1, COLEC12, COMMD10, COMMD2, COMMD7, COP1, COPA1 COPS4, COPZ1, COQ10A, COQ2, COQ5, COQ6, CORO2A, CORO6, COTL1, COVA1, COX17, COX18, COX19, COX4I2, COX5A, COX6A2, COX7B2, CP, CPB1, CPD, CPE, CPEB2, CPEB4, CPLX2, CPM1 CPN1, CPNE3, CPNE7, CPNE8, CPNE9, CPO, CPS1, CPSF6, CPVL, CPXM2, CR2, CRABP1, CRB1, CREB1, CREB3L1, CREB3L2, CREB3L3, CREB5, CREG1, CREG2, CREM1 CRIM1, CRISP3, CRISPLD1, CRLS1, CROP, CRSP2, CRSP8, CRTC2, CRTC3, CRYAA, CRYGC1 CRYGD1 CRYGS1 CSDE1, CSF1R, CSF3R, CSNK1A1, CSNK1A1L, CSNK1D, CSRP1, CSRP2BP, CSRP3, CSS3, CST3, CTAGE1, CTAGE5, CTCFL, CTDSP2, CTGF, CTGLF1, CTGLF5, CTHRC1, CTNNB1, CTNNBL1, CTNND1, CTPS, CTSB, CTSC, CTSH, CTSK, CTSL, CTSL2, CTSO1 CTTNBP2, CTXN3, CUBN1 CUEDC1, CUEDC2, CUGBP2, CUL1, CUL3, CUL4A, CUTL1, CWF19L2, CXADR1 CXCL2, CXCL3, CXCL5, CXCL9, CXCR7, CXorf15, CXorf21, CXorf23, CXorfθ, CXXC4, CXXC5, CXXC6, CYB561, CYB5A, CYB5D1, CYB5R4, CYCS1 CYFIP2, CYGB, CYLN2, CYP19A1, CYP1A1, CYP26C1, CYP27A1, CYP2A6, CYP2A7, CYP2C18, CYP2C19, CYP2C9, CYP2R1, CYP2S1, CYP2U1, CYP3A4, CYP4A11, CYP4B1, CYP7A1, CYR61, CYSLTR2, CYYR1, DAB2, DAB2IP, DACT2, DAK, DAOA, DAP1 DAPK1, DAPK2, DARS1 DAZL, DBC1, DBX2, DCAKD, DCBLD1, DCC1 DCP1A, DCP1B, DCP2, DCTD, DCTN2, DCTN6, DCUN1D1, DCUN1D3, DCUN1D4, DDAH1, DDB2, DDC, DDEF1, DDHD2, DDH, DDI2, DDIT4, DDO, DDR2, DDX25, DDX3X, DDX5, DDX50, DDX52, DDX58, DEF6, DEFA3, DEFA5, DEFB112, DEGS1, DENND1A, DENND1B, DENND2D, DENND3, DENND4A, DEPDC6, DEPDC7, DERA1 DERL1, DEXI1 DFNA5, DGAT1, DGAT2, DGKA1 DGKB1 DGKI1 DHCR24, DHFR, DHRS3, DHRS8, DHRSX1 DHX15, DHX29, DHX35, DIAPH1, DIAPH2, DIAPH3, DID01, DI01, DI03, DIP13B, DIP2B, DIP2C, DIRC2, DISC1, DISP1, DIXDC1, DKFZp434N035, DKFZp686l15217, DKFZp686K16132, DKFZp686L1814, DKFZp779B1540, DKK1, DLC1, DLD1 DLEC1, DLG1, DLG2, DLG5, DLGAP1, DLGAP2, DLGAP4, DLX1, DMD, DMGDH, DMTF1, DMXL1, DMXL2, DNAH1, DNAH10, DNAH11; DNAH7, DNAHL1, DNAJB13, DNAJB14, DNAJB7, DNAJC11, DNAJC12, DNAJC13, DNAJC15, DNAJC18, DNAJC19, DNAJC5, DNAJC5B, DNAJC9, DNAL1, DNAPTP6, DNASE1L3, DNMBP, DNMT3A, DOC1, DOCK10, DOCK11, DOCK4, DOCK5, DOCK7, D0CK9, DOK4, D0T1L, DPP10, DPP4, DPPA3, DPPA4, DPT, DPYD, DPYS, DPYSL2, DPYSL3, DPYSL4, DR1, DRD2, DRP2, DSC2, DSCR1, DSCR1L1, DSCR2, DSG1, DSG2, DSG4, DSP1 DST, DTL, DTNA, DTNB1 DTNBP1, DTX2, DUOX2, DUPD1, DUSP1, DUSP15, DUSP16, DUSP18, DUSP21, DUSP22, DUSP5, DUSP7, DUSP9, DUT1 DYDC2, DYNC1I2, DYNC1LI1, DYNLL2, DYRK2, DZIP1, E2F3, E2F7, E2F8, EBAG9, EBI2, EBP, EBPL, ECE1, ECHDC1, ECHDC2, ECM2, EDAR1 EDC3, EDD1, EDEM1, EDEM3, EDG1, EDN1, EDN3, EEA1, EEF1A1, EEF1G, EEF2K, EEFSEC, EFCAB1, EFCAB2, EFCAB3, EFCAB4B, EFCBP1, EFEMP1, EFHA1, EFHC2, EFHD2, EFNA1, EFNB1, EFNB2, EFTUD1, EGF1 EGFLAM, EGFR, EGLN1, EGLN3, EGR1, EHBP1, EHD1, EHD4, EHF, EID1, EIF1AX, EIF2AK3, EIF2B2, EIF3S10, EIF4A2, EIF4B, EIF5A2, EIF5B, ELAC2, ELAVL3, ELF1, ELF4, ELK3, ELK4, ELL2, ELMO1, ELM02, EL0VL2, EL0VL5, EL0VL6, ELOVL7, ELP3, EMG1, EMILIN2, EML1, EML4, EMP1, ENAH, EN0SF1, ENPEP, ENPP1, ENPP2, ENPP3, ENPP6, ENPP7, ENTHD1, ENTPD5, ENTPD7, ENY2, EP300, EPAS1, EPB41, EPB41L1, EPB41L2, EPB41L3, EPB41L4B, EPDR1, EPHA10, EPHB3, EPHX2, EPPB9, EPS15, EPS8, EPSTM, ERAL1, ERBB2, ERCC3, ERG, ERGIC1, ERGIC2, ERN1, ERRFI1, ESD, ESF1, ESRRG, ESSPL, ESX1, ETAA1, ETF1, ETFA, ETFB, ETFDH, ETHE1, ETNK1, ETNK2, ETS1, ETS2, ETV1, ETV3, ETV4, ETV6, EVL, EXOC5, EXOC6, EXOSC8, EXOSC9, EXT1, EXTL3, EYA2, EYA3, F11R, F13A1, F13B, F2, F2R, F2RL1, F3, F5, FA2H, FABP1, FABP2, FABP6, FADS1, FAH, FAHD1, FAHD2A, FAIM, FAIM3, FAM100B, FAM101A, FAM104B, FAM105A, FAM105B, FAM107B, FAM109A, FAM110C, FAM113B, FAM114A1, FAM120AOS, FAM120B, FAM124A, FAM124B, FAM125B, FAM13A1, FAM13C1, FAM19A4, FAM20A, FAM20B, FAM20C, FAM35A, FAM36A, FAM38B, FAM3B, FAM3C, FAM40A, FAM40B, FAM43A, FAM45B, FAM46A, FAM46C, FAM47A, FAM49A, FAM50B, FAM54A, FAM60A, FAM62C, FAM63B, FAM70B, FAM76A, FAM78A, FAM78B, FAM7A2, FAM82A, FAM82C, FAM83B, FAM83C, FAM83D, FAM83F, FAM84B, FAM86A, FAM86B1, FAM86C, FAM8A1, FAM91A1, FAM92B, FAM9A, FAM9B, FAM9C, FANCC, FARP2, FARS2, FARSLB, FASTKD1, FASTKD3, FAT, FBLIM1, FBLN5, FBN1, FBP1, FBP2, FBXL10, FBXL11, FBXL14, FBXL17, FBXL18, FBXL2, FBXL20, FBXL21, FBXO16, FBXO17, FBXO25, FBXO27, FBXO28, FBXO31, FBXO32, FBXO33, FBXO38, FBXO39, FBXO6, FBXW10, FBXW4, FBXW7, FCAMR, FCER1A, FCER1G, FCGR1B, FCGR2A, FCHO2, FCHSD2, FCMD, FCRL3, FCRLB, FDFT1, FEM1C, FER1L3, FEZ2, FEZF2, FGA, FGB, FGD4, FGD5, FGF12, FGF14, FGF19, FGF2, FGF23, FGF8, FGF9, FGFR1OP, FGFR2, FGG, FGL1, FGL2, FHL1, FHL2, FHL5, FILIP1, F1P1L1, FKHL18, FKSG44, FLCN, FLJ10154, FLJ10159, FLJ10847, FLJ10986, FLJ11171, FLJ11506, FLJ12118, FLJ12331, FLJ13197, FLJ13236, FLJ14154, FLJ16124, FLJ16237, FLJ16641, FLJ16793, FLJ20054, FLJ20152, FLJ20184, FLJ20273, FLJ20294, FLJ20366, FLJ20674, FLJ20920, FLJ21062, FLJ21075, FLJ21127, FLJ21865, FLJ22374, FLJ22531, FLJ23834, FLJ23861, FLJ25371, FLJ25439, FLJ25476, FLJ25530, FLJ25680, FLJ25801, FLJ27505, FLJ30277, FLJ30934, FLJ30990, FLJ31196, FLJ31951, FLJ32310, FLJ32312, FLJ32549, FLJ32784, FLJ32894, FLJ34870, FLJ35773, FLJ35776, FLJ35779, FLJ36004, FLJ37357, FLJ37953, FLJ38377, FLJ38964, FLJ39198, FLJ39531, FLJ39653, FLJ39739, FLJ39779, FLJ39822, FLJ40142, FLJ40243, FLJ40296, FLJ40432, FLJ41603, FLJ42117, FLJ42133, FLJ42280, FLJ42562, FLJ42842, FLJ42953, FLJ42957, FLJ43505, FLJ44006, FLJ44048, FLJ44290, FLJ44796, FLJ45079, FLJ45139, FLJ45187, FLJ45244, FLJ45248, FLJ45337, FLJ45422, FLJ45557, FLJ45721, FLJ45803, FLJ45831, FLJ46210, FLJ46257, FLJ90709, FLNB, FLNC, FLT1, FLVCR, FMNL2, FMO2, FMO3, FMO4, FMO5, FMOD, FN1, FN3K, FNBP1, FNDC3B, FNDC6, FNDC7, FNIP1, FOLH1, FOS1 FOXA2, FOXB1, FOXC1, FOXD1, FOXD2, FOXD3, FOXD4, FOXD4L1, FOXD4L2, FOXD4L4, FOXE3, FOXJ1, FOXJ3, FOXL1, FOXM1, FOXN1, FOXN2, FOXO3A, FOXP1, FOXP4, FOXQ1, FRAS1, FREM2, FRMD1, FRMD3, FRMD5, FRMD6, FRMD7, FRMPD2, FRRS1, FRS2, FRY, FRZB, FSD1L, FSD2, FSIP1, FSIP2, FTHL17, FTS, FUCA2, FUT11, FUT4, FUT8, FVT1, FYN, FZD10, FZD4, FZD5, FZD7, G3BP1, G6PC2, GAB2, GADD45A, GAL3ST1, GALC, GALK1, GALM, GALNT10, GALNT11, GALNT14, GALNT2, GALNT3, GALNT5, GALNT6, GALNT8, GALNTL1, GALNTL5, GALP, GAN, GANAB, GANC, GAP43, GAPVD1, GARNL4, GAS2, GAS6, GATA1, GATA3, GATA4, GATA5, GATA6, GATAD1, GATAD2A, GATM, GATS, GBA3, GBGT1, GBP1, GCC2, GCH1, GCLC, GCM1, GCNT2, GCNT3, GCNT4, Gcomi, GCSH, GDA, GDF10, GDF7, GDF9, GDI2, GDPD4, GDPD5, GEM, GEMIN6, GENX-3414, GFRA1, GFRA4, GFRAL, GGH1 GGT2, GHDC, GHR, GHRH, GIMAP7, GINS2, GIT2, GJA1, GJA5, GJA7, GJB6, GK, GK5, GLCCM, GLCE, GLDC, GLG1, GLI4, GLIS3, GLP1R, GLRX, GLRX5, GLS, GLS2, GLT1D1, GLTP, GLTSCR1, GLUD1, GLUL, GLULD1, GLYATL2, GLYCTK, GMCL1, GMFB, GMNN, GMPPB, GMPR, GMPR2, GNA11, GNA12, GNA13, GNAO1, GNAQ, GNAS, GNAT2, GNG12, GNG3, GNG4, GNGT2, GNPNAT1, GNPTAB, GNRH1, GNRH2, GOLPH2, GOLPH3, GOLPH3L, GOLT1A, GORASP2, GOT2, GP5, GPA33, GPAM, GPATC1, GPATC2, GPBP1L1, GPC3, GPC5, GPD1, GPD1L, GPD2, GPHB5, GPIAP1, GPM6A, GPR101, GPR109B, GPR110, GPR116, GPR119, GPR124, GPR125, GPR126, GPR128, GPR132, GPR133, GPR135, GPR137B, GPR148, GPR15, GPR157, GPR160, GPR161, GPR176, GPR177, GPR21, GPR31, GPR37L1, GPR55, GPR63, GPR68, GPR85, GPR88, GPR89A, GPRASP1, GPRC5A, GPRC5C, GPSM2, GPSN2, GPT2, GPX3, GPX4, GPX6, GRAMD2, GRAMD3, GRB10, GRB14, GRB7, GREM1, GRHL2, GRIA1, GRID1, GRIK4, GRIN2A, GRIN2B, GRIN3A, GRINL1A, GRK5, GRK7, GRLF1, GRPEL1, GRPEL2, GRTP1, GSC, GSDML, GSH1, GSH2, GSN, GSR1 GSTA1. GSTA2, GSTA5, GSTCD, GSTK1, GSTO1, GSTP1, GTDC1, GTF2A1, GTF2H5, GTF2IRD1, GTPBP4, GUCA1B, GUCA1C, GUCA2B, GUCY2C, GUCY2D, GULP1, GYPA, H1F0, H2AFB1, H2AFY, H2AFZ, H2-ALPHA, HABP2, HACE1, HACL1, HADH, HAK, HAO2, HAPLN 1, HAVCR1, HBLD1, HCCS, HCN3, HCN4, HCP1, HCRTR2, HDAC11, HDAC7A, HDAC9, HDC, HDDC2, HDGF, HDGFL1, HDHD3, HEBP2, HECA, HECTD1, HECW1, HECW2, HELZ1 HEMK1, HEMK2, HEPH, HERC1, HERPUD1, HERPUD2, HERV- FRD, HES1, HES3, HEXB, HEXIM1, HEXIM2, HFE2, HIBADH, HIC1, HIC2, HIGD2A, HILS1, HINT1, HIP2, HIPK1, HIPK2, HIPK4, HISPPD2A, HIST1H2BI, HIST1H2BL, HIST1H3G, HIST1H4B, HIST1H4C, HIST1H4G, HIST1H4H, HIST1H4K, HIST2H2BE, HIST3H2BB, HIST4H4, HIVEP1, HIVEP2, HIVEP3, HK1, HK2, HKDC1, HLA-A, HLX1, HMBOX1, HMG20A, HMGA2, HMGB1, HMGCLL1, HMGCS1, HMGN3, HMGN4, HMHB1, HMOX1, HMP19, HNF4A, HNF4G, HNRPA3, HNRPF, HNRPH2, HNRPH3, HNRPLL, HNRPU, HNT, HOM-TES-103, HORMAD2, HOXA13, HOXA3, HOXB2, HOXB4, HOXC10, HOXC11, HOXC4, HOXC9, HOXD1, HOXD8, HPCAL1, HPGD1 H-plk, HPR, HPRT1, HPS3, HPS4, HPS5, HPSE2, HRASLS2, HRB, HRBL, HRG, HRH2, HRH4, HS2ST1, HS3ST4, HS3ST5, HSD17B13, HSD17B6, HSD3B1, HSD3B2, HSP90AB1, HSPA1A, HSPA4, HSPB3, HSPB8, HSPBAP1, HSPC049, HSPC159, HSPG2, HSPH1, HTR3A, HTR3B, HYAL4, HYDIN, HYI, IARS2, IBRDC3, IBTK, ICA1, ICEBERG, ICMT1 ICOS, ID1, ID2, ID3, ID4, IDH3A, IDS, IER2, IER3, IER5L, IFI35, IFIT1L, IFIT2, IFIT3, IFIT5, IFNAR2, IFNE1, IFT122, IFT20, IFT52, IFT88, IGF1R, IGF2BP1, IGF2BP3, IGFBP2, IGFBP4, IGFL4, IGSF1, IGSF2, IGSF21, IGSF3, IGSF4, IGSF9, IHH, IHPK1, IHPK2, IK, IKZF2, IKZF4, IL10, IL10RB, IL12RB1, IL12RB2, IL13, IL16, IL17RE, IL18, IL18BP, IL18RAP, IL1R1, IL1RAPL1, IL22, IL22RA1, IL22RA2, IL23A, IL27RA, IL28RA, IL2RA, IL31RA, IL6R, IL8RA, IL9R, ILDR1, IMP3, IMP4, IMPA1, IMPA2, IMPAD1, IMPG1, ING1, ING2, INHBE, INPP1, INPP4A, INPP4B, INPP5F, INSIG1, INSIG2, INSM1, INSM2, INSR, INTS12, INTS3, INTS4, INTS6, IPF1, IPMK, IPO7, IQCK, IQGAP2, IQGAP3, IQSEC1, IQSEC3, IRAKI BP1, IRAK3, IREB2, IRF2, IRF2BP2, IRF5, IRF8, IRS1, IRS2, IRX5, ISG20, ISL2, ISOC1, ITCH, ITFG1, ITGA11, ITGA2, ITGA2B, ITGAD, ITGAM, ITGAV, ITGB1, ITGB1BP2, ITGB3, ITGB4BP, ITGB5, ITGB8, ITIH1, ITIH2, ITIH5, ITM2B, ITPKA, ITPKC, ITPR1, ITSN1, IVD, IVNS1ABP, IWS1, IXL, IYD, JAG1, JAK1, JAKMIP2, JARID1A, JARID1B, JARID2, JAZF1, JDP2, JMJD1A, JMJD1C, JMJD2A, JMJD2C, JMJD5, JOSD1, JOSD3, JPH2, JUB, JUP, KALRN, KATNAL2, KATNB1, KBTBD1, KBTBD11, KBTBD2, KBTBD7, KBTBD8, KCMF1, KCNA1, KCNA2, KCNA4, KCNA6, KCNB1, KCNC4, KCND3, KCNE3, KCNG1, KCNIP2, KCNIP4, KCNJ11, KCNJ16, KCNJ2, KCNJ8, KCNK1, KCNK10, KCNK5, KCNMA1, KCNMB2, KCNMB3, KCNN3, KCNQ1, KCNQ3, KCNRG, KCNS2, KCTD10, KCTD13, KCTD16, KCTD3, KCTD4, KCTD6, KCTD7, KDR, KHDRBS2, KHK, KIAA0040, KIAA0143, KIAA0152, KIAA0195, KIAA0232, KIAA0240, KIAA0241, KIAA0247, KIAA0251, KIAA0256, KIAA0265, KIAA0319, KIAA0355, KIAA0367, KIAA0427, KIAA0460, KIAA0586, KIAA0664, KIAA0701, KIAA0738, KIAA0746, KIAA0773, KIAA0774, KIAA0776, KIAA0802, KIAA0922, KIAA0980, KIAA1005, KIAA1009, KIAA1026, KIAA1033, KIAA1128, KIAA1160, KIAA1161, KIAA1189, KIAA1191, KIAA1217, KIAA1267, KIAA1303, KIAA1324, KIAA1333, KIAA1370, KIAA1377, KIAA1411, KIAA1429, KIAA1456, KIAA1467, KIAA1505, KIAA1542, KIAA1546, KIAA1598, KIAA1600, KIAA1604, KIAA1718, KIAA1727, KIAA1737, KIAA1772, KIAA1794, KIAA1804, KIAA1826, KIAA1913, KIAA1919, KIAA1958, KIAA2013, KIAA2018, KIDINS220, KIF13A, KIF13B, KIF1C, KIF21A, KIF25, KIF2A, KIF4A, KIF5B, KIF6, KIFAP3, KIFC3, KIR3DX1, KIRREL, KL, KLB, KLC4, KLF12, KLF13, KLF2, KLF3, KLF4, KLF5, KLF6, KLF7, KLHDC2, KLHDC4, KLHDC5, KLHDC6, KLHL13, KLHL15, KLHL2, KLHL20, KLHL23, KLHL24, KLHL25, KLHL8, KMO, KNG1, KPNA2, KPNA3, KPNA4, KRAS, KREMEN1, KRT12, KRT18, KRT19, KRT20, KRT31, KRT39, KRT8, KRT80, KRTAP15-1, KRTAP26-1, KRTAP8-1, KSR1, KTN1, Kua, KU-MEL-3, KYNU, L3MBTL, L3MBTL3, L3MBTL4, LACE1, LAMA3, LAMA4, LAMB1, LAMB3, LAMB4, LAMC1, LAMC2, LAP3, LARP1, LARP4, LARP5, LARS, LARS2, LASP1, LASS2, LASS5, LASS6, LAT1 LATS2, LAX1, LBH1 LBR, LBX2, LBXCOR1, LCE2D, LCMT1, LCOR, LCP1, LCT, LCTL, LDB2, LDHA, LDHAL6B, LEAP2, LECT2, LEMD3, LENG9, LEO1, LEPROT1 LEPROTL1, LFDH, LGALS14, LGALS3, LGALS4, LGALS8, LGR4, LGR5, LHCGR, LHFPL2, LHFPL5, LHPP1 LHX3, LHX9, LIAS, LIF, LIFR, LIG4, LIMA1, LIMS3, LIN9, LIPA, LIPC, LIPF, LIX1, LIX1L, LMBRD2, LMCD1, LMNB1, LMOD3, LMTK2, LNX1, LNX2, LOC112714, LOC123876, LOC124446, LOC130355, LOC145757, LOC148696, LOC149134, LOC149950, LOC150383, LOC151760, LOC152485, LOC153222, LOC158572, LOC196752, LOC196913, LOC200261, LOC202459, LOC220594, LOC220686, LOC222967, LOC255374, LOC283537, LOC283551 , LOC284009, LOC284402, LOC284751, LOC284757, LOC285016, LOC285074, LOC285382, LOC285498, LOC285908, LOC286334, LOC338809, LOC339745, LOC339977, LOC340156, LOC340204, LOC340529, LOC347487, LOC348174, LOC375133, LOC375748, LOC387601, LOC387646, LOC387680, LOC387882, LOC387911, LOC388323, LOC388335, LOC388503, LOC388692, LOC388965, LOC389286, LOC389432, LOC399706, LOC399900, LOC400120, LOC400451, LOC400566, LOC400968, LOC401152, LOC401233, LOC401286, LOC401431, LOC401589, LOC401720, LOC401898, LOC440093, LOC440313, LOC440570, LOC440742, LOC440905, LOC441108, LOC441177, LOC441193, LOC441257, LOC441268, LOC441294, LOC442247, LOC51233, LOC54103, LOC552891, LOC642265, LOC642980, LOC643866, LOC643923, LOC644083, LOC645745, LOC646962, LOC650293, LOC653107, LOC90826, LOC91461, LOC92196, LOH12CR1, LONRF3, LOR, LOX, LPAAT-THETA, LPGAT1, LPHN3, LPIN1, LPIN2, LPIN3, LPP, LPXN, LRAP, LRAT, LRIG1, LRIG3, LRP1, LRP10, LRP2, LRP6, LRPPRC, LRRC1, LRRC16, LRRC17, LRRC18, LRRC2, LRRC28, LRRC31, LRRC4, LRRC40, LRRC48, LRRC52, LRRC61, LRRC8C, LRRC8D, LRRFIP1, LRRFIP2, LRRK1, LRRN3, LRRN5, LRRTM1, LRRTM2, LRTM2, LSM14A, LSM14B, LSM2, LSM6, LSP1, LTBP1, LTBP3, LTBP4, LTV1, LUM, LUZP1, LUZP2, LUZP4, LY6G5C, LY75, LYCAT, LYK5, LYN, LYNX1, LYPD1, LYPD6, LYPLAL1, LYRM2, LYRM5, LYSMD1, LYZL1, LYZL2, LYZL4, LZTR2, MACF1, MADD, MAFB, MAGEA4, MAGED1, MAGED2, MAGED4, MAGEF1, MAGEH 1, MAGM1 MAGI3, MALL, MAMDC1, MAML1, MAML3, MAN1A1, MAN1A2, MAN2C1, MANSC1, MAOB, MAP1LC3B, MAP2K2, MAP2K6, MAP3K13, MAP3K14, MAP3K15, MAP3K7IP2, MAP3K7IP3, MAP3K8, MAP3K9, MAP4K3, MAP4K4, MAP4K5, MAP6, MAPK14, MAPK6, MAPK8, MAPKAPK2, MAPRE1, MAPRE3, MARCH1, MARCH3, MARCH8, MARCKS, MARCKSL1, MARK1, MARK2, MARS2, MARVELD2, MARVELD3, MASP1, MAST2, MAT2B, MATN 1, MATN2, MATR3, MAX, MBD2, MBD5, MBIP, MBL2, MBNL1, MBNL2, MBNL3, MBOAT2, MBOAT5, MBP, MCART2, MCART6, MCC1 MCL1, MCM10, MCM8, MCMDC1, MCOLN3, MCTP1, MCTP2, MDFIC, MDM1, MDM2, MDM4, ME1, ME2, MED11, MED18, MED31, MED4, MED9, MEF2A, MEF2C, MEIS1, MEIS2, MEOX1, MEOX2, MEP1A, MEP1B, MERTK, MESDC1, MESP1, MESP2, METRNL, METHOD, METT5D1, METTL7A, METTL7B, METTL8, MFAP3L, MFGE8, MFHAS1, MFNG, MFSD1, MFSD2, MFSD4, MGAM, MGAT1, MGAT2, MGAT3, MGAT4A, MGAT5B, MGC11332, MGC13057, MGC21644, MGC21881, MGC23985, MGC24039, MGC26963, MGC29671, MGC33556, MGC33657, MGC33846, MGC34646, MGC35361, MGC39715, MGC4172, MGC42174, MGC4268, MGC44328, MGC45491, MGC50559, MGC51025, MGC52498, MGC61571, MGC87631, MGLL, MGMT, MIA2, MICAL2, MIF, MINK1, MINPP1, MIST, MITF, MKI67, MKI67IP, MKKS, MKL1, MKL2, MKS1, MLC1, MLH3, MLLT10, MLNR, MLSTD1, MLSTD2, MLXIPL, MMACHC, MMD, MME1 MMP15, MMP16, MMP17, MMP20, MMRN2, MND1, MNS1, MNT, M0AP1, MOBKL2C, MOCOS, M0CS1, MOGAT1, M0GAT2, M0RC1, MOS, M0SC2, M0SPD1, MPL, MPP5, MPP6, MPPED2, MPV17L, MPZ1 MPZL1, MRC1L1, MRGPRF, MRGPRX1, M-RIP, MRLC2, MRPL1, MRPL13, MRPL14, MRPL45, MRPL55, MRPS10, MRPS22, MRVM, MS4A10, MS4A8B, MSH5, MSI2, MSL2L1, MSRB2, MST4, MSX2, MT1F, MTA3, MTAP, MTDH, MTERFD3, MTF2, MTFMT, MTFR1, MTIF3, MTMR10, MTMR11, MTMR12, MTMR8, MTRF1L, MTRR, MTSS1, MTTP, MTUS1, MUC13, MUC20, MUSK, MUTED, MVP, MX2, MXD1, MYB, MYBPC1, MYBPHL, MYCBP2, MYCBPAP, MYCL1, MYCN, MYF6, MYH10, MYH14, MYH4, MYLIP, MYO10, MYO15A, MY018A, MY018B, MY01B, MY01D, MY01E, MYO1G, MY06, MYO7A, MYOC, MYOCD, MYOM1, MY0Z2, MYPN, MYST2, MYST3, MYST4, MYT1, N4BP1, N4BP2, NAALAD2, NAALADL2, NAB1, NACAL1 NAG6, NAG8, NANS, NAP1L4, NAP1L5, NAPE-PLD, NARG2, NARS2, NAT2, NAT5, NAT8B, NAT8L, NAV2, NAV3, NBEA, NBL1, NBPF1, NBPF11, NBPF15, NBPF3, NBPF4, NBR1, NCAM1, NCAPG2, NCF2, NCK2, NCKAP1, NC0A1, NCOA2, NC0R2, NDEL1, NDFIP1, NDFIP2, NDP1 NDST1, NDUFA8, NDUFA9, NDUFB4, NDUFV2, NEBL, NEDD1, NEDD4, NEDD9, NEIL3, NEK10, NEK11, NEK6, NELL2, NENF, NE01, NET1, NEU1, NEUROD1, NEUR0D4, NEUR0D6, NEXN, NF1, NFAM1, NFAT5, NFATC3, NFATC4, NFE2L2, NFIA1 NFKB1, NFKBIA, NFKBIZ1 NFRKB1 NFX1, NFXL1, NFYB, NFYC, NGB, NGFB, NGRN, NHLH2, NHLRC2, NHS, NID1, NID2, NIN, NINJ2, NIPBL, NIPSNAP1, NIT1, NIT2, NKAP, NKD1, NKIRAS1, NKIRAS2, NKPD1, NKX2-2, NKX3-1, NLF2, NLGN3, NLK, NMB, NME6, NMNAT3, NMT2, NMUR2, NNMT, NODAL, N0L5A, N0M01, NOMO2, N0M03, NOPE, N0S1AP, NOS2A, NOSTRIN, N0TCH2, NOTCH2NL, N0TCH4, NOVA1, N0X1, N0X3, NP, NPAL1, NPAL2, NPAS3, NPC2, NPEPPS, NPHP1, NPL1 NPNT, NPR2, NPS, NPSR1, NPTN1 NPTX2, NPY, NR1H4, NR1I3, NR2C1, NR2C2, NR2F1, NR2F6, NR4A2, NR5A1, NR6A1, NRBF2, NRG1, NRG2, NRIP1, NRIP2, NRP1, NRP2, NRSN1, NRXN3, NSFL1C, NSMCE1, NSMCE2, NSUN5B, NSUN5C, NSUN6, NSUN7, NT5C2, NT5DC1, NT5DC3, NT5E, NTF3, NTNG1, NTSR2, NUAK2, NUB1, NUCKS1, NUDCD3, NUDT12, NUDT13, NUDT16, NUDT4 NUDT9, NUF2, NUFIP2, NUMA1, NUMB, NUP205, NUTF2, NXN, NXPH2, NXPH3, NYX, OAF, OAT, OBFC1, OBFC2A, OCA2, OCLN, ODC1, ODF1, OFCC1, OFD1, OGDH, OGDHL, 0GF0D2, OGG1, OIT3, 0LIG3, 0LR1, OMG, 0NECUT1, OPCML, OPN1SW, 0PN3, OPRK1, OPRM1, OPTN, OR10J1, OR10J5, OR10P1, OR13C5, 0R13F1, OR13H1, OR1D2, 0R1L1, 0R1L3, 0R1L4, 0R2B11, OR2B3, 0R2F1, OR2G3, OR2H2, OR2L2, OR2S2, OR4D2, OR4D9, OR4E2, OR4F17, OR4F29, OR4F4, OR4F5, OR52K2, OR5B12, OR5M11, OR5U1, OR6A2, 0R6B1, OR6S1, OR6V1, OR8D2, OR8G2, OR8G5, OR9Q2, 0RC2L, OSBP2, OSBPL10, 0SBPL1A, OSBPL3, 0SBPL5, 0SBPL6, 0SBPL8, 0SBPL9, OSM, 0SR2, OSTalpha, OSTbeta, OSTM1, OTOF, OTOP3, OTUD6B, 0TUD7B, OVOL1, OVOL2, 0XGR1, OXR1, P2RY1, P2RY12, P2RY4, P2RY5, P2RY6, P4HA1, P4HA3, PABPC4, PACRG, PACS 1, PACSIN2, PACSIN3, PADI2, PAG1, PAGE4, PAH, PAK1, PAK2, PAK6, PAK7, PALM2-AKAP2, PAM, PAMCI, PAN3, PANK1, PANK3, PANX1, PAOX, PAP2D, PAPD1, PAPD4, PAPD5, PAPLN, PAPOLA, PAPOLG, PAPPA2, PAPSS1, PAPSS2, PAQR3, PAQR8, PAQR9, PARD3, PARD3B, PARD6B, PARG, PARL1 PARN, PARP1, PARP11, PARP12, PARP15, PARP16, PARP2, PARP4, PARP8, PAX7, PBEF1, PBX1, PBX3, PC, PCBD1, PCBP1, PCCA, PCCB, PCDH1, PCDH7, PCDH8, PCDHGC5, PCGF2, PCGF5, PCM1, PCMTD2, PCNX, PCNXL2, PCSK1, PCSK2, PCSK5, PCSK6, PCSK9, PCTK3, PCYT1B, PCYT2, PDC, PDCD2, PDCD6, PDCL2, PDE1C, PDE3A, PDE4B, PDE6A, PDE6C, PDE6H, PDE7A, PDE8A, PDF, PDGFB, PDGFC, PDGFD, PDGFRL, PDHX, PDIA5, PDK1, PDK3, PDLIM2, PDLIM3, PDLIM5, PDPK1, PDPR1 PDSS1, PDXK, PDXP1 PDZD2, PDZD3, PDZD8, PDZK1IP1, PDZK5B, PDZRN3, PEBP1, PECAM1, PECR, PELH, PELI2, PER1, PERLD1, PERP1 PEX11G, PEX26, PEX5, PFAAP5, PFKFB1, PFKFB2, PFKM, PFKP, PFN2, PGAM4, PGBD5, PGCP, PGD1 PGDS, PGEA1, PGF1 PGK2, PGLYRP2, PGM2, PGM2L1, PGM3, PGPEP1, PGRMC2, PHACTR2, PHC2, PHC3, PHEX, PHF1, PHF10, PHF12, PHF15, PHF16, PHF17, PHF20, PHF20L1, PHF21A, PHF3, PHGDH, PHLDA3, PHLDB3, PHLPP, PHLPPL, PHOSPHO1, PHYH, PHYHIPL, PI3, PIB5PA, PICALM, PIG38, PIGC, PIGK, PIGL, PIGM, PIGR, PIGY, PIGZ, PIK3AP1, PIK3C2A, PIK3C2G, PIK3CA, PIK3CB, PIK3R1, PIK3R4, PIM1, PIN4, PIP3-E, PIP5K1B, PIP5K2A, PIP5K2B, PIP5K3, PISD, PITPNA, PITPNM2, PITX1, PITX2, PITX3, PIWIL2, PIWIL4, PJA2, PKD1L1, PKD1L2, PKD2L2, PKHD1L1, PKIB1 PKIG1 PKN2, PKP2, PKP4, PLA2G12B, PLA2G2A, PLA2G2E, PLA2G4A, PLA2G6, PLAC1, PLAC2, PLAGL1, PLAU1 PLB1, PLCB1, PLCE1, PLCG1, PLCH1, PLCXD2, PLCZ1, PLD1, PLEKHA1, PLEKHA5, PLEKHA6, PLEKHA7, PLEKHA8, PLEKHB1, PLEKHB2, PLEKHC1, PLEKHF2, PLEKHG1, PLEKHG3, PLEKHG6, PLEKHH1, PLEKHJ1, PLEKHM1, PLG1 PLGLB1, PLGLB2, PLIN, PL0D2, PLS1, PLXDC2, PLXNA2, PLXNA4B, PLXNC1, PMAIP1, PMFBP1, PMM1, PMP22CD, PMS2L5, PMVK1 PNLDC1, PNLIPRP2, PNMA2, PNMA3, PNOC, PNPLA1, PNPLA3, PNPLA8, PNPO, PNPT1, PNRC1, P0FUT1, POGZ1 P0LA1, P0LDIP3, P0LE4, POLI, POLN, POLQ, P0LR1D, P0LR1E, P0LR2C, POLR2E, POLR3B, P0LR3H, P0M121, POMP, P0N2, POP4, POPDC3, POR, P0T1, POU2AF1, P0U2F1, POU3F1, POU3F2, POU4F1, P0U6F1, POU6F2, PPA1, PPAP2A, PPAP2B, PPAPDC1B, PPARBP, PPARD, PPARG, PPARGC1B, PPCDC, PPCS, PPEF2, PPFIA1, PPIAL4, PPIB, PPID, PPIG, PPIL4, PPIL5, PPM1A, PPM1B, PPM1E, PPM1K, PPM1L, PPM2C, PPP1CB, PPP1R12A, PPP1R12C, PPP1R14A, PPP1R14C, PPP1R2, PPP1R9A, PPP1R9B, PPP2R1B, PPP2R2A, PPP2R2B, PPP2R3A, PPP2R5A, PPP2R5D, PPP3CB? PPTC7, PPYR1, PQLC1, PQLC2, PRDM1, PRDM10, PRDM8, PRDX1, PRDX4, PRDX6, PREP, PRF1, PRG1, PRG2, PRH1, PRICKLE1, PRICKLE2, PRKAA1, PRKAA2, PRKAG2, PRKAR1A, PRKAR2B, PRKCA, PRKCBP1, PRKCD, PRKCI, PRKG2, PRKRIR, PRL, PRLHR, PRM1, PRMT1, PROC, PR0CA1, PRODH, PR0M1, PR0S1, PR0X1, PRPF38B, PRPF39, PRPSAP1, PRPSAP2, PRR13, PRR15, PRR17, PRR3, PRR6, PRR8, PRRG4, PRRT1, PRSS12, PRSS16, PRSS23, PRSS3, PRSS35, PRSS8, PRTFDC1, PRTG, PRUNE, PSCD4, PSCDBP, PSD3, PSD4, PSEN1, PSKH2, PSMAL, PSMB3, PSMB4, PSMD14, PSPC1, PSTPIP2, PTBP2, PTCD2, PTCHD3, PTDSS1, PTEN, PTER, PTGES, PTGFRN, PTGIS, PTH, PTHLH, PTHR2, PTK2B, PTMS, PTP4A1, PTP4A2, PTPDC1, PTPLAD1, PTPN11, PTPN14, PTPN2, PTPN21, PTPN23, PTPN9, PTPRJ, PTPRM, PTPRN2, PTPRO, PTPRR, PTRH2, PTTG1IP, PTTG2, PUM1, PUNC, PVR, PVRL1, PVRL4, PXMP2, PXMP4, PYGB, PYG01, QDPR, QKI, QPCT, QRICH1, QSCN6, RAB11FIP1, RAB11FIP2, RAB11FIP4, RAB17, RAB1A, RAB20, RAB27A, RAB31, RAB32, RAB33A, RAB35, RAB37, RAB38, RAB3B, RAB3D, RAB3GAP1, RAB3GAP2, RAB3IP, RAB43, RAB5A, RAB5C, RAB6C, RAB7L1, RAB8B, RAB9, RABGAP1L, RABGEF1, RABIF, RABL3, RAD18, RAD51L1, RAD54L, RAD9B, RAF1, RAM, RALB1 RALGPS1, RALGPS2, RALY, RAMP1, RAN, RANBP17, RANBP2, RANBP3, RANBP5, RANGNRF, RAP1A, RAP1B, RAP2A, RAP2B, RAPGEF1, RAPGEF4, RAPH1, RARA, RARB, RARSL, RASA3, RASAL2, RASGEF1C, RASGRF1, RASGRF2, RASL10B, RASL11B, RASL12, RASSF3, RASSF4, RASSF6, RAVER1, RAVER2, RB1CC1, RBBP8, RBCK1, RBJ, RBKS, RBM11, RBM12B, RBM24, RBM25, RBM26, RBM28, RBM34, RBM39, RBM41, RBM5, RBM9, RBMS1, RBMXL1, RBP2, RBP4, RBPMS, RBPSUH, RC74, RCBTB1, RCC2, RCHY1, RCL1, RCN3, RCOR1, RCSD1, RDH12, RDH14, RDHE2, RDM1, RDX, REEP1, REEP3, REEP6, REG4, REP15, REPS1, RERE, REST, REV3L, REXO1L1, REXO2, RFC1, RFFL, RFK, RFWD2, RFXDC1, RGL1, RGN, RGPD1, RGPD2, RGPD5, RGPD7, RGS1, RGS13, RGS18, RGS2, RGS21, RGS22, RGS3, RGS7BP, RGS8, RGS9, RHBDD3, RHBG, RHEB, RHO, RHOBTB2, RHOBTB3, RHOC, RHOF, RHOH, RHOT1, RHOU1 RHPN2, RIC8B, RICS, RICTOR1 RIMBP2, RIN2, RIOK1, RIOK2, RIOK3, RIPK3, RIPK4, RKHD2, RKHD3, RMM, RMND5A, RNASE1, RNASE9, RNASEH2B, RNASEL, RND1, RNF10, RNF11, RNF113B, RNF121, RNF128, RNF135, RNF138, RNF157, RNF168, RNF182, RNF183, RNF186, RNF19, RNF2, RNF32, RNF44, RNF6, RNF7, RNPEP, ROBO1, ROBO2, ROCK2, ROD1, ROR1, RORA, ROS1, RP11-311P8.3, RP13-360B22.2, RP1L1, RP3-473B4.1, RPA1, RPA3, RPAIN, RPIA, RPIB9, RPL10L, RPL17, RPL34, RPL4, RPL9, RPLPO, RPN2, RPS10, RPS12, RPS26, RPS27, RPS27L, RPS6KA6, RPUSD4, RRAGC, RRBP1, RREB1, RRM2, RRN3, RS1, RSBN1, RSBN1L, RSC1A1, RSF1, RSL1D1, RSN, RSPO3, RSPRY1, RTN1, RTN4, RTN4RL1, RTTN, RUNDC2B, RUNX1, RUNX2, RUSC2, RUVBL1, RWDD2, RWDD3, RXFP2, RXFP3, RXRA, RYBP, RYR1, S100A1, S100A10, S100G, S100P, S100Z, SAA4, SACM1L, SACS, SAE1, SALL1, SALL4, SAMD11, SAMD13, SAMD14, SAMD3, SAMD4A, SAMD8, SAP30, SAP30L, SAPS3, SAR1B, SASH1, SASP, SAT1, SAT2, SC5DL, SCAMP1, SCAMP2, SCAND2, SCAP, SCARA3, SCARB1, SCARB2, SCD5, SCGB1A1, SCGN, SCIN, SCMH1, SCML4, SCN3A, SCN8A, SCOC, SCP2, SCRT2, SCTR, SCUBE2, SCYL1, SCYL1BP1, SCYL3, SDC2, SDC4, SDCBP2, SDHB, SDK1, SDPR, SDR-O, SEC13L1, SEC14L1, SEC14L2, SEC24A, SEC24C, SEC61A1, SEC61A2, SEC61G, SECISBP2, SEH1L, SELE, SELI, SELL, SELPLG, SELS, SEMA3A, SEMA3G, SEMA4B, SEMA4D, SEMA4G, SEMA6A, SEMA6D, SENP2, SENP5, SENP6, SENP8, SEPHS1, SEPHS2, SEPP1, SERF1A, SERGEF1 SERINC2, SERINC3, SERINC5, SERPINA13, SERPINA3, SERPINA7, SERPINA9, SERPINB1, SERPINB9, SERPINE2, SERPINH1, SERTAD2, SERTAD3, SERTAD4, SESN1, SESN3, SESTD1, SETBP1, SETD2, SETD3, SETD4, SF3B3, SFMBT1, SFPQ, SFRP5, SFRS10, SFRS15, SFRS2IP, SFRS3, SFRS4, SFRS6, SFT2D1, SFT2D2, SFTPA2, SFXN1, SFXN3, SFXN5, SGCB, SGEF, SGOL1, SGPL1, SGPP1, SGPP2, SH2D1A, SH2D3A, SH2D4B, SH2D6, SH3BP4, SH3BP5, SH3BP5L, SH3D19, SH3GL1, SH3GLB1, SH3KBP1, SH3PX3, SH3PXD2A, SH3RF1, SH3RF2, SH3TC2, SHANK2, SHBG, SHC1, SHE, SHF, SHFM1, SH0C2, SHPRH, SHQ1, SHROOM3, SHR00M4, Sl, SIAE, SIAH1, SIAH2, SIDT1, SIGLEC12, SIL1, SIM1, SIN3A, SIPA1L1, SIPA1L2, SIPA1L3, SIRPA, SIRT1, SIX1, SKAP1, SKAP2, SKIL, SKP1A, SLA1 SLA/LP, SLAMF1, SLAMF6, SLAMF9, SLC10A2, SLC10A5, SLC11A2, SLC12A1, SLC12A3, SLC12A6, SLC12A7, SLC12A8, SLC13A2, SLC13A3, SLC13A5, SLC14A2, SLC15A1, SLC16A1, SLC16A10, SLC16A13, SLC16A4, SLC16A5, SLC16A7, SLC17A3, SLC17A4, SLC17A8, SLC19A2, SLC1A1, SLC1A3, SLC1A5, SLC1A7, SLC20A1, SLC20A2, SLC22A1, SLC22A16, SLC22A2, SLC22A9, SLC23A3, SLC24A1, SLC24A5, SLC25A14, SLC25A15, SLC25A16, SLC25A20, SLC25A24, SLC25A25, SLC25A3, SLC25A30, SLC25A33, SLC25A36, SLC25A37, SLC25A43, SLC25A44, SLC25A46, SLC26A2, SLC26A3, SLC26A8, SLC27A2, SLC28A1, SLC28A2, SLC2A1, SLC2A14, SLC2A2, SLC2A3, SLC2A7, SLC2A9, SLC30A1, SLC30A10, SLC30A4, SLC30A5, SLC33A1, SLC34A1, SLC35A5, SLC35B4, SLC35D1, SLC35D2, SLC35F2, SLC35F5, SLC36A2, SLC36A4, SLC37A1, SLC37A2, SLC37A4, SLC38A2, SLC39A10, SLC39A11, SLC39A14, SLC39A5, SLC3A1, SLC40A1, SLC41A1, SLC41A2, SLC41A3, SLC43A1, SLC43A2, SLC44A1, SLC44A3, SLC45A3, SLC4A4, SLC4A7, SLC4A8, SLC5A1, SLC5A10, SLC5A12, SLC5A4, SLC5A9, SLC6A11, SLC6A20, SLC6A4, SLC6A6, SLC6A7, SLC6A9, SLC7A11, SLC7A14, SLC7A5, SLC7A6, SLC7A7, SLC7A8, SLC9A2, SLC9A3R1, SLC9A4, SLC9A8, SLCO2A1, SLC02B1, SLC03A1, SLCO4A1, SLIT1, SLIT3, SLMAP, SMA4, SMAD2, SMAD3, SMAD5, SMAD6, SMAD7, SMAF1, SMAP1L, SMARCA1, SMARCA4, SMARCB1, SMARCD1, SMC3, SMEK1, SMNDC1, SMOC1, SMOC2, SMOX, SMPD3, SMPDL3A, SMPX1 SMS, SMTNL2, SMUG1, SMURF1, SMURF2, SMYD2, SMYD4, SMYD5, SNAM, SNAPAP1 SNCAIP, SNF1LK2, SNFT1 SNIP, SNRK1 SNRPC, SNTB1, SNTG2, SNX10, SNX12, SNX13, SNX14, SNX24, SNX4, SNX7, SNX9, SOAT1, SOAT2, SOCS5, SOCS6, SOCS7, SOD2, SOLH, SORBS1, SORBS2, SORBS3, SORCS2, SORD, SORL1, SORT1, SOS1, SOSTDC1, SOX1, SOX13, SOX14, SOX30, SOX5, SOX6, SP1, SP3, SP5, SP6, SP8, SPAG17, SPAG5, SPAG6, SPAG9, SPAM1, SPANXN4, SPARC, SPATA1, SPATA12, SPATA13, SPATA20, SPATA8, SPATA9, SPATS2, SPECC1, SPECC1L, SPFH1, SPG21, SPG7, SPHAR, SPINK1, SPINK5, SPINK5L2, SPINT2, SPIRE1, SPN, SPOCD1, SPOCK2, SPOCK3, SPON1, SPOP, SPPL2A, SPPL2B, SPPL3, SPR, SPRED1, SPRED2, SPRY1, SPRY2, SPRY4, SPTAN1, SPTBN1, SPTBN2, SPTBN5, SPTLC2, SQLE, SRD5A2, SRD5A2L, SREBF1, SRFBP1, SRG, SRGAP1, SRGAP2, SRI, SRPK1, SRPK2, SRPX2, SSBP3, SSPN, SSR1, SSX2IP, ST14, ST18, ST3GAL2, ST3GAL3, ST3GAL6, ST5, ST6GAL1 , ST6GALNAC1 , ST6GALNAC3, ST8SIA4, STAMBPL1 , STARD13, STARD4, STARD5, STARD6, STAT5B, STAT6, STAU1, STC2, STEAP3, STEAP4, STIM2, STK10, STK11IP, STK17A, STK17B, STK3, STK31 , STK32A, STK35, STK38, STK38L, STK39, STK40, STOM, STON2, STOX2, STRA8, STRAP, STRBP, STRN, STRN3, STS-1, STT3B, STX1B2, STX3, STX6, STX7, STXBP4, STXBP5, STXBP6, STYX, SUB1, SUCLG2, SUCNR1, SUDS3, SUHW4, SULF2, SULT1A4, SULT1B1, SULT1E1, SUPT4H1, SUPT7L, SURF4, SUSD1, SUSD4, SUV39H1, SUV39H2, SV2B, SVIL, SVOP1 SYCP2, SYCP3, SYNCRIP, SYNE1, SYNGAP1, SYNJ2, SYNJ2BP, SYNPO2, SYNPR, SYPL1, SYT1, SYT11, SYT13, SYT15, SYT16, SYT3, SYT6, SYT7, SYT9, SYTL2, SYTL3, SYTL5, TAAR1, TAAR2, TAC1, TAC4, TACC2, TACR2, TACSTD 1, TAF 1 A, TAF4, TAGLN3, TAIP-2, TAL1, TAL2, TANC1, TANK, TAOK3, TARBP1, TASP1, TATDN2, TATDN3, TBC1D14, TBC1D16, TBC1D19, TBC1D2, TBC1D23, TBC1D2B, TBC1D4, TBC1D7, TBC1D8, TBC1D8B, TBC1D9, TBC1D9B, TBCC, TBCCD1, TBCE, TBK1, TBL1X, TBL1XR1, TBN, TBPL1, TBPL2, TBRG1, TBX15, TBX19, TBX4, TBXAS1, TCEA1, TCEAL8, TCF20, TCF23, TCF4, TCF7L1, TCFL5, TCHHL1, TCL6, TCN1, TCP10, TCP10L2, TCP11L1, TCP11L2, TEAD1, TEC, TECTA, TECTB, TEKT3, TEP1, TERF2, TES, TEX10, TEX101, TEX12, TEX14, TEX2, TFAP2A, TFAP2C, TFB1M, TFDP1, TFDP2, TFEB1 TFEC, TFF1, TFF2, TFF3, TFG, TFPI2, TG1 TGFBI1 TGFBR2, TGFBR3, TGFBRAP1, TGIF, TGIF2, TGM2, TGOLN2, THAP2, THAP9, THBS2, THEM2, THEM4, THEX1, THNSL1, THOC1, THOC3, TH0C7, THRAP1, THRAP2, THRB, THSD1, THUMPD3, THY1, TIAL1, TIAM1, TIAM2, TICAM2, TIE1, TIGD2, TIGD3, TIMM8A, TIMP4, TINAG1 TINP1, TIPRL, TJAP1, TJP1, TJP2, TLE1, TLN2, TLR2, TLR7, TLX1, TM2D1, TM2D2, TM4SF1, TM4SF11, TM4SF20, TM9SF3, TM9SF4, TMBIM1, TMC1, TMC7, TMCC1, TMCC3, TMCO1, TMCO2, TMED10, TMED4, TMED8, TMEM1, TMEM10, TMEM100, TMEM105, TMEM106A, TMEM106B, TMEM108, TMEM117, TMEM12, TMEM125, TMEM126A, TMEM128, TMEM132B, TMEM135, TMEM139, TMEM140, TMEM142A, TMEM144, TMEM150, TMEM154, TMEM156, TMEM158, TMEM161B, TMEM165, TMEM166, TMEM16F, TMEM16J, TMEM16K, TMEM17, TMEM171, TMEM173, TMEM174, TMEM177, TMEM180, TMEM19, TMEM2, TMEM20, TMEM23, TMEM24, TMEM26, TMEM30B, TMEM33, TMEM37, TMEM38B, TMEM40, TMEM41A, TMEM44, TMEM45A, TMEM45B, TMEM48, TMEM49, TMEM5, TMEM50B, TMEM51, TMEM56, TMEM60, TMEM62, TMEM63A, TMEM67, TMEM69, TMEM79, TMEM87B, TMEM9, TMEM92, TMEM93, TMEM97, TMEM98, TMEPAI, TMIGD1, TMOD3, TMPO, TMPRSS11B, TMPRSS12, TMPRSS13, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMTC3, TNFAIP2, TNFAIP3, TNFAIP8, TNFRSF10B, TNFRSF11A, TNFRSF19, TNFRSF1A, TNFRSF25, TNFRSF8, TNFSF10, TNFSF12-TNFSF13, TNFSF13B, TNFSF15, TNFSF18, TNFSF4, TNFSF5IP1, TNIK, TNIP1, TNIP2, TNIP3, TNK1, TNK2, TNKS2, TNMD1 TNN, TNNC2, TNNI3K, TNP1, TNPO1, TNPO3, TNR, TNRC4, TNRC6B, TNRC6C, TNS1, TNS3, TNXB, TOB1, TOB2, TOM1L2, TOMM34, TOMM40, TOMM7, TOP1, TOPBP1, TOR1B, TOR3A, TP53I13, TP53INP1, TP53INP2, TP73L, TPCN1, TPCN2, TPD52, TPM1, TPM3, TPMT, TPP1, TPPP1 TPT1, TRA2A, TRAF3, TRAF3IP2, TRAF3IP3, TRAK1, TRAM2, TRAPPC4, TRERF1, TREX1, TRIB1, TRIB3, TRIM14, TRIM16, TRIM16L, TRIM2, TRIM24, TRIM25, TRIM27, TRIM3, TRIM32, TRIM33, TRIM36, TRIM37, TRIM39, TRIM40, TRIM42, TRIM43, TRIM44, TRIM59, TRIM69, TRIM71, TRIM8, TRIM9, TRIO, TRIP6, TRIT1, TRPA1, TRPC4, TRPC4AP, TRPC6, TRPC7, TRPM3, TRPM6, TRPM7, TSC22D1 , TSC22D2, TSGA2, TSHZ1, TSKU, TSNARE1, TSPAN13, TSPAN15, TSPAN2, TSPAN3, TSPAN32, TSPAN33, TSPAN7, TSPAN8, TSPAN9, TSPYL4, TSR1, TSSK3, TTBK1, TTC1, TTC13, TTC14, TTC17, TTC21B, TTC22, TTC26, TTC30A, TTC31, TTC6, TTC7A, TTC7B, TTL1 TTLL2, TTLL6, TTLL9, TTMB, TTR1 TTRAP1 TUBAL3, TUBG1, TUBG2, TULP4, TWF2, TXLNA TXLNB1 TXN, TXNDC11 , TXNDC13, TXNDC2, TXNDC5, TXNDC8, TXNL5, TYSND1 , TYW3 UACA, UAP1, UBAP1, UBAP2L, UBC, UBE2D4, UBE2H, UBE2I, UBE2L3, UBE2R2, UBE2W, UBE3A, UBE4A, UBE4B, UBL3, UBN1, UBR1, UBTD2, UCHL5, UCK1, UCK2, UCP3, UFM1, UGCG, UGCGL1, UGP2, UGT2A3, UGT2B7, UGT8, UHRF1, UHRF2, UIMC1, ULBP2, ULK1, ULK2, UMODL1, UNC45A, UNC5B, UNC84B, UNC93A, UNG, UNG2, UNQ473, UNQ5830, UNQ830, UNQ9433, UNQ9438, UPK1B, UPK3A, UPP2, URG4, USH1C, USH2A, USH3A, USP10, USP13, USP18, USP25, USP30, USP31, USP36, USP38, USP39, USP40, USP44, USP47, USP49, USP54, USP8, USP9X, USPL1, UTP14C, UTP15, UTRN, UTX, UVRAG1 UXS1, VAMP3, VANGL1, VAPA, VAPB, VARSL1 VAV2, VAV3, VAX1, VCL, VCPIP1, VCX, VCX3A, VDAC3, VEGFA1 VEZF1, VEZT, VGLL3, VIL1, VIL2, VILL, VIPR1, VIPR2, VKORC1L1, VLDLR, VPREB1, VPS13D, VPS37A, VPS37B, VPS37C, VPS39, VPS41, VPS4B, VPS52, VPS54, VPS8, VSIG4, VSIG8, VSNL1, VWCE, VWF, WASF2, WBSCR23, WDFY1, WDFY3, WDR20, WDR23, WDR26, WDR3, WDR36, WDR41 , WDR42A, WDR42B, WDR49, WDR52, WDR59, WDR60, WDR61, WDR63, WDR65, WDR66, WDR71, WDR72, WDR75, WDR78, WDR81, WDSUB1, WEE1, WFDC2, WHDC1L1, WHSC1, WIF1, WIPF1, WISP1, WNK1, WNK2, WNK4, WNT11 , WNT5A, WNT8B, WSB1 , WSB2, WT1 , WWC1 , WWC2, WWC3, WWOX, WWP2, WWTR1, XBP1, XCL2, XG1 XKR8, XKRX1 XPNPEP1, XPNPEP2, XPO4, XPO7, XRN1, XYLT1, YAF2, YAP1, YBX1, YEATS4, YIPF6, YPEL2, YPEL5, YTHDF3, YWHAE, YWHAG1 YWHAH, YWHAQ, YWHAZ1 YY1, ZADH1, ZAK1 ZBED1, ZBED2, ZBTB10, ZBTB16, ZBTB2, ZBTB20, ZBTB33, ZBTB40, ZBTB41, ZBTB46, ZBTB48, ZBTB7B, ZBTB8OS, ZC3H12B, ZC3H14, ZC3H15, ZC3H6, ZC3H7B, ZC3HAV1, ZCCHC10, ZCCHC11, ZCCHC12, ZCCHC13, ZCCHC14, ZCCHC6, ZCCHC7, ZCCHC8, ZCCHC9, ZCD1, ZCRB1, ZDHHC11, ZDHHC13, ZDHHC14, ZDHHC21, ZDHHC3, ZFAND2A, ZFAND2B, ZFAND3, ZFAT1, ZFHX1B, ZFP14, ZFP161, ZFP36, ZFP36L2, ZFP41, ZFP91, ZFPM1, ZFR1 ZFYVE20, ZFYVE27, ZHX2, ZHX3, ZKSCAN1, ZMAT3, ZMIZ1, ZMYND12, ZNF137, ZNF143, ZNF148, ZNF160, ZNF180, ZNF182, ZNF184, ZNF187, ZNF200, ZNF202, ZNF215, ZNF217, ZNF219, ZNF223, ZNF232, ZNF234, ZNF238, ZNF248, ZNF267, ZNF271, ZNF291, ZNF3, ZNF31, ZNF313, ZNF326, ZNF331, ZNF33B, ZNF34, ZNF341, ZNF346, ZNF354B, ZNF366, ZNF367, ZNF384, ZNF395, ZNF415, ZNF436, ZNF444, ZNF445, ZNF451, ZNF462, ZNF488, ZNF503, ZNF507, ZNF508, ZNF513, ZNF518, ZNF552, ZNF553, ZNF557, ZNF563, ZNF565, ZNF567, ZNF57, ZNF574, ZNF585B, ZNF587, ZNF592, ZNF599, ZNF608, ZNF609, ZNF622, ZNF641, ZNF644, ZNF650, ZNF652, ZNF664, ZNF692, ZNF704, ZNF706, ZNF710, ZNF740, ZNF746, ZNF750, ZNF775, ZNF783, ZNF786, ZNF79, ZNF92, ZNFX1, ZNRF2, ZP2, ZRANB1, ZSCAN2, ZSWIM2, ZSWIM3, ZUBR1, ZW10, ZZEF1 orZZZ3.
In yet a further preferred embodiment, the gene is selected from the group of genes regulated on the transcriptional level by the HNF4α inducing agent Aroclor 1254, wherein the gene is preferably selected from the group of genes according to Table 35.
Table 35 - list of genes regulated on the transcriptional level by the HNF4α inducing agent Aroclor 1254: ABCA1, ABCC3, ABP1, ACE2, ACOX1, ACSL5, ACY3, ACYP2, ADH4, AFF1, AFP, AGR2, AGT, AGXT2, AHSG, AIG1, AKAP7, ALB, ALDH6A1, ALDOB, AMICA1, AMMECR1, ANKRD9, ANKZF1, ANTXR2, ANXA4, AP1S3, APOA1, APOA4, APOB, APOBEC1, APOC3, APOM, AQP3, ARHGAP18, ARL4C, ASGR1, ATAD4, ATXN1, AXIN2, BCL2L14, BCMP11. BMP4, BPHL, BTNL3, C10orf114, C11orf54, C12orf28, C12orf59, C15orf48, C17orf61, C17orf76, C1orf115, C1orf163, C1orf19, C20orf75, C3orf26, C9orf52, CA9, CAPN3, CCND2, CD55, CDC25A, CDC6, CDKN1A, CEACAM1, CEACAM6, CEL, CES1, CFB, CG018, CGI-115, CHORDC1, CLDN2, CMTM8, COL6A1, COTL1, CPS1, CTSB, CYP1A1, CYP27A1, CYP2C9, CYR61, DDIT4, DEPDC7, DI01, DIP2C, DKK1, DPP4, DTL1 DUSP9, ECM2, EDN1, EEF1A1, EFNA1, ENPP7, EPHX2, ERBB3, ETHE1, EVA1, F11R, F2, F2R, FABP1, FAM110C, FAM13A1, FAM20C, FBP1, FGA, FGB, FGG, FGL1, FHIT, FLJ20273, FLJ20920, FLJ42562, FMO5, FOXD1, FOXQ1, FRMD3, FRY, GATM, GBA3, GJB1, GLRX, GLS, GLULD1, GNRH2, GOLT1A, GOSR2, GPA33, GPC3, GPR133, GPR157, GPR160, GRK5, GSN1 GSTA1, GUCY2C, HAL1 HAVCR1, HEPH, HHLA2, HIST2H2BE, HKDC1, HNF4A, HNMT1 HSD17B2, HSPH1 , IGFBP2, IGSF4, IHH, IL6R, INSIG1 , INSIG2, JMJD1A, KCNJ8 KLB, KLC4, KLHL24, KNG1 , KRT20, LCT, LGALS14, LGR5, LHPP1 LIPA, LMCD1 , LOC283537, LOC388323, LOC401152, LOX, MAF, MALL1 MAOB, MAP4K4, MAP6, MBNL3, MCF2L, MCM10, MEP1A, METT10D, METTL7A, MGC24039, MGC33657, MGC4172, MGLL, MLN, MLXIPL, MUC13, MUC20, MYO1A, NAT2, NDRG1 , NR1 I3, NR5A2, NRP2, ODC1 , OIT3, OLR1 , OSTα, OSTβ, P4HA1 , PAG1 , PAPSS2, PCK1 , PCTK3, PCYT1 B, PDK1 , PDZK1 IP1 , PECAM1 , PGCP, PGPEP1 , PHLDA1 , PI3, PIGZ, PIPOX, PLA2G12B, PLD1 , PLOD2, PNRC1 , PODXL, P0LR3H, PPP1 R2, PRDM1 , PRLR, PRODH, PRSS23, PRSS35, RALGPS1 , RASL11 B, RDH5, REEP6, RHOF1 RNASE4, RNF183, RNF19, ROR1 , S100G, S100P, SAMD4A, SAT2, SC4M0L, SCD5, SDCBP2, SEMA6A, SEPP1 , SERPINA1 , SERPINA10, SERPINA6, SERPINC1 , Sl, SIAE, SLC11A2, SLC15A1 , SLC16A4, SLC17A4, SLC1A3, SLC23A3, SLC26A3, SLC39A5, SLC3A1 , SLC44A1 , SLC5A1 , SLC5A9, SLC6A4, SLC7A5, SLC7A6, SLC02B1 , SOAT2, SORL1 , ST6GAL1 , STOM, STS-1 , SULF2, SULT1C1 , SULT1 E1 , SULT2A1 , SYNCRIP, SYT7, TBC1 D4, TBC1 D8, TCF4, TEP1 , TFEC, TFF1 , TFF2, TFF3, TIMP3, TJP2, TM4SF20, TMCC1 , TMEM12, TMEM45A, TMEM92, TMPRSS4, TNFRSF19, TNFSF10, TNS1 , TOB1 , TSC22D2, TSR1 , TTC26, TTR, TTRAP, TUBAL3, UGT2A3, UNC93A, UPB1 , VAV3, WNK4, WNT11 , WWC1 , XPNPEP2, YPEL2, ZC3H6 and ZNF557
Within the context of diagnosis, the first aspect of the invention is, in one example, directed to the use, in particular the in vitro use, of
(A) a human gene, or
(B) the coding region of (A), or
(C) the template strand of a (A) or (B), wherein (C) is preferably a recombinant DNA molecule, or
(D) the coding strand of (A) or (B), wherein (D) is preferably a recombinant DNA molecule, or
(E) the gene product encoded by (A) - (D), or
(F) DNA or RNA sequences hybridizing with (C), and encoding a polypeptide having the biological activity of (E)1) for the diagnosis of colorectal cancer, wherein (A) is selected from one of the groups of genes described herein and, in particular, wherein the level of expression of (A) in a biological sample of a subject suffering from colon cancer is detected by measuring the level of (E) or (F) in the sample, preferably by the use of antibodies directed against (E) or by the use of primers directed against (F), and wherein a level of (E) or (F), being significantly higher or lower than the level of (E) or (F) in a control sample, is indicative of an overexpresssion of (A) in the biological sample, and wherein the subject, is then diagnosed as suffering from adenocarcinoma of the colon.
The invention is thus also directed to the use of an antibody directed against a gene product encoded by a human gene selected from one of the groups of genes described herein, in particular of the group described in claim 1 , for the diagnosis, prognosis and/or treatment monitoring of metabolic and tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer, preferably colorectal adenocarcinoma, or said antibody is used for the preparation of a diagnostic agent for for the diagnosis, prognosis and/or treatment monitoring of metabolic and tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer, preferably colorectal adenocarcinoma.
Within the inventive context, antibodies are understood to include monoclonal antibodies and polyclonal antibodies and antibody fragments (e.g., Fab, and F(ab')2) specific for one of said polypeptides. Polyclonal antibodies against selected antigens may be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, goats, rabbits, mice, rats, chicken or preferably of eggs derived from immunized chicken. Monoclonal antibodies may be generated using conventional techniques (see Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are incorporated herein by reference).
The invention is thus further directed to the use of primer sequences, preferably primer pairs, directed against the mRNA of a human gene selected from one of the groups of genes described herein, in particular of the group described in claim 1 , for the diagnosis, prognosis and/or treatment monitoring of metabolic and tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer, preferably colorectal adenocarcinoma, or said primer sequences are used for the preparation of a diagnostic agent for the diagnosis, prognosis and/or treatment monitoring of metabolic and tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer, preferably colorectal adenocarcinoma, wherein the production of adequate primer sequences and the use thereof, e.g. in a quantitative RT-PCR, is known to the person skilled in the art.
Within the context of the invention the use of a primer or of primers, such as a primer pair may be, selected from Table M1 is particularly preferred.
The first aspect of the invention is accordingly directed to the use of a human gene, in particular the coding region thereof, or of a gene product encoded thereby or of an antibody directed against said gene product or of RNA or DNA sequences, which hybridize to said gene and which code for a polypeptide having the function of said gene product, as a biomarker in the diagnosis, prognosis and/or treatment monitoring of metabolic and tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer, wherein the gene is selected from one of the groups described herein, in particular to the group described in claim 1 , and the use is preferably performed for monitoring the therapeutic treatment of a patient suffering from a metabolic and/or tumorous disease, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer. Within the context of using said biomarkers a method for diagnosing, prognosing and/or staging metabolic and tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer and/or monitoring the treatment of at least one of said diseases, is provided by
(a) measuring the level of expression of at least one biomarker, wherein the biomarker is a gene selected from one of the groups of genes described herein, in particular of the group described in claim 1 in a patient or in a sample of a patient suffering from or being susceptible to a metabolic and/or tumorous disease, and
(b) comparing the level of said at least one biomarker in said patient or in said sample to a reference level of said at least one biomarker.
Accordingly, the first aspect of the invention may also be used in a method of qualifying the HNF4α activity in a patient suffering or being susceptible to metabolic and/or tumorous disease, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer or for classifying a patient suffering from or being susceptible to at least one said diseases, comprising determining in a sample of a subject suffering from or being susceptible to one of said diseases the level of at least one biomarker, wherein the biomarker is the gene product encoded by a gene selected from one of the groups of genes described herein, in particular of the group described in claim 1 , and/or the biomarker is the mRNA sequence encoding the gene product of said selected gene, and wherein the sample level of the at least one biomarker being significantly higher or lower than the level of said biomarker(s) in the sample of a subject without a disease associated with increased activity of HNF4α is indicative of induced HNF4α activity in the subject.
Such method of qualifying the HNF4α activity in a patient suffering from metabolic and/or tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer to a method of treating such diseases preferably comprises administering a drug identified by the first aspect of the invention, or by the second aspect of the invention as described hereinafter, or administering a HNF4α activity modulator, wherein the level of the at least one biomarker being significantly higher or lower than the level of said biomarker(s) in a subject without cancer associated with increased activity of HNF4α is indicative that the subject will respond therapeutically to a method of treating cancer comprising administering said drug or administering a HNF4α activity modulator.
Such methods of qualifying the HNF4α activity may be also used for monitoring the therapeutically response of a patient suffering from metabolic and/or tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer to a method of treating at least one of said diseases comprising administering a drug identified by the first aspect of the invention, or by the second aspect of the invention as described hereinafter, or administering a HNF4α activity modulator, wherein the level of the at least one biomarker before and after the treatment is determined, and a significant decrease or increase of said level(s), preferably a decrease or increase to the normal level(s), of the at least one biomarker after the treatment is indicative that the subject therapeutically responds to the administration said drug or to the administration of the HNF4α activity modulator.
In a preferred embodiment, preferably a RT-PCR (RT = real time) is performed for the afore mentioned methods of qualifying the HNF4α activity , in particular by using the kit described hereinafter.
Within the context of diagnosis of the first aspect, the invention is also directed to a composition for qualifying the HNF4α activity in a patient suffering or being susceptible to a metabolic and/or tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer or for classifying a patient suffering from or being susceptible to at least one of said diseases, wherein the composition comprises an effective amount of at least one biomarker, and wherein the biomarker is a gene selected from one of the groups of genes described herein, in particular of the group described in claim 1 , and/or the biomarker is the gene product encoded by said gene, and/or wherein the composition comprises an effective amount of an antibody directed against said gene product.
Within the context of the invention it is thus understood that the gene according to the invention, or the DNA sequences hybridizing to said gene and encoding a polypeptide having the function of the gene product of said gene, may be part of a recombinant DNA molecule for use in cloning a DNA sequence in bacteria, yeasts or animal cells. Within the context of the invention it is further understood that the gene according to the invention, or the DNA sequences hybridizing to said gene and encoding a polypeptide having the function of the gene product of said gene, may be part by a vector. The invention is thus also directed to the use of a vector for the therapy and/or diagnosis of metabolic and/or cancerous diseases and/or to screen for and to identify drugs against metabolic and/or cancerous diseases, such as diabetes mellitus and/or colorectal cancer may be, wherein the vector comprises a gene selected from one of the goups of genes described herein, in particular the group according to claim 1 , or the vector comprises DNA sequences hybridizing to said gene and encoding a polypeptide having the function of the gene product of said gene.
In particular, the composition for qualifying the HNF4α activity is used for the production of a diagnostic agent, in particular of a diagnostic standard.
In a preferred embodiment, this composition is used for the production of a diagnostic agent for qualifying the HNF4α activity in a patient suffering or being susceptible to a metabolic and/or tumorous disease, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer or for classifying a patient suffering from or being susceptible to at least one of said diseases. In another preferred embodiment this compositon is used for the production of a diagnostic agent for predicting or monitoring the response of a patient suffering from a metabolic and/or tumorous disease, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer to a method of treating at least one of said diseases with a drug, in particular a drug identified according to the first aspect of the invention , or according to the second aspect of the invention as described hereinafter, and/or with a HNF4α activity modulator.
Accordingly the first aspect of the invention is, in another example, directed to the use, in particular the in vitro use, of
(A) a human gene, or
(B) the coding region of (A), or
(C) the template strand of a (A) or (B), wherein (C) is preferably a recombinant DNA molecule, or
(D) the coding strand of (A) or (B), wherein (D) is preferably a recombinant DNA molecule, or
(E) the gene product encoded by (A) - (D), or
(F) DNA or RNA sequences hybridizing with (C), and encoding a polypeptide having the biological activity of (E)1) for the screening of drugs directed against adenocarcinoma of the colon, wherein (A) is selected from one of the groups of genes described herein and, in particular, wherein a biological sample of a subject suffering from adenocarcinoma of the colon, such as a histological sample may be, is contacted with a drug to be tested, and wherein the level of expression of (A) is detected in the sample by measuring the level of (E) or (F) in the sample, preferably by the use of antibodies directed against (E) or by the use of primers directed against (F), and wherein a level of (E) or (F), being significantly lower or higher in the sample contacted with the drug to be sceened than the level of (E) or (F) in a control sample of the same subject, is indicative of a downregulation or upregulation of (A) in the biological sample, contacted with the drug, and wherein the drug is then identified as a drug to be used in the medication of said subject, preferably in a medication for normalizing the level of (A) to the level of (A) in a healthy individual.
Accordingly the first aspect of the invention is, in a further example, directed to the use, in particular the in vitro use, of
(A) human gene, or
(B) the coding region of (A), or
(C) the template strand of a (A) or (B)1 wherein (C) is preferably a recombinant DNA molecule, or
(D) the coding strand of (A) or (B), wherein (D) is preferably a recombinant DNA molecule, or
(E) the gene product encoded by (A) - (D), or
(F) DNA or RNA sequences hybridizing with (C), and encoding a polypeptide having the biological activity of (E)1) for the identification of drugs directed against adenocarcinoma of the colon, wherein (A) is selected from the group of human chromosomal genes as described herein and, in particular, wherein a biological sample of a subject suffering from adenocarcinoma of the colon, such as CACO2 cells may be, is contacted with a drug to be tested, and wherein the level of expression of (A) is detected in the sample by measuring the level of (E) or (F) in the sample, preferably by the use of antibodies directed against (E) or by the use of primers directed against (F), and wherein a level of (E) or (F), being significantly lower or higher in the sample contacted with the drug to be sceened than the level of (E) or (F) in a control sample of the same subject, is indicative of a downregulation or upregulation of (A) in the biological sample contacted with the drug, and wherein the drug is then identified as a drug to be used in the medication of adenocarcinomas of the colon, preferably in a medication for normalizing the level of (A) to the level of (A) in a healthy individual.
Still according to the first aspect of the invention the gene is preferably selected from the group of genes identified by the method according to the second aspect of the invention.
Within the first aspect of the invention and/or within the second aspect of the invention, as described hereinafter, an agent selected from the group consisting of: the agonists, antagonists, drugs, agents, antiestrogens, and compounds according to Tables 36-54, sulfonlyurea derivates, and Aroclor 1254, is used and/or tested and/or screened as the drug and/or as the HNF4α activity modulator.
As disclosed by Mehlmann et al. Toxicology 5 89-95 (1975), the administration of Aroclor 1254 decreases the activity of pyruvate carboxylase (PC), phosphoenolpyrutvate carboxykinase (PEPCK) and glucose-6-phosphatase in diabetic liver.
Since the rate of hepatic gluconeogenesis is increased dramatically in the diabetic state, concomitant with increases in the activities of all gluconeogenic enzymes, i.e. phosphoenolpyrutvate carboxykinase, fructose-1 ,6-bisphosphatase, glucose-6-phosphatase and pyruvate carboxylase, Arocolor 1254 is thus a, at least putative, drug against diabetes mellitus. For comparison, also the administration of insulin to diabetic rats brings the increased amount of pyruvate carboxylase back to the normal level, as disclosed by Wallace & Jitrapakdee Biochemical Journal 340 1-16 (1999).
Further, it is known from the prior art that Aroclor 1254 can inhibit tumor growth in rats, as described, for example, by the article "Inhibition of tumor growth in rats by feeding a polychlorinated biphenyl, Aroclor 1254" by Kervliet & Kimeldorf in Bull Environ Contam 1977 Aug , 18 (2) :243-6. Aroclor 1254 is thus a, at least putative, drug against cancerous diseases.
Consequently, Aroclor 1254 is thus a, at least putative, drug against metabolic diseases, in particular against diabetes mellitus, and/or against cancerous diseases.
The second aspect of the invention provides a method for a genomewide identification of functional binding sites at targeted DNA sequences bound to DNA complexes in cells, healthy and diseased tissues and/or organs, wherein the method comprises, or preferably consists of, the steps of a. chromatin immunoprecipitation and b. DNA-DNA hybridisation for the c. de novo identification of gene targets.
In a preferred embodiment the method according to invention is used for the identification of the functional binding sites of a protein of interest, preferably of a transcription factor, and/or the method is characterized in that a. the chromatin immunoprecipitation comprises or consists of two rounds of sequential chromatin immunoprecipitation, b. a genome wide tiling array is used for the DNA-DNA hybridisation, and/or c. the de novo identification of gene targets comprises reducing the number of false enhancer-gene associations.
In another preferred embodiment the method according to invention is used for determining a region of ChIP enrichment in the immunoprecipitated sample (enrichment site), with respect to a control or to genomic DNA, preferably a HNF4α binding site, and/or the method is characterized in that a. an anti HNF4α antibody is used for the immunoprecipiation b. a human or murine array with a genome-wide 20-50 bp resolution is used, and/or c. all genes, which are separated from their associated enhancer by a CTCF-binding site, are removed from the group of the de novo identified genes (target list).
In a further preferred embodiment the method according to invention is used for mapping the in vivo enrichment sites' of a specific protein of interest, and/or the method is characterized in that a. Caco-2 cells are used, b. human tiling arrays with a genome-wide 35 bp resolution are used, and/or c. all genes, which are separated from their associated enhancer by the CTCF-binding site according to matrix 2, are removed from the target list.
In particular, it is preferred if the method according to the invention further comprises the use of DNA-sequences and/or genes identified by the second aspect of the invention to screen for and to identify novel drug targets for the treatment of metabolic and cancerous diseases, preferably comprising the use according to the first aspect of the invention.
In another preferred embodiment of the method according to the invention novel identified sequences and genes encoding DNA are used.
In yet another preferred embodiment of the method according to the invention polypeptides encoded by novel identified sequences and genes are used.
Still further according to the second aspect of the invention it is preferred, if
-Caco-2 cells cultures treated with Aroclor 1254 are used,
-Protein A Sepharose is used for the chromatin immunoprecipitation
- a quantile normalization is performed and subsequently the raw data is analyzed for enriched regions by three independent algorithms, in particular TAS, MAT and Tilemap, and the results from all three algorithms are intersected,
- parameters for enrichment site (ES) detection are further improved based on the frequency of motives of the protein of interest, in particular of HNF4α -motives, within the enriched regions, which is determined by application of the MATCH algorithm,
- the distribution of the binding sites of the protein of interest, in particular the HNF4α binding sites, relative to transcription start sites (TSS) is analyzed, and the distance from the center of each ES to the closest TSS of a RefSeq gene is determined, - the enrichment allows an easy 'de novo' identification of the DNA binding motif of the protein HNF4α motif,
- regions of 500bp surrounding the identified binding sites are analyzed for DNA binding motifs of the protein of interest, in particular for HNF4α motives
- wherein the ChIP regions are analyzed for overrepresented motifs, in particular using motif analysis programs, such as from Biobase and Genomatix, as well as the tool CEAS -transcription factors cross-talking with the protein of interest, in particular with HNF4α, are determinded
-a relative increase of the frequency of binding motifs for AP1 , GATA, ER and HNFI a, and/or a negative relationship between HNF4α and CART binding motifs is observed.
- the genomic position of the highest scoring motif of the protein of interest (highest likelihood ratio) , in particular of the HNF4α motif (highest likelihood ratio), within the ChIP regions is retrieved and extended for 500 nucleotides to both flanks, and within these sequences, other enriched motifs are detected and preferably the distance to the DNA- binding motif of the protein of interest, in particular to the HNF4α motif, is calculated
- ER motifs co-locate with HNF4α motifs,
- HNF1 , AP1 an GATA motifs have their highest enrichment in a distance of 20 to 60 nucleotides to the DNA-binding motifs of the protein of interest, in particular to the HNF4α motifs,
- all RefSeq genes with a TSS separated by less than 100000 nucleotides from these binding sites are selected for associating the binding sites of the protein of interest, in particular the HNF4α binding sites, identified by the ChlP-chip approach with target genes,
- the potential RefSeq target genes of the protein of interest, in particular of HNF4α, are identified,
- the following parameter are chosen for ES identification with the different programs: for MAT bandwidth = 200, maximum gap = 300, minimum probes = 8, P-value < 0.00001 and MAT score > 5, for Tilemap truncation =-1000000, 1000000, transform = none, GAP<=300/probes between peaks <=5, minimum length 200nt/5 probes, region summary method = HMM (A peak 28 probes on average, cutoff 0,5), FDR = left tail and FDR < 0.015, and for TAS bandwidth = 400, P-value < 0.01 , minimum run = 200 and maximum gap = 250,
- the resulting regions are intersected using the Galaxy tool and after intersection resulting enriched regions shorter than 200 nt are removed,
- the ChIP regions are scanned for transcription factor motifs using position-specific score matrices (PSSM) from TRANSFAC, in particular 533 well-defined PSSM,
- for each matrix, all PSSM matches with cutoff scores from 5.0 (90% of relative entropy) up to 12.0, in increments of 0.5 are considered,
- at each cutoff level, the resulting two sets of motifs are tested for significance and minimum change (with respect to control) of 1.5-fold,
- association of binding sites identified by ChlP-chip with RefSeq annotated genes is performed with the software tool CisGenome, - ES are associated with all RefSeq genes with transcript coding regions within 100000 nt from the center of the ES using Cisgenome,
- all genes with an TSS further than 10OOOOnt from the center of the ES were removed from this list,
-genomic positions of insulator sites retrieved from Kim et al., (2007) are used and converted to the hg18 assembly,
- it is tested if an insulator site is located between ES and the associated TSS, and if this is the case, then the association is removed,
- all RefSeq genes associated with an ES are joined into a single list, which then is used for Gene Ontology categorization, and/or
-gene Ontology categorization is performed with GOFFA and DAVID, -de novo identified genes associated with the DNA binding sites of the protein of interest , in particular with the HNF4α binding sites, are grouped by Ontology terms, in particular in genes in metabolic and cancerous disease,
- the de novo identified genes involved in metabolic processes (lipid metabolism, organic acid metabolism, carboxylic acid metabolism, phosphate, alcohol and carbohydrate metabolism), in particular genes coding for steroid metabolism, are grouped,
- the de novo identified genes involved in developmental (e.g., kidney development) and differentiation categories are grouped,
- the de novo identified genes related to transport, especially lipid transport, are grouped,
- the de novo identified genes involved in signaling, in particular insulin receptor signaling, and regulation of cellular processes are grouped
- the de novo identified genes involved in regulation of different signaling pathways are grouped, and/or
- the de novo identified genes involved in cell death and apoptosis are grouped.
In particular, it is preferred if the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to develop new medications for the treatment of disease as a result of HNF4α dysfunction.
Preferably, the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to develop new drug candidates for treatment of metabolic and tumorous diseases by interfering with the activity of polypeptides, as described herein, encoded by novel identified sequences and genes are used for the purpose of normalizing its activity and to restore a healthy condition.
In another preferred embodiment, the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to optimize novel chemical entities for the purpose of drug development. In yet another preferred embodiment, the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to identify drugs targeting chromatin and its regulation by interfering with protein-DNA, protein-protein and multiprotein- DNA complexes for the purpose of normalizing gene activities in metabolic and cancerous diseases.
In a particular preferred embodiment, the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to identify drugs targeting carbohydrate metabolism in metabolic and tumorous diseases for the purpose of its normalization, wherein a gene, in particular the coding region thereof, selected from the group consisting of
ACLY, ACO1 , ACO2, ADPGK, AKR1A1 , ALDH2, ALDOB, AMDHD2, APOA2, ARPP-19, ARSB, B3GAT1 , B4GALT1 , B4GALT4, B4GALT5, B4GALT6, B4GALT7, C9orf103, CARKL, ChGn, CHST12, CHST13, CHST3, CHST9, CMAS, COG2, DLST, EXT1 , FBP1 , FBP2, FLJ10986, FN3K, FUCA2, FUT4, FUT8, G6PC, GALK1 , GALM, GALNT3, GALNT4, GALNT5, GALT, GANAB, GANC, GBA3, GBGT1 , GCNT2, GCNT3, GENX-3414, GK, GLCE, GMDS1 GNPDA1 , GNS, GPD1 , GPD2, GSK3B, GUSB, GYS2, H6PD, HECA, HEXB, HIBADH, HK1 , HK2, HKDC1 , HS3ST4, HS3ST5, IDH1 , IDH3A, IDS, IMPA1 , INSR, IRS2, ITIH1 , ITIH2, ITIH3, ITIH5, KHK, KIAA0100, KL, KLB1 LARGE, LCT, LCTL, LDHA, LDHAL6B, MAN2A1 , MAN2C1 , MANBA, MDH2, ME1 , ME2, ME3, MGAM, MGAT1 , MGAT2, MGC40579, MMP15, MMP16, MMP17, MMP2, MMP20, NAALADL2, NAGK, NANS, NDST1 , OGDH1 PC, PCK1 , PCK2, PDK1 , PDK3, PDK4, PFKFB1 , PFKFB2, PFKFB3, PFKM, PFKP, PGAM4, PGD, PGK2, PGLYRP2, PGM1 , PGM2, PGM2L1 , PGM3, PHKB1 PKM2, PMM1 , POFUT1 , PPARD, PPP1 CB, PPP1 R2, PRKAA1 , PRKAB1 , PTEN1 PYGB, PYGL, RBKS, RPIA1 SDHB1 Sl, SLC2A2, SLC2A3, SLC2A8, SLC35A2, SLC35A3, SLC35D1 , SLC37A4, SLC3A1 , SMA4, SORD, SPAM1 , ST3GAL2, ST3GAL6, ST6GAL1 , ST8SIA2, ST8SIA4, SUCLA2, SUCLG1 , SUCLG2, SULF2, TFF1 , UAP1 , and UGP2 is used or a gene product encoded by a gene selected from said group of genes, in particular encoded by the coding region of said gene, is used, or RNA sequences, which hybridize to said gene and which code for a polypeptide having the function of said gene product, are used.
In a particular preferred embodiment, the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to identify drugs targeting lipid metabolism in metabolic and tumorous disease for the purpose of its normalization, wherein a gene, in particular the coding region thereof, selected from the group consisting of A4GALT, ABCA1 , ABCD2, ABCG1 , ABHD4, ACAA2, ACACA, ACADM, ACADS, ACAT2, ACBD3, ACLY, ACOT1 , ACOT11 , ACOT12, ACOT2, ACOT7, ACOX1 , ACOX2, ACSL1 , ACSL3, ACSL4, ACSL5, ACSL6, ACSS2, ADIPOR2, ADM, AGPAT1 , AGPAT2, AGPAT3, AKR1 B10, AKR1C1 , AKR1C3, AKR1C4, AKR1 D1 , ALDH3A2, ALOX5AP, ALOXE3, ANGPTL3, AOAH, APOA1 , AP0A2, AP0A4, APOB, APOBEC1 , APOC3, APOF, APOM, ASAH1 , ASAH2, ATP8B1, AYTL2, B3GALT1, B4GALNT2, B4GALT4, BAAT, BTN2A1, BTNL3, C11orf11, C14orf1, CD36, CD74, CDC91L1, CDS1, CDS2, CEL, CERK, CHKA, CLU, CMAS, COQ2, CPNE3, CPNE7, CPT2, CRLS1, CROT, CUBN, CYP19A1, CYP2J2, CYP3A4, CYP3A5, CYP4A11, CYP4F3, CYP51A1, CYP7A1, DCTN6, DEGS1, DGAT1, DGKD, DHCR24, DHRS3, DHRS8, DPAGT1, EBP, EBPL, EHHADH, ELOVL2, ELOVL4, ENPP6, ENPP7, ETNK1, FABP1, FABP2, FABP5, FABP6, FADS1, FDFT1, FDX1, FLJ25084, GALC, GPAM, GPX4, HACL1, HADH, HADHA, HADHB, HAO2, HEXB, HMGCR, HMGCS1, HNF4A, HPGD, HSD17B2, HSD17B3, HSD17B4, HSD17B6, HSD3B1, HSD3B2, IHPK3, IMPA1, LARGE, LASS2, LASS5, LIPA, LIPC, LIPF, LOC340204, LPGAT1, LRP1, LRP2, LRP5, LYPLA2, MGC26963, MGLL, MGST2, MGST3, MIF, MTMR10, MTMR11, MTMR12, MTMR2, MTMR4, MTTP, MVK, NANS, NPC1, NPC2, NR1H4, NR1I2, NR2F2, NR5A1, OSBP, OSBP2, OSBPL10, OSBPL11, OSBPL1A, OSBPL3, OSBPL5, OSBPL6, OSBPL8, OSBPL9, PAFAH2, PBX1, PC, PCCA, PCCB, PCSK9, PCYT1A, PCYT1B, PCYT2, PDE3A, PDSS1, PECl, PECR, PEMT1 PGDS, PHYH, PIGC, PIGF, PIGH, PIGK, PIGL, PIGM, PIGY, PIGZ, PIK3C2A, PIK3R1, PIK4CB, PIP5K2A, PISD, PITPNA, PITPNB, PLA2G12B, PLA2G2A, PLA2G2E, PLA2G4A, PLA2G4D, PLA2G6, PLB1, PLCB1, PLCE1, PLCG1, PLCH1, PLCZ1, PLD1, PLIN, PMVK, PNLIPRP1, PNLIPRP2, PNPLA3, PNPLA8, PPAP2A, PPAP2B, PPARD, PPARG, PPP2CA, PPP2R1B, PRDX6, PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG2, PRLR, PSAP, PTDSS1, PTEN, PTGES, PTGIS1 RBP3, RDH12, SAMD8, SC4MOL, SC5DL, SCAP, SCARB1, SCD, SCD5, SCP2, SEC14L2, SELI, SERINC2, SERPINA3, SGPL1, SHH, SLC27A2, SLC27A3, SMPD1, SMPD3, SOAT1, SOAT2, SORL1, SQLE, SRD5A2, SREBF1, ST3GAL6, STARD4, STARD5, SULT1A1, SULT1B1, SULT1E1, SULT2A1, TBXAS1, TMEM23.TNFRSF1A, TPP1, TRERF1, UCP3, UGCG, UGT1A1, UGT2B11, UGT2B7, UGT8, VLDLR, WWOX, YWHAH, and ZNF202 is used or a gene product encoded by a gene selected from said group of genes, in particular encoded by the coding region of said gene, is used, or RNA sequences, which hybridize to said gene and which code for a polypeptide having the function of said gene product, are used.
In a further preferred embodiment, the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to identify drugs targeting intracellular signaling in metabolic and tumorous disease for the purpose of its normalization, wherein a gene, in particular the coding region thereof, selected from the group consisting of ABL1, ABL2, ABR, ABRA1 ACOT11, ACR, ADCY5, ADCY6, ADCY8, ADCY9, ADIPOR2, ADORA2A, ADORA2B, ADORA3, ADRA1B, ADRA1D, ADRB1, AGTR1, AKAP13, AKAP7, AMBP, ANP32A, APBB2, ARF1, ARF6, ARFGEF2, ARHGAP29, ARHGAP5, ARHGAP6, ARHGEF1, ARHGEF10L, ARHGEF11, ARHGEF12, ARHGEF17, ARHGEF18, ARHGEF3, ARHGEF5, ARHGEF7, ARID1A, ARL1, ARL4A, ARL4C, ARL4D, ARL5A, ARL5B, ARL8B, ASB1, ASB13, ASB14, ASB2, ASB4, ASB7, ASB9, ASIP, ATP2C1, AVPR1A, BCAR3, BCL10, BIRC2, BLNK, BRAF, BRCA1, C20orf23, C5AR1, CALCR, CALCRL, CAP1, CARD10, CARD4, CARHSP1, CBLB1 CCKAR, CCM2, CCNE1, CCR1, CDC42BPA, CDK7, CENTD3, CERK, CERKL1 CFL1, CFLAR1 CHN1, CHN2, CHP1 CHRM1, CHRM2, CNIH, CNIH3, CNIH4, CORO2A, CRKL, CRSP2, CRSP6, CSK, CTNNB1, DAB2IP, DAPK1, DAPK2, DAPP1, DDAH1, DEPDC7, DERL1, DGKA, DGKB, DGKD, DGKH, DGKI, DIRAS3, DLG5, DNMBP, DRD2, DRD5, DSCR1, DSCR1L1, DUSP16, DUSP22, DUSP6, DUSP9, DVL3, DYNC1LI1, EDD1, EDG2, EDNRB, EGF1 EGFR, ELMO1, ERBB2, ERN1, ESR1, F2, F2R, FARP2, FGD2, FGD4, FGD5, FGF2, FHL2, FLJ20184, FLJ30934, FLJ38964, FLJ41603, FRK, FYN, G3BP, GADD45B, GADD45G, GAP43, GAPVD1, GBF1, GCC2, GEM, GHRH, GJA1, GLP1R, GNAQ, GNB1L, GPR89A, GRAP, GRB10, GRB14, GRB2, GRB7, GRK5, GRLF1, GTPBP4, GUCY2C, GUCY2D, HIPK2, HIST4H4, HMOX1, HRH2, HS1BP3, ICK, IFNAR2, IGF1, IHPK3, IL10, IL22RA2, IQGAP2, IQGAP3, IQSEC1, IQSEC3, IRAKI BP1, ITSN1, JAK1, KALRN, KIAA1804, KRAS, KSR1, KSR2, LAT, LATS 1, LATS2, LAX1, LGALS9, LHCGR, LTB4R2, LYN, MAGI3, MAP3K11, MAP3K13, MAP3K4, MAP3K7IP2, MAP3K9, MAP4K3, MAP4K4, MAP4K5, MAPK13, MAPK14, MAPK8, MAPKAPK2, MARK1, MARK2, MBIP, MC3R, MCF2L, MCTP1, MCTP2, MED4, MFHAS1, MGC39715, MINK1, MIST, NCK2, NCOA1, NCOA3, NCOA4, NDFIP1, NEK11, NEK6, NET1, NF1, NFAM1, NFKBIA, NKIRAS1, NKIRAS2, NLK, NMUR2, NOTCH2, NPR2, NPY, NRAS, NRIP1, NUDT4, OPRK1, OPRM1, OTUD7B, P2RY1, P2RY2, P2RY4, P2RY6, PAK1, PARD3, PARK7, PDCD11, PDK1, PDZD8, PIK3C2A, PIK3C2G, PIK3CB, PIK3R1, PIP5K3, PLCB1, PLCE1, PLCG1, PLCH1, PLCZ1, PLD1, PLEK2, PLEKHG1, PLEKHG2, PLEKHG3, PLEKHM1, PLK2, PPAP2A, PPARBP, PPARGC1B, PPM1A, PPP2CA, PPP2R1B, PRDX4, PRKAA1, PRKAR1A, PRKAR2B, PRKCA1 PRKCD, PRKCE, PRKCI, PRKCSH, PRKCZ, PRLR1 PSCD4, PSD3, PSD4, PSEN1, PTEN, PTPLAD1, PTPN11, RAB10, RAB11A, RAB17, RAB1A, RAB20, RAB27A, RAB30, RAB31, RAB32, RAB33A, RAB35, RAB37, RAB38, RAB3B, RAB3D, RAB43, RAB4A, RAB5A, RAB5C, RAB6C, RAB7, RAB7L1, RAB8B, RAB9A, RABIF1 RABL3, RAF1, RALB, RALGPS1, RALGPS2, RAN, RANBP3, RAP1A, RAP1B, RAP2A, RAP2B, RAPGEF1, RAPGEF4, RASA1, RASA2, RASA3, RASAL2, RASGEF1C, RASGRF1, RASGRF2, RBJ, RBM9, RGL1, RGS1, RGS3, RGS9, RHEB, RHOBTB2, RHOC, RHOF, RHOH, RHOT1, RIN2, RIPK1, RND1, RND3, ROCK1, ROCK2, RP1L1, RPS6KA5, RRAGC, RREB1, S100A1, SAC, SAR1B, SCAP, SDCBP2, SELS, SGEF, SGK2, SH2D1A, SH2D3A, SH2D4A, SH2D6, SH3BP5, SH3PXD2A, SHANK2, SHC1, SHE, SHF, SHOC2, SIAH2, SLA, SLC20A1, SMAD2, SNF1LK2, SNX1, SNX10, SNX11, SNX12, SNX13, SNX14, SNX19, SNX24, SNX27, SNX3, SNX4, SNX5, SNX7, SNX9, SOCS5, SOCS6, SOCS7, SOS1, SPAG5, SPRED1, SPRED2, SRPK1, SRPK2, SSTR1 , STAT5B, STAT6, STK17A, STK17B, STK3, STK38, STK38L, STK4, STMN4, SYNGAP1, TACR1, TAOK3, TBK1, TEC, TESK2, TFG, THRAP1, TIAM1, TIAM2, TMED4, TMEPAI, TMPRSS6, TNFAIP3, TNFRSF10B, TNFRSF19, TNFRSF1A, TNFSF10, TNFSF15, TNIK, TNK2, TNS1, TNS3, TRAF3IP2, TRAF5, TRAF6, TRIO, TRIP6, TSSK3, TULP4, UBE2V1, USH1C, VAPA, VAV2, VAV3, VIPR1, WASF2, WNK1, WNK2, WNK4, WSB1, WSB2, YES1, YWHAH1 ZAK, and ZDHHC13 is used or a gene product encoded by a gene selected from said group of genes, in particular encoded by the coding region of said gene, is used, or RNA sequences, which hybridize to said gene and which code for a polypeptide having the function of said gene product, are used. In another preferred embodiment, the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to identify drugs targeting cell cycle and cell proliferation in metabolic and tumorous disease for the purpose of its normalization , wherein a gene, in particular the coding region thereof, selected from the group consisting of
ABM, ABL1, ACHE, ACPP1 ACTN4, ADRA1B, ADRA1D, AGGF1, AHR, AIF1, ALS2CR19, ANAPC4, ANKRD15, APBB1, APBB2, APC, AR, AREG, ARHGEF1, ATM, ATPIF1, AXIN1, B4GALT7, BCAR1, BCAR3, BCL10, BCL2, BCL6, BHLHB3, BIN1, BRCA1, BRCA2, BRIP1, BTC, BTG1, BTG2, BUB1, C10orf46, C10orf9, C2orf29, C9orf127, CABLES1, CCL14, CCNA2, CCND1, CCND2, CCNE1, CCNE2, CCNH, CCNJL, CCNL1, CCNT1, CCNT2, CCRK, CD160, CD164, CD28, CD3E, CD74, CD86, CDC14A, CDC2, CDC25A, CDC25B, CDC25C, CDC37L1, CDC6, CDK10, CDK3, CDK4, CDK5R1, CDK5RAP3, CDK7, CDKL1, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CDKN3, CDT1, CDV3, CENPF, CEP250, CETN2, CETN3, CHAF1A, CHES1, ChGn, CHRM1, CHRM4, CITED2, CLASP1, COL18A1, CREG1, CRTAM, CSF1, CSF1R, CSK, CTCFL, CUL1, CUL3, CUL4A, CXCL5, CYR61, DAB2, DBC1, DCC, DCTN2, DHCR24, DIP13B, DIRAS3, DLEC1, DLG1, DLG3, DLG5, DST, DUSP1, DUSP22, DUSP6, E2F3, E2F7, EDD1, EDN1, EGF, EGFR, ELF4, ELN, EML4, EMP1, ENPEP, ENPP7, EP300, EPS15, EPS8, ERBB2, ERBB2IP, ERG, ERN1, ESCO1, ESR1, ETS1, EXT1, F2, F2R, FABP6, FGA, FGB, FGF2, FGF8, FGF9, FGFR1OP, FGG, FHIT, FLCN, FLJ16793, FLJ40432, FLT1, FRK, G0S2, GAB1, GADD45A, GAS2, GAS6, GMNN, GNRH1, GRLF1, HBP1, HDAC6, HDAC7A, HDAC9, HDGF, HECA, HEXIM1, HIC1, HIPK2, HK2, HOXC10, HPGD, IGF1, IGF1R, IGFBP4, IL10, IL12RB1, IL12RB2, IL18, IL28RA, IL2RA, IL6R, IL9R, ING1, INHBA, INSIG1, IRF1, IRF2, IRS2, ISG20, JAG1, KATNB1, KIAA0367, KIAA0376, KIF25, KITLG, KLF4, KPNA2, KRAS, LAMB1, LAMC1, LATS1, LATS2, LIF, LIG4, LMO1, LRP1, LRP5, LTBP2, LYN, LZTS2, MACF1, MAD2L1, MAP2K6, MAP3K11, MAPK13, MAPK6, MAPRE1, MAPRE2, MAPRE3, MCC, MCM3, MCM8, MCRS1, MDM2, MDM4, MET, MIF, MKI67, MLH3, MNAT1, MNT, MOS, MPL, MSH5, MTSS1, MUTYH, MXD1, MXM, MYB, MYC, NBL1, NCK2, NDP, NEDD9, NEK11, NEK2, NEK6, NF1, NFYC, NIPBL, NME1, NOTCH2, NPY, NR6A1, NRAS, NRD1, NRP1, NUMA1, OPRM1, OSM, PAM, PAPD5, PARD3, PARD6B, PBEF1, PBK, PDCD4, PDF, PDGFB, PDGFRA, PEMT, PFDN1, PGF, PIK3CB, PIM1, PLAGL1, PLCB1, PLG, PMP22, POLA, POLS, POU3F2, PPAP2A, PPARD, PPM1D, PPP1CB, PPP1R9B, PPP2CA, PPP2R1B, PPP3CB, PPP6C, PRDX1, PRKCA, PRKG2, PRKRIR, PRL, PRM1, PROK1, PSMD1, PTEN, PTHLH, PTK2B, PTMA, PTMS, PTP4A1, PTPRC, PTTG1, QSCN6, RAD17, RAD50, RAD51, RAD51L1, RAD54L, RAD9B, RAF1, RAN, RAP1A, RASSF4, RB1CC1, RBL1, RBM5, RBM9, RCBTB1, RCC2, RECK, RFP, RGS2, RINT1, RPS27, RSN, RUNX3, S100A6, SASH1, SCIN, SEPT11, SEPT3, SEPT4, SEPT7, SESN1, SESN3, SGOL1, SH3BP4, SHC1, SHH, SIAH1, SIAH2, SKP2, SLAMF1, SLC12A6, SMARCB1, SMC3, SMPD3, SPAG5, SPHAR, SSR1, SSTR1, STARD13, STIM1, STRN3, SUPT3H, SYCP2, SYCP3, TACC1, TADA3L, TAL1, TBC1D8, TCF7L2, TCFL5, TERF2, TFDP1, TFDP2, TGFB3, TGFBI, TGFBR2, THY1, TM4SF4, TNFRSF11A, TNFRSF8, TNFSF13B, TNFSF15, TNFSF4, TOB1, TOB2, TSGA2, TSPAN2, TSPAN3, TUBG1, TXLNA, TXN, UBE2V1, UHRF1, UHRF2, UNC84B, UNG2, USP8, UTP14C, VEGF, VIPR1, WEE1, WT1, WWOX, XRN1, YWHAG, YWHAH, YWHAQ, ZAK, ZFP36L2, ZW10, andZZEFI is used or a gene product encoded by a gene selected from said group of genes, in particular encoded by the coding region of said gene, is used, or RNA sequences, which hybridize to said gene and which code for a polypeptide having the function of said gene product, are used.
In a particular preferred embodiment, the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to identify drugs targeting programmed cell death in metabolic and tumorous disease for the purpose of its normalization, wherein a gene, in particular the coding region thereof, selected from the group consisting of ABL1, ACIN1, ACTN1, ACTN4, ADORA2A, AHR, ALB, AMID, AMIG02, ANXA4, ANXA5, APOE, APP1 ASAH2, ATG5, AXIN1, BAD, BAG2, BAG3, BBC3, BCAR1, BCL10, BCL2, BCL2A1, BCL2L1, BCL2L10, BCL2L11, BCL2L14, BCLAF1, BID, BIRC2, BIRC3, BIRC4, BMF, BRAF, BRCA1, BRE, BTG1, CARD10, CARD4, CASP10, CASP3, CASP6, CBX4, CD28, CD3E, CD74, CDC2, CDK5R1, CDKN1A, CDKN2A, CEBPG, CFL1, CFLAR, CIAS1, CLU, COP1, CRADD, CROP, CRTAM, CTNNBL1, CTSB, CUL1, CUL3, CUL4A, CYCS, DAD1, DAP, DAPK1, DAPK2, DCC, DHCR24, DIDO1, DNASE1, DNASE1L3, DUSP22, EBAG9, EDAR1 EFHC1, EGLN3, ELMO1, ELMO2, EP300, ERCC3, ERN1, F2, F2R, FAIM, FAIM3, FASTKD1, FOXL2, FOXO1A, FOXO3A, GADD45A, GADD45B, GADD45G, GAS2, GPR65, GSTP1, HBXIP, HIPK2, HMGB1, ICEBERG, IER3, IGF1R, IHPK2, IHPK3, IL10, IL18, IL2RA, INHBA, KIAA0367, KNG1, MAGEH1, MAGI3, MCL1, MDM4, MIF, MOAP1, MRPS30, MTP18, NCKAP1, NEK6, NFKB1, NFKBIA, NME1, NME6, NOTCH2, NRG2, NTF3, NUAK2, NUDT2, OPA1, PAK1, PAWR, PAX7, PDCD10, PDCD11, PDCD2, PDCD4, PDCD6, PECR, PERP, PHLDA1, PHLPP, PIM1, PLAGL1, PLG, PPARD, PPP2CA, PPP2R1B, PRF1, PRKAA1, PRKCA, PRKCE, PRKCZ, PRLR, PROC, PRODH, PSEN1, PTEN, PTH, PTK2B, PTPRC, PTRH2, RAF1, RASA1, RFFL1 RHOT1, RIPK1, RIPK3, RNF7, ROCK1, RP6-213H19.1, RRAGC, RTN4, RUNX3, RYBP, SCARB1, SCIN, SEMA4D, SEMA6A, SEPT4, SERPINB9, SGK, SGPL1, SH3GLB1, SIAH1, SIAH2, SIRT1, SMNDC1, SNRK, STK17A, STK17B, STK3, STK4, TAIP-2, TAX1BP1, TEGT1 TESK2, THY1, TIA1, TIAL1, TIMP3, TLR2, TNFAIP3, TNFAIP8, TNFRSF10B, TNFRSF11B, TNFRSF19, TNFRSF1A, TNFRSF21, TNFRSF25, TNFSF10, TNFSF15, TNFSF18, TP53INP1, TP73L, TPT1, TRAF3, TRAF5, TRAF6, TRIB3, TXNDC5, UBE4B, UNC5B, VDAC1, VEGF, YWHAG, YWHAH, ZAK, ZBTB16, ZDHHC16, and ZNF346 is used or a gene product encoded by a gene selected from said group of genes, in particular encoded by the coding region of said gene, is used, or RNA sequences, which hybridize to said gene and which code for a polypeptide having the function of said gene product, are used.
In a particular preferred embodiment, the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to identify drugs targeting cell morphogenesis and cell / organ development in metabolic and tumorous disease for the purpose of its normalization , wherein a gene, in particular the coding region thereof, selected from the group consisting of
ABLIM1, ABTB2, ACHE, ACIN1, ACTL6A, AGGF1, AHSG, ALX4, AMELX, AMIGOi, AMOT, ANGPTL3, ANKH, ANXA2, APBB1 , APBB2, APOE, AR, ARF6, ARHGEF11 , ARTS-1 , ASH2L, ATP10A, ATPIF1, AXIN1, BCAR1, BCL11A, BHLHB3, BMP2, BMP3, BMP4, BMP6, BMP7, BMP8A, BMP8B, BRAF, BRD8, BTG1, BVES, CACNB2, CALCR, CAMK2D, CANX, CAP1, CAPN3, CART1, CASC5, CASQ2, CCL4, CCM2, CD164, CD1D, CD74, CD86, CD9, CDC42EP4, CDC42EP5, CDH11, CDK5R1, CDK5RAP2, CDK5RAP3, CDX2, CEACAM1, CEBPA, CEBPG, CENPF, CENTD3, CHRDL2, CITED2, CLASP1, CLEC3A, CNTN4, COL11A2, COL12A1, COL18A1, COL1A1, COL1A2, COL2A1, COL9A1, COVA1, CRB1, CREG1, CRIM1, CSDE1, CSF1, CSRP3, CTGF, CYR61, DAZL, DCC, DDX5, DGAT1, DGKD, DLC1, DLG1, DMAP1, DMD, DVL3, DZIP1, EBAG9, EBP, EGFR, ELN, EMP1, EPAS1, ERBB2, ERBB2IP, ESR1, ETS1, ETS2, EVL, EXT1, EYA2, FABP1, FARP2, FBLIM1, FBN1, FCMD, FEZ2, FGD2, FGD4/FGD5, FGF2, FGFR2, FHL1, FLNB, FLT1, F0XL2, FOXN1, FOXO3A, FRZB, GAP43, GAS2, GAS6, GATA4, GATA6, GDF10, GHR1 GJA1, GLCE, GNAO1, GRLF1, HAND1, HBEGF, HCCS, HDAC7A, HDAC9, HECA, HEY1, HILS1, HMGCR, HOXA13, HSD17B3, IFRD1, IGF1, IGFBP2, IGFBP4, IHPK2, IL10, IL18, ING1, ING2, INHBA, IPF1, ITCH, ITGA11, ITGA2, ITGB1BP2, JAG1, KAZALD1, KDR, KIRREL3, KITLG, KL, KLF6, KRT19, LAMB1, LARGE, LECT2, LHCGR, LHX3, LIMA1, LTBP4, LYN, MAFB, MAP7, MARK2, MATN1, MBNL1, MBP, MEF2A, MEF2C, MKKS, MKL2, MPZ, MSX1, MSX2, MTSS1, MUSK, MYF6, MYH10, MYH14, MYST3, NCOA4, NEDD9, NET1, NFAM1, NKX2-2, NOTCH2, NOTCH4, NR5A1, NRCAM, NRD1, NRP1, NRP2, NRXN3, NTNG1, OKL38, OSM, PAPPA2, PAPSS1, PAPSS2, PARD3, PARD6B, PAX1, PAX2, PBX1, PBX3, PDLIM5, PGF, PHEX, PHGDH, PITX1, PITX2, PITX3, PLEKHC1, PLG, POU3F1, P0U4F1, POU6F1, PPARD, PPP1R9B, PPP2CA, PPP2R1B, PRDX1, PRELP, PRKCI1 PRL1 PTEN, PTH1 PTPRC1 QSCN6, RAB3D, RASA1, RHOH, RND1, ROBO1, ROBO2, RPS6KA3, RRAGC, RTN4, RTN4RL1, RTN4RL2, RUNX1, RUNX2, RUVBL1, S100A6, SCGB1A1, SCIN1 SEMA6A, SGCB1 SGCE1 SHC1, SHH, SIAH1, SIRT1, SIX1, SLIT1, SMARCA1, SNAH1 SNRK1 SOCS5, SOCS6, SOCS7, S0RT1, SOX6, SOX9, SPAG6, SPARC, SPINK5, SPN1 SPRY2, SRD5A2, SRI1 STIM2, SVIL, SYCP3, TAGLN3, TBX3, TCF12, TC0F1, TGFB3, THY1, TINAG, TLE1, TLE3, TMEM97, TMPRSS6, TNFAIP2, TNFRSF11B, TNN, TNP1, TNR1 TPD52, TRAPPC4, TSSK3, TUFT1, UBE3A, UTRN, VCL, VCX3A, VEGF1 VIL2, WISP1, WISP3, XRN2, YEATS4, YWHAH, ZBTB16, ZNF160 and ZNF22. is used or a gene product encoded by a gene selected from said group of genes, in particular encoded by the coding region of said gene, is used, or RNA sequences, which hybridize to said gene and which code for a polypeptide having the function of said gene product, are used.
In yet a further preferred embodiment, the use according to the first aspect of the invention or the method according to the second aspect of the invention is performed to select drug candidates of an antisense molecule, ribozyme, triple helix molecule or other new chemical entities targeting the chromatin.
Within the context of diagnosis and therapy, the invention further comprises a kit for qualifying the HNF4α activity in a patient suffering or being susceptible to metabolic and/or tumorous disease, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer or for classifying a patient suffering from or being susceptible to at least one of said diseases, in particular for predicting or monitoring the response of a patient to suffering from at least one of said diseases by a method of treating metabolic and/or tumorous diseases comprising administering a HNF4α activity modulator, comprising at least one standard indicative of the level of a biomarker selected from the groups of genes described herein, preferably the group according to claim 1 , or from the group of gene products encoded by said groups of genes in normal individuals or individuals having metabolic and/or tumorous disease associated with increased HNF4α activity, and instructions for the use of the kit, and, preferably, wherein the at least one standard comprises an indicative amount of at least one gene selected from one of the groups of the genes and/or at least one gene product encoded thereby.
In particular it is preferred if the kit according to the invention further comprises at least one primer or primer pair specifically hybridizing with the mRNA of a biomarker selected from one of the groups of genes and/or at least one antibody specific for a biomarker selected from the group of gene products encoded by said genes, and reagents effective to detect said biomarker(s) in a serum sample.
Within the context of the therapy according to the first aspect of the invention, a medicament for the treatment of metabolic and tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer is provided, wherein the medicament comprises a composition that decreases the expression or activity of a HNF4α modulated gene selected from one of the groups of genes described herein, preferably from the group specified in claim 1.
Within the context of the therapy also an SiRNA composition is provided, wherein the siRNA composition reduces the expression of a de novo identified HNF4α modulated gene selected from one of the groups of genes described herein, preferably from the group specified in claim
1.
The present invention thus employs SiRNA oligonucleotides directed to said genes specifically hybridizing with nucleic acids encoding the gene products of said genes and interfering with gene expression of said genes. Preferably, the siRNA composition comprises siRNA (double stranded RNA) that corresponds to the nucleic acid ORF sequence of the gene product coded by one of said human genes or a subsequence thereof; wherein the subsequence is 19, 20, 21 , 22, 23, 24, or 25 contiguous RNA nucleotides in length and contains sequences that are complementary and non-complementary to at least a portion of the mRNA coding sequence.
The nucleotide sequences and siRNA according to the invention may be prepared by any standard method for producing a nucleotide sequence or siRNA, such as by recombinant methods, in particular synthetic nucleotide sequences and siRNA is preferred.
Further, an antisense composition is provided, wherein the antisense composition comprises a nucleotide sequence complementary to a coding sequence of a HNF4α modulated gene selected from one of the groups of genes described herein, preferably from the group specified in claim 1.
In this regard, the term "coding sequence" is directed to the portion of an mRNA which actually codes for a protein. The term "nucleotide sequence complementary to a coding sequence" in particular is directed to an oligonucleotide compound, preferably RNA or DNA, more preferably DNA, which is complementary to a portion of an mRNA, and which hybridizes to and prevents translation of the mRNA. Preferably, the antisense DNA is complementary to the 5' regulatory sequence or the 5' portion of the coding sequence of said mRNA. It is preferred that the antisense composition comprises a nucleotide sequence containing between 10-40 nucleotides , preferably 12 to 25 nucleotides, and having a base sequence effective to hybridize to a region of processed or preprocessed human mRNA.
In particular, the composition comprises a nucleotide sequence effective to form a base-paired heteroduplex structure composed of human RNA transcript and the oligonucleotide compound, whereby this structure is characterized by a Tm of dissociation of at least 450C.
Preferably, the siRNA composition and/or the antisense composition is/are used for the preparation of a medicament, in particular for the preparation of a medicament for preventing, treating, or ameliorating metabolic and tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer.
Within the context of using drugs according to the invention it is preferred, if said siRNA composition and/or said antisense composition a composition is used. Within the context of using a HNF4α acitivity modulator according to the invention, it is preferred, if a nucleic acid, in particular DNA, hybridizing with the expression regulatory sequence of matrix 1 is used.
Further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
Detailed description
Because of its master regulatory function and its role in malignant disease a new method was probed were particularly interested to search genome-wide for genes regulated by HNF4α. Here a comprehensive genome-wide scan for the de novo identification of HNF4α gene targets by use of the ChlP-chip human genome tiling arrays is reported. With high resolution (on average 35 bp) it was possible to map greater than 17000 binding sites for HNF4α in a human intestinal adeno-carcinoma cell line. Notably, the enterocyte-like Caco-2 cell line has been widely used as a model for intestinal epithelial cells (Delie and Rubas, 1997) and was found to express a high level of HNF4α. This cell line differentiates into enterocytes upon confluence (Soutoglou et al., 2002). Furthermore, in differentiated Caco-2 cells HNF4α protein expression is comparable to its expression in liver (Niehof and Borlak, 2008). Therefore, Caco-2 cells are an interesting system to study the HNF4α gene regulatory network.
The study according to the invention is the first genome-wide approach to identify nearby 6600 RefSeq genes targeted by HNF4α. The importance of promoter-distal regions in HNF4α mediated transcriptional control is highlighted, and a basis for elucidation of transcriptional networks formed by cooperating transcription factors acting in concert with HNF4α is offered. Finally, based on induction of HNF4α protein expression and a genome-wide transcriptome analysis, good agreement between de novo identified genes and their expression in Caco-2 cells is demonstrated. Taken collectively, the studies according to the invention will help to determine the role of HNF4α gene regulatory networks in cancerous and metabolic diseases and can therefore be applied for an identification of new drug targets in the development of genome based medicines.
Results:
ChIP experiments were performed with Caco-2 cell culture as described by Niehof et al. (2005). An antibody with high specificity against HNF4α (Santa Cruz sc 6556x) was used for the IP. Enrichment of two known positive controls, binding sites in the promoter regions of HNFIa and AGT, was confirmed by quantitative real time PCR. Total input DNA from three independent biological replicates, which served as a control, was diluted to 1 ng/μl, and amplified in parallel with IP-DNA from three independent biological replicates following a PCR amplification protocol optimized for unbiased amplification. After labeling, the samples were hybridized to Affymetrix Human tiling 2.0R arrays with a genome- wide 35 bp resolution. After quantile normalization, raw data were analyzed for enriched regions by three independent algorithms (TAS (Affymetrix), MAT (Johnson et al., 2006) and Tilemap (Ji et al., 2005)). Initial cut off criteria were determined based on the detection of a weakly enriched positive control (OTC). Parameters for enrichment site (ES) detection were further improved based on the frequency of HNF4α -motives within the enriched regions, which was determined by application of the MATCH algorithm (KeI et al., 2003). To increase confidence in the identified intervals, results from all three algorithms were intersected. However, the overlap of ES identified by the three approaches was very high (Fig. S1), and differences partially due to the use of different repeat libraries. Intersecting of the different data sets lead to the identification of 17.561 enrichment sites, which agrees well with the high number of ES described by Rada-lglesis et al. (2005).
To determine the reliability of the identified ES, 15 ES were randomly chosen and enrichment in the primary IP-DNA was confirmed by realtime PCR (Fig. 1). For all selected sites, enrichment could be confirmed, indicating a low false discovery rate.
Among the identified ES were many HNF4α binding sites described in literature and present in the Biobase database, including /WT (R00114), GCC (R08885), PCK (R12074), APOB (R01612), CYP2C9 (R15905), AKR1C4 (R13037), ACADM (R15923) or CYP27A1 (R15917). In several cases, like SHBG (R15941), enrichment sites were found within a few hundred basepairs relative to the reported binding sites. Other binding sites described in literature, like ALDH2 (R15845), could not be confirmed. Quantification by real time PCR showed that the ALDH2 site was also not enriched in the primary IP-DNA. This does not necessarily mean that these sites are not bound by HNF4α in Caco-2 cells, as interacting factors may mask the HNF4α epitope.
However, as HNF4α binding sites described in literature were identified in many different tissues/cell lines, it must be expected that some are not bound in Caco-2 cells, as accessibility of binding sites depends on chromatin organization, which in turn depends on the cell type. This is supported by ChlP-chip publications on other transcription factors, where different cell types were tested in parallel, and significant differences in DNA binding sites in these different cell types were reported (e.g. Xu et al., 2007).
HNF4α binds predominantly to enhancer elements
To analyze the distribution of HNF4α binding sites relative to transcription start sites (TSS), the distance from the center of each ES to the closest TSS of a RefSeq gene was determined. In the study according to the invention, an ~4.7-fold overrepresentation of promoter-proximal ES was observed (Table 1 ; Fig. 2). However, only 4 % of the ES mapped to promoter-proximal regions, and 3 % of all RefSeq promoters are bound (Table 1). This agrees well with the findings reported by Rada-lglesias et al. (2005), but contradicts results reported by Odom et al. (2004), where >12% of all promoters were bound by HNF4α. The overall distribution of ES is very similar to that observed in genome-wide ChlP-chip for another member of the nuclear receptor family, i. e. the estrogen receptor (Carroll et al., 2006). A significant lack of preference for binding to 5' promoter-proximal regions has also been reported for other transcription factors, e.g. p53, cMyc or p63 (Table 7; Cawley et al., 2004; Wei et al., 2006; Yang et al., 2006). Thus, some transcription factors like E2F1 show a clear preference for binding to 5' promoter- proximal regions (Bieda et al., 2006), but accumulating evidence is highly suggestive for promoter-proximal regions to constitute only a small fraction of mammalian gene regulatory sequences. Indeed, members of the nuclear receptor family clearly display higher activity at enhancer rather than promoter binding sites, as evidenced by the invention and other investigators (e.g. Bolton et al., 2007). Consequently, studies based on promoter sequence containing arrays can be misleading.
Indeed, for many transcription factor, binding sites are located in first introns. Therefore the frequency of binding sites in introns of RefSeq annotated genes was analyzed in more detail (Fig. 2). A clear overrepresentation for binding sites in the first introns was observed, but less so for second introns, while binding sites in third (or more distant) introns were not significantly enriched as compared to the random control set.
The distribution of identified ES across the chromosomes shows a clear under representation for the Y chromosome (Table 2). Noteworthy, even when excluding the sex chromosomes, the ES/gene ratio varied greater than 6 fold between the chromosomes. Therefore, further the chromosomal distribution of ES was analyzed, and it was found that they are not randomly distributed, rather clusters are formed (Fig. 3a). These clusters are not related to differences in the gene density within these regions, as shown for on chromosome 10 (Fig. 3b).
The HNF4α motif is highly enriched within the ChIP regions:
Known HNF4α binding motifs are highly abundant within the ChIP regions. Using stringent criteria to minimize false positives, an up to > 14-fold enrichment was detected for different HNF4α matrices compared to the genomic background (Table 3). This enrichment allowed an easy 'de novo' identification of the HNF4α motif: Among 5 motifs identified by a Gibbs motif sampler, 2 motifs represented HNF4α binding sites (Fig. 4).
When the regions of 500bp surrounding the 17561 identified binding sites were analyzed for HNF4α motifs with settings to minimize false negatives, 23145 motifs more than in random control sequences (i.e. above background) were counted. This equals 1.32 motifs / ChIP region. Further, in 98.1% of all ChIP regions at least one motif was detected. Minimize false negatives (MinFN) cut off criteria of the Transfac matrix were set to a value that provides recognition of 90% of binding sites used to create the matrix, accepting 10% false negatives. As the fraction of sequences without detected motifs is significantly smaller than 10%, it is possible that these sequences may also contain HNF4α binding sites. This indicates that most of the ChIP regions are enriched due to direct binding of HNF4α.
Then the binding sites reported by Rada-lglesias et al. (2005) were analyzed by the same approach, and 1.13 motifs / Chip region (500bp surrounding the 194 identified binding sites) were counted. This is significantly less than seen with the data set according to the invention. Therefore, the use of high resolution tiling arrays enabled better identification of high quality binding sites.
Rada-lglesias et al. (2005) speculated that the identified ES within 5000 nt from the closest transcription start site are due to indirect interactions of HNF4α with other transcription factors. Consequently, they developed a model where HNF4α binds to distant (>5000 nt) enhancer elements, that create chromatin loops by interacting with other, promoter-bound transcription factors. Unfortunately, this model is based on less than 1 % of genomic sequences. However, using the high statistical power of the genome-wide data according to the invention, indeed a higher representation of HNF4α binding motifs was shown in promoter-distal regions compared to promoter-proximal regions (Figure S3a). Further, regions with a low enrichment, indicated by a high P-Value given by the detection algorithm, show a higher percentage of promoter- proximal binding sites, and a lower number of HNF4α motifs, than regions with higher enrichment (Figure S3b). Therefore, it was demonstrated that often HNF4α binding to promoter- proximal regions is indirect (due to interaction with other transcription factors), and therefore weaker. However, as HNF4α motifs are still enriched in promoter-proximal regions it seems likely that HNF4α can act as a promoter binding as well as an enhancer binding transcription factor.
Enhancer elements defined by HNF4α display high conservation
An analysis of the average conservation of the HNF4α binding sites identified in this study was performed by use of the tool CEAS (Ji et al., 2006), which extends binding sites in both directions, and calculates the average conservation score for each nucleotide (Fig. 5a). Nucleotides in the center, were an enhancer element could be expected, show a two times higher conservation than those at the ends of the plot (genomic background). The increased conservation supports the idea that the detected HNF4α binding sites are situated within evolutionary maintained enhancer elements. When the same analysis was performed by aligning the ES peak position detected by MAT, instead of the center position of the ES, the conservation peak was even more clearly defined. The sharp drop at a distance of ~250 nucleotides to the peak position may be suggestive for these enhancer elements to have a size of ~500 nucleotides.
Subsequently, the distribution of HNF4α motifs around the peak and center positions of the ChIP regions was analyzed (Fig. 5b). Again, distribution of motifs around the peak position showed less scattering than the distribution around the center position, supporting that the peak position gives a better prediction of the actual binding site. An enrichment of motifs above background was restricted to a region of 600-1000 nucleotides around the peak position.
Transcription factors cross-talk with HNF4α
To identify transcription factor binding sites that might form composite modules together with detected HNF4α binding sites, the ChIP regions for overrepresented motifs were analyzed, using motif analysis programs from Biobase and Genomatix, as well as the tool CEAS. Among the motifs with the highest fold enrichment are matrices similar to the HNF4α binding motif, e. g. the binding motifs for COUP-TF, PPAR or LEF1 (Table 3; Fig. 6). These transcription factors are known to compete with HNF4α for common binding sites (Galson et al., 1995; Hertz et al., 1995; Hertz et al., 1996; Dongol et al., 2007; For motif similarity see also Kielbasa et al., 2005). However, many motifs dissimilar to HNF4α, e.g. the binding motifs for HNF1 , AP1 , GATA transcription factors or CREB, where also enriched within the ChIP regions (Table 3; Supplementary tables S1 , S2 and S3; Fig. 7). Significant overrepresentation of these motifs has been further confirmed with different sets of ChIP regions defined by high- and low stringency cut-off criteria (data not shown). The corresponding transcription factors can therefore be expected to form composite modules with HNF4α. If these factors act combinatorial with HNF4α, it could be further expected that the frequency of their motifs increases with decreasing distance to the HNF4α binding sites. Therefore, the frequency of their motifs relative to the HNF4α binding sites was analyzed identified in this study.
A histogram of the density of binding motifs for the transcription factors AP1 , GATA, ER and HNF1 relative to the peak position of the regions detected by ChlP-chip was created (Fig.8). This histogram shows that the enrichment of these motifs is restricted to a region of a few hundred basepairs around the peak position, supporting the idea that they are part of enhancer elements defined by HNF4α. Interestingly, besides relative increase of the frequency of binding motifs for AP1 , GATA, ER and HNFIa, a negative relationship between HNF4α and CART binding motifs was also observed. It is tempting to speculate that this significant drop in CART motif frequency is of regulatory importance for HNF4α.
Other analyzed motifs showed only a slight correlation between the number of motifs and the distance to the peak position, although they were clearly enriched or depleted in ChIP regions compared to background controls (e.g. USF, HNF6, CREB). Future studies need to delineate their cooperativity with HNF4α, even though their motifs are wider spread and no clear peak formation is detectable.
Additionally, sequence similarity of HNF4α and estrogen receptor (ER) binding motifs at flanking sequences was observed (Fig. 9). Enrichment of ER motifs, and possibly other motifs as well, could therefore be coincidental for enrichment of HNF4α. To analyze the probability of cooccurrences of enriched motifs, and to get a better idea about the distances between AP1 , GATA, ER and HNF4α binding sites, it was aimed to locate the exact HNF4α binding sites by motif analysis. The genomic position of the highest scoring HNF4α motif (highest likelihood ratio) within the ChIP regions was retrieved and extended for 500 nucleotides to both flanks. Within these sequences, other enriched motifs were detected and the distance to the HNF4α motif (i.e. the centre) was calculated (Fig. 10). As expected, most ER motifs co-locate with HNF4α motifs, leading to the high peak at the center. In contrast, HNF1 , AP1 an GATA motifs have their highest enrichment in a distance of 20 to 60 nucleotides to the HNF4α motifs, and are underrepresented in the center due their mutual exclusion by the presence of the HNF4α motif. As the ER motif partially overlaps with the HNF4α motif, it is difficult to judge whether enrichment within the ChIP regions is due to a functional connection between the two factors. Recently, a genome-wide map of ER binding sites was obtained (Carroll et al, 2006). Binding sites for ER and HNF4α were compared, and a considerable overlap was found (Fig. 1 1). Using the low stringency set of HNF4α binding sites binding, 15% of the ER binding sites were also targeted by HNF4α, supporting the idea of cooperation between HNF4α and the ER nuclear receptor.
A genome-wide ChlP-chip scan reveals HNF4α's master function
As mentioned above, ChlP-chip experiments were employed by Rada-lglesias et al (2005) to identify HNF4α binding sites within the ENCODE regions in HepG2 hepatoma cells. Likewise, a ChlP-chip protocol was used by Odom et al. (2004) to identify binding sites within promoter regions in human hepatocytes and pancreatic islets. Here the genomewide approach according to the invention was compared with the aforementioned studies to identify regions which overlap amongst these platforms, and the data according to the invention was compared to the binding sites reported in these publications (Fig. 12; Table 7).
Of the 194 enrichment sites reported by Rada-lglesias et al (2005), 76 overlapped with 244 enrichment regions identified according to the invention within the ENCODE region. This equals to 39 % agreement as compared to an overlap with a random control group of 1 %. Nonetheless, in the study according to the invention and that of Rada-lglesias different cells were used and the platforms and protocols employed may not bedirectly comparable. Furthermore, in the study of Odom et al. (2004) 1553 HNF4α bound promoter sequences were reported for hepatocytes. In the study according to the invention, and when compared with the sequences used on the Hu13K arrays of Odom et al., a total of 575 binding sites were detected (Fig 12; Table 7). Of these, 200 binding sites overlapped with the data according to the invention. Therefore 13 % of the promoter binding sites proposed by Odom et al. are confirmed, but no evidence for the remaining ~ 1300 HNF4α binding sites suggested by Odom et al. was obtained. Given the fact that the authors proposed a 16% false discovery rate the need for quality controls becomes apparent. Furthermore, the overlap with a random control group of 5% is comparably high. Actually, of HNF4α binding sites reported in pancreatic islets only 9 % could be confirmed for Caco-2 cells. This is much less significant than the overlap obtained with the data reported by Rada-lglesias et al.(2005). Search for insulators in HNF4α targeted genes
Most enhancers appear to be promiscuous and can regulate multiple genes (West et al., 2005). Additionally, the genes with the TSS closest to the enhancer are not necessarily the ones regulated by this enhancer (Blackwood, 1998). Enhancer action can thus take place over hundreds of kilobases (Dekker et al.2008), and even cases of inter-chromosomal regulation by enhancers are known (Spilianakis et al., 2005). Nonetheless, most known enhancer elements are within 100000 nt of their respective TSS.
Activity of an enhancer is defined by enhancer-blocking insulators. If an active insulator is placed between an enhancer and a promoter, no communication between them, and therefore no activation of transcription by the enhancer, is possible. In vertebrates, binding of the protein CTCF to an insulator element is required for insulator function. Recently, genome-wide data of insulator regions became available. Kim et al. (2007) identified 13.804 CTCF binding sites. Furthermore, they compared insulator activity between different cell types, and found that CTCF localization is largely invariant.
To associate the HNF4α binding sites identified by the ChlP-chip approach according to the invention with target genes, all RefSeq genes with a TSS separated by less than 100000 nucleotides from these binding sites were selected. It was then considered reasonable to reduce the number of false enhancer-gene associations by removing all genes from the target list according to the invention which were separated from their associated enhancer by a CTCF- binding site originally identified by Kim et al. (2007). With this approach 5936 potential RefSeq target genes of HNF4α were identified.
Ontology of de novo identified HNF4α gene targets
Based on RefSeq annotations it was possible to group de novo identified genes associated with HNF4α binding sites by Ontology terms. As expected, many genes identified in the present study are involved in different metabolic processes (lipid metabolism, organic acid metabolism, carboxylic acid metabolism, phosphate, alcohol and carbohydrate metabolism), with the highest significance for genes coding for steroid metabolism. Categories related to transport, especially lipid transport, were also significantly overrepresented. The pivotal role of HNF4α in the regulation of a wide range of metabolic and transport processes (e.g., fatty acid and cholesterol metabolism) is well known (Watt et al., 2003; Schrem et al., 2002). Likewise, significant overrepresentation of developmental (e.g., kidney development) and differentiation categories was found. HNF4α is well known to have an important role in development (Sladek et al., 1990; Sladek et al, 1993), and is an essential driver for epithelial differentiation (Watt et al., 2003). Needless to say, HNF4α ko mice die around E 8_5 due to deficient organ development. In general, HNF4α can be seen as a regulator of an epithelial phenotype, and several lines of evidence suggest HNF4α to play an essential role in activation of the expression of genes encoding cell junction molecules (Spa'th and Weiss, 1998; Chiba et al., 2003; Parviz et al., 2003; Satohisa et al., 2005; Battle et al., 2006).
Another overrepresented GO category is insulin receptor signaling. HNF4α is well known to control the insulin secretory pathway (Miura et al., 2006), and was linked to rare monogenic disorder, maturity-onset diabetes of the young (MODY), confirming its role in insulin signaling (Yamagata et al., 1996). The overrepresentation of GO categories representing well known functions of HNF4α supports the general biological significance of the association of binding sites according to the invention with target genes.
Furthermore, overrepresentation of categories involved in signaling and regulation of cellular processes was found. Few direct targets in these categories are known for HNF4α, however, the role of HNF4α in development is likely to involve regulation of different signaling pathways. Also a highly significant overrepresentation of the categories cell death and apoptosis was found, which might be related to HNF4α -functions in development. Indeed, genes from these categories might be important in context of a tumor suppressor activity of HNF4α which was recently suggested (Grigo et al., 2008). Overall, the data according to the invention can help to identify genes in metabolic and cancerous disease.
Correlation of genome-wide HNF4α ChlP-chip and gene expression data
Besides ChlP-chip, the most common approach to identify targeted genes is to analyze changes in transcript abundance caused by its increased or diminished expression or activity. Several investigators have thus used this approach to identify genes targeted by HNF4α. Among these, three publications used human cells (Lucas et al, 2005: HEK293 human embryonic kidney cells; Naiki et al., 2002: HUH7 human hepatoma cells and Sumi et al., 2007: HepG2 human hepatoma cells). Surprisingly, the authors reported that HNF4α activity influenced the transcript levels of only a small number of genes. However, it is known from yeast, that most transcription factors bind under 'non-activating' conditions, and that often transcriptional regulation is not mediated at the level of DNA binding alone (Harbison et al., 2004). A further limitation of expression studies is that they only recover target genes that respond to analyzed transcription factor under the chosen, often unphysiological experimental conditions, e. g. exaggerated expression etc.. Furthermore, such expression analyses cannot differentiate between direct and indirect targets.
Therefore, to confirm functional binding sites of de novo identified HNF4α gene targets, Caco-2 cell cultures were treated with Aroclor 1254, a known HNF4α inducer (Borlak et al., 2001). Indeed, after Aroclor 1254 treatment of Caco-2 cells, binding of the HNF4α protein to the HNF1 promoter was increased (Niehof and Borlak, 2008). Caco-2 cells were treated with Aroclor 1254 for 48 and 72 hours. Strong induction of HNF4α was confirmed by western blot analysis (Fig. 12) and increased binding activity by EMSA, and genome-wide expression profiling by microarray analyses was performed to search for regulated genes. Using stringent criteria (llogarithmized signal ratio| > 1.5, both after 48 and 72 hours), 536 unique RefSeq-annotated genes were identified to be regulated (Suppl. table 4). Of these, 383 genes were upregulated and 153 genes were downregulated. These were compared to the list of potential targets identified by the ChlP-chip study according to the invention, and a highly significant overlap of 63 % or 336 genes was found (Table 6). This stresses the importance of HNF4α in the Aroclor 1254 response, and supports the biological significance of HNF4α binding sites identified by ChlP-chip tiling arrays.
Finally, published data for HNF4α targets based on its overexpression in mammalian cell lines were compared. Essentially, a high overlap, ranging from 65 to 94 %, of the genes identified by expression profiling experiments was found (Table 6). Interestingly, the highest overlap was found with the targets identified by Sumi et al. (2007). The approach used in this study differs from the others in the point that targets were not only identified by overexpression of HNF4α, but also by knock down by si'RNA, and only genes increased by overexpression and decreased by siRNA were reported as target genes. Therefore, targets reported here might be considered more reliable.
Discussion
For technical as well as practical reason, research on trans-acting factors and their corresponding c/s-elements focused on promoter-proximal binding sites. With the availability of
ChlP-chip assays, genome-wide scans for transcription factor binding sites becomes feasible.
This highly significant improvement will translate to better understanding of basic mechanisms of transcriptional control and an identification of promoter distal binding sites in facilitating RNA processing events.
In the study according to the invention it was focused on the transcription factor HNF4α, as this protein plays a critical role in liver development and its metabolic competence. This Zinc-finger protein is also of highest interest in drug discovery and cancer research, as highly malignant hepatocellular carcinomes could be reverted to a less aggressive phenotype by overexpression of HNF4α (Lazarevich et al., 2004). For the first time an unbiased, genome-wide set of HNF4α binding sites is reported, and a valuable resource for interrogating HNF4α gene regulatory networks is established.
In the study according to the invention, it was possible to evidence > 95% of the HNF4α binding sites to be located in promoter-distal regions, a distribution similar to that described for the estrogen receptor (Carrol et al., 2006). Therefore, ChlP-chip assays focusing only on promoter regions (e.g. Odom, 2004 or Cheng, 2006) are limited.
Furthermore, an analyses of HNF4α motifs within the regions identified show an enrichment of motifs in regions confined to ~600 bp, which demonstrates the high accuracy in identification of
HNF4α binding sites achieved by the use of high resolution tiling array.
The use of genome-wide ChlP-chip tiling arrays enabled an identification of an unexpected high number of HNF4α binding sites. Even with stringent criteria, i.e. reproducible identification of enriched regions by three different algorithms, and stringent exclusion of repetitive elements, > 17000 binding sites for HNF4α were identified. The high number of HNF4α motifs which were counted within these ChlP-chip identified regions further supports that the vast majority of these sites are direct HNF4α binding sites. Additionally, it is shown that the HNF4α binding sites identified in this study are conserved between species (see conservation score reported in fig. 5), which is an accepted indicator of the biological relevance of sequences. Enhancer elements are constituted by clusters of binding sites for different transcription factors (Michelson, 2002). The fact that it was possible to identify a highly significant enrichment or even depletion of binding motifs of several other transcription factors in the close vicinity of the detected HNF4α binding sites, together with the conservation across species, stresses that the identified binding sites are biologically functional enhancer elements.
The regions surrounding the HNF4α binding sites were analyzed for overrepresentation of transcription factor binding motifs from different databases with different algorithms, and it was shown for several motifs to be significantly overrepresented in these regions. Moreover, there are also motifs which are significantly underrepresented, such as CART. Based on a thorough and detailed analysis, it was possible to evidence a close relationship between HNF4α and AP1 , GATA, ER or HNF1 binding sites. The close association of these motifs enabled to predict composite models formed by the corresponding transcription factors on bound enhancer elements. While single cases of synergistic action of HNF4α with HNF1 (Fourel et al.,1996), ER (Harnish et al., 1996) or GATA transcription factors (Sumi et al., 2007; Alrefai et al., 2007) are already known, to the best of our knowledge the data according to the invention is the first report to suggest a collaborative action of HNF4α and AP1. While over 95% of the identified binding sites are promoter distal sequences identified as enhancer elements, promoter proximal sites are highly enriched compared to random controls. This suggests that HNF4α can also interact directly with the basal transcriptional machinery. The actual number of promoter-proximal binding sites will even be higher, as transcription start sites only of RefSeq annotated genes were included.
In the past enhancers could hardly be identified by available methods; their genome-wide mapping is only possible through the advent of ChlP-chip technology. One of the most promising aspects of future research will therefore be the integration of all the transcription factor binding sites into a genome-wide map of enhancer elements, like it is currently done within the ENCODE regions (See The ENCODE Project Consortium, 2007). Current methods for association of enhancers and their target genes are mostly restricted to CCC or related methods, which allow only the analysis of single enhancer elements, and are technically challenging (Dekker, 2006). In recent publications, association of promoter-distal transcription factor binding sites with their target genes is mostly based on the search for the closest transcription start site or the closest gene. In this regard, the importance of insulator elements in determining enhancer activity has just been unravelled; Their position can be analyzed genome-wide (Dorman et al., 2007; Kim et al., 2007). Therefore, available data on insulator elements was used to narrow down the number of genes associated with the HNF4α binding sites identified in the study according to the invention. Based on such analysis ~6000 RefSeq annotated gene targets were determined.
Because of its master function knowledge on HNF4α targeted genes will help to identify novel drug targets for the treatment of cancerous and metabolic disease. This principle has been demonstrated by the invention and other investigators based on identification of novel HNF4α gene targets in liver cancer and metabolic diseases (Niehof and Borlak, 2005; Niehof and Borlak, 2005; Niehof and Borlak, 2008)
Methods
Caco-2 Cell Culture and Aroclor 1254 treatment
Caco-2 cells were obtained from and cultivated as recommended by Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), seeded with a density of 4 X 106 cells/75 cm2 flask, and harvested after 11 days. Aroclor 1254 treatment was performed as described in Borlak et al. (2001).
Chromatin immunoprecipitation (ChIP)
All chromatin immunoprecipitation (ChIP) procedures were carried out as described by Weinmann et al. (2001) with some modifications. The samples were sonicated on ice until cross-linked chromatin was fragmented to approximately 0.2 to 1.6 kilobase pairs. Protein A Sepharose CLB4 (Amersham Biosciences, Freiburg, Germany) was blocked with 1 mg/ml bovine serum albumin and washed extensively before use. Chromatin preparations were precleared by incubation with "blocked" Protein A-Sepharose for 1 h at 4°C. Precleared chromatin from 2.5 X 107 cells was incubated with 1 μg of HNF4α antibody and rotated at 4°C overnight. After recovering of immunocomplexes, extensive washing, and elution, two samples were pooled for a second immunoprecipitation step with the HNF4α antibody. Two rounds of sequential chromatin immunoprecipitations (Weinmann et al., 2001) were used to increase purity and specificity of target DNA for ChlP-cloning and for validation of ChlP-derived clones. Other investigators used a single immunoprecipitation step with obvious limitations for validation of targets (Horak et al., 2002; Tomaru et al., 2003).
[Caco-2 cell culture, ChIP and chromatin preparation were performed as previously described (Niehof et al., 2005), with the exception that the blocking steps With herring sperm DNA were omitted. Aroclor 1254 treatment was performed as described in Borlak et al. (2001).].
ChlP-chip assay
High specificity of the antibody against HNF4α (Santa Cruz sc 6556x) used for the IP was confirmed by Western blot analysis. Enrichment of two known positive controls, binding sites in the promoter regions of HNF1 and AGT, was confirmed by quantitative real time PCR, to ensure that the ChIP was successful. The three samples showing the highest enrichment were selected for ChlP-chip. Total input DNA from three independent biological replicates, which served as a control, was diluted to 1ng/μl, and amplified in parallel with IP-DNA from the three independent biological replicates. Amplifikation was performed according to the Affymetrix protocol, however, cycle number and amount of taq polymerase have been optimized for unbiased amplification. For fragmentation and labeling of the amplified DNA, the GeneChlP WT Double-Stranded DNA Terminal Labeling Kit (P/N 900812) from Affymetrix was used. Fragmentation success was confirmed with the Agilent Bioanalyzer. The labeled samples were hybridized to Affymetrix Human tiling 2.0R arrays with a 35 base pair resolution.
Raw data (CEL-files generated by GCOS after scanning) were analyzed for enriched regions by three independent algorithms, TAS (Affymetrix/Cawley et al., 2004), MAT (Johnson et al., 2006) and Tilemap (Ji et al., 2005). Initial cut off criteria were determined based on the detection of a weakly enriched positive control (OTC). Parameters for enrichment site (ES) detection were further improved based on the rate of HNF4α -motives within the enriched regions, which was determined by application of the MATCH algorithm (KeI et al., 2003). Finally, following parameter were chosen for ES identification with the different programs: for MAT bandwidth = 200, maximum gap = 300, minimum probes = 8, P-value < 0.00001 and MAT score > 5, for Tilemap truncation =-1000000, 1000000, transform = none, GAP<=300/probes between peaks <=5, minimum length 200nt/5 probes, region summary method = HMM (A peak 28 probes on average, cutoff 0,5), FDR = left tail and FDR < 0.015, and for TAS bandwidth = 400, P-value < 0.01 , minimum run = 200 and maximum gap = 250. Resulting regions were intersected using the Galaxy tool (Giardine et al., 2005). After intersection resulting enriched regions shorter than 200 nt were removed.
ChIP and enrichment validation by real-time PCR
ChIP was performed as previously described (Niehof et al., 2005). ChIP-DNA from three independent experiments (122, 124 and f3) was used for enrichment validation. Realtime PCR was performed on the Light Cycler (Roche Diagnostics, Mannheim, Germany) with the following conditions denaturation at 94°C for 120 s, extension at 720C for different times, and fluorescence at different temperatures. Primer sequences, annealing times and temperatures, extension times and fluorescence temperatures are summarized in Table M1. The reaction was stopped after a total of 45 cycles, and at the end of each extension phase, fluorescence was observed and used for quantification within the linear range of amplification. Δct-values were calculated versus diluted total input, and normalization, i. e. calculation of ΔΔct-values was performed using a β-actin negative control, HNF1 upstream. No enrichment of on negative control against the other was observed. Sequence conservation analysis
Enrichment site centers (left) or Peak positions detected by MAT (right) were extend to 200 bp in both directions, and analyzed with CEAS (Ji et al., 2006) for conservation and motif content. For conservation analysis, CEAS extends the 200 bp genomic regions to 3000 bp, and calculates for each nucleotide the average conservation score, based on the high-quality phast- Cons (Siepel et al., 2005) information from the UCSC GoldenPath genome Resource. The average conservation scores were plotted against the nucleotides position.
Analysis of ChlP-chip enriched regions for overrepresented motifs
Sequence analyses for the detection of TF binding motifs were performed with MATCH (KeI et al., 2003) and Cisgenome (http://www.biostat.jhsph.edu/~hji/cisgenome/). Further, identification of enriched motifs within ChlP-chip detected regions was performed by use of CEAS (Ji et al., 2006) and Genomatix Region Miner (http://www.genomatix.de/index.html).
Correlation of HNF4α binding sites to RefSeq annotated genes and Gene Ontology categorization
Association of binding sites identified by ChlP-chip with RefSeq annotated genes was performed with the software tool CisGenome (http://www.biostat.jhsph.edu/~hji/cisgenome/). ES were associated with all RefSeq genes with transcript coding regions within 100000 nt from the center of the ES using Cisgenome (http://www.biostat.jhsph.edu/~hji/cisgenome/). All genes with an TSS further than IOOOOOnt from the center of the ES were removed from this list. Genomic positions of insulator sites where retrieved from Kim et al., (2007), and converted to the hg18 assembly at http://main.g2.bx.psu.edu/. Subsequently, it was tested if an insulator site was located between ES and the associated TSS, and if this was the case, then the association was removed. All RefSeq genes associated with an ES where joined into a single list, which then was used for Gene Ontology categorization. Gene Ontology categorization was performed with GOFFA (Tong et al., 2003) and DAVID (Dennis et al., 2003).
To analyze enrichment of binding sites in introns, a list of all RefSeq genes and their intron/exon structure was obtained from http://genome.ucsc.edu/. The number of ChIP regions and of regions from the random control set, which overlap introns, was determined using the Galaxy tool (http://g2.trac.bx.psu.edu/).
Expression profiling:
Total RNA was isolated using QIAGEN's RNeasy total RNA isolation kit according to the manufacturer's recommendations. 10 μg of total RNA were prepared for hybridization using the respective Affymetrix kits according to the manufacturer's recommendations. Samples were hybridized to Affymetrix U133Plus2.0 genechip arrays. GCOS 1.4 was used to calculate the level of differential expression at each time point relative to 0 h.
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Aroclor experiments:
To confirm functional binding sites of de novo identified HNF4α gene targets, Caco-2 cell cultures were treated with Aroclor 1254, a known HNF4α inducer36. After Aroclor 1254 treatment of Caco-2 cells, binding of the HNF4α protein to the HNF1 promoter was increased7. Caco-2 cells were treated with Aroclor 1254 for 48 and 72 hours, respectively. Strong induction of HNF4α was confirmed by western blot analysis and increased binding activity was observed by EMSA (Fig. 7). A genome-wide expression profiling was performed by microarray analyses to search for regulated genes. Using stringent criteria, 536 RefSeq-annotated genes were identified to be regulated (Supplementary Table 4). Of these, 383 genes were upregulated and 153 downregulated. These were compared to the list of potential targets identified by ChlP-chip, and it was found a significant overlap of 63 % or 336 genes (Supplementary Table 6). This stresses the importance of HNF4α in the Aroclor 1254 response, and supports the biological significance of HNF4α binding sites identified by ChlP-chip.
References
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Figure legends:
Fig. 1 : HNF4α ChIP was performed and enrichment of novel HNF4α binding sites detected by ChlP-chip was confirmed by real time PCR. ChIP-DNA from three independent experiments was used. Normalization was performed using a β-actin negative control, and the values are shown as fold enrichment versus total input. HNF1 upstream is a second negative control, located upstream of the known HNF4α binding site in the HNF1 promoter, and is used to confirm the β-actin negative control.
Fig. 2: Distribution of ES relative to RefSeq loci. a) Location of HNF4α binding sites relative to the closest transcription start sites (TSS) of RefSeq genes, compared to random distribution. The Y-axis gives the number of binding sites, and the X-axis the distance to closest TSS in nucleotides, b) Overrepresentation of HNF4α ES in first, second an third introns of RefSeq annotated genes, relative to a random control regions.
Fig. 3: A) Each chromosome was divided into 150 'bins', and within each bin the number of ES was counted. In the grey line chart, the number of HNF4α binding sites within each bin is represented as a single data point. Below each chromosome the minimum and maximum number of binding sites located in a single bin is given. B) The distribution of HNF4α binding sites (grey line chart, upper half) is compared to the distribution of known genes on chromosome 10. Green arrows mark two gene-sparse regions, in which also hardly ES are found. The red arrows mark two region with a high number of HNF4α binding sites and a low number of genes. Analyses where performed using the Ensembl tool Karyoview ( http://www.ensembl.org/Homo_sapiens/karyoview ).
Fig. 4: HNF4α ChlP-chip enrichment sites allow easy 'de novo' prediction of the HNF4α binding motif. After analysis of the sequence of regions enriched by HNF4α ChlP-chip with Gibbs motif sampler, the HNF4α-motif, as described by the TRANSFAC matrices M01031or M00134, was actually detected two times, with the second motif presenting only a half site.
Fig. 5: a) Conservation of all HNF4α binding sites (blue line). Enrichment site centers (blue) or Peak positions (red) were extend to 1000 bp in both directions, and for each nucleotide the average conservation score, based on the high-quality phast-Cons (Siepel et al., 2005) information from the UCSC GoldenPath genome Resource, was calculated. The average conservation scores were plotted against the nucleotides position. Analyses where performed with CEAS (Ji et al., 2006). b) HNF4α motifs in the area of 1000 bp surrounding enrichment site peak or center positions were detected with MATCH, using cut offs to minimize false positives. The distance of the center of detected motifs to the peak or center position of the enrichment sites was calculated. A Histogram was created using bins of 50 nucleotides around the center or peak positions. The blue line shows the deviation of HNF4α motifs relative to the enrichment site center, the red line shows the deviation relative to the peak position.
Fig. 6: Computational screens were performed for motifs which are enriched within the detected HNF4α ChlP-chip enriched regions. Binding sites were defined as the area of 300 surround the peak positions. Besides the expected overrepresentation of HNF4α-motifs, an enrichment of HNF4α-similiar motifs like COUP-TF, PPAR or LEF was found. Similarity of the motifs is visualized by using Weblogo depiction (http://weblogo.berkeley.edu/). A complete list of enriched motifs can be found in Table 3.
Fig. 7: As described in Fig. 6, HNF4α-dissimilar motifs are displayed. Dissimilarity is visiualized by using Weblogo.
Fig. 8: Distribution of AP1 , CART, ER, GATA2, HNF1 and SREBP motifs within the regions enriched by HNF4α-ChlP, relative to the peak position (represented as 0). Enrichment site centers were extend to 500 nt in both directions, and Motifs were detected by use of the MATCH algorithm using cutoff criteria to minimize the sum of false positives and false negatives. Regions were segmented into bins of 25 nt, and the number of occurrences of the different motifs within each bin was counted. Fig. 9: Display of the overlap between the binding motifs of HNF4α and the estrogen receptor by use of Weblogo illustrations. Both motifs show an partial overlap.
Fig. 10: Plot of the relative distance of HNF4α motifs to other motifs enriched in the ChIP region. Within ChIP regions the most conserved HNF4α motifs where identified. The sequences of the 500 nucleotides surrounding these most conserved HNF4α motifs where retrieved and analyzed for those motifs of other TF that were also enriched in the ChIP regions. Then, the distance between these motifs and the HNF4α motif was calculated using Cisgenome for motif detection, and plotted as histogram, using bins of 20 nt. The HNF4α motif is found at the center, reaching from nt -6 to nt +6.
Fig. 11 : Overlap between estrogen receptor (ER) binding sites and HNF4α binding sites. The high stringency set of ER binding sites identified by ChlP-chip, described in Carrol et al. (2006), was obtained. Thereafter, the percentage of ER binding sites which were identified in this study to by also bound by HNF4α was calculated, and displayed in a bar chart. To give an estimation of random background, further the overlap of ER binding sites was calculated with the random control regions described herein.
Fig. 12: For Rada Iglesias, the Random Control group (600nt) was used. For Odom et al, a number of promoter regions from the Huk13 array used in their study equal to the number of promoters they detected as binding sites was selected by random. This was necessary, because Odom et al. tested only promoter regions, and these regions are highly enriched within binding sites identified in this study. Therefore, comparison to the Random Control group (600nt) would have a high negative bias.
Fig. S1 : Venn diagram of overlap between ES identified by variable algorithms. ES were identified by use of three different algorithms. Although different parameter settings (e. g. band width of 200, 300 and 400 nucleotides) and different algorithms were used, the overlap was surprisingly high. The Venn diagram was calculated using the intersect function of Galaxy (Giardine et al., 2005).
Figure S2: HNF4α induction in Caco-2 cells. a) HNF4α Western blotting of 20 μg Caco-2 cell extract. A clear induction of HNF4α protein expression can be seen after 48 and 72h of ArocloM 254 treatment. b) Electrophoretic mobility shift assays with 2.5 μg Caco-2 cell nuclear extract and oligonucleotides corresponding to the A-site of the HNF1_ promoter (HNFI pro) as 32P labeled probes. In supershift assays an antibody directed against HNF4α (+) was added. Binding of HNF4α is significantly increased after 72h of Araclor1254 induction. Figure S3: Promoter-proximal ES might be indirect HNF4α binding sites, a) Bootstrapping analysis of HNF4α binding motif (matrix M01031) occurrences in promoter-proximal and promoter distal regions. 100 promoter-proximal ES (-138 to -2 relative to the TSS) were compared to 100 promoter-distal ES (-24972 to -23489 relative to the TSS) by the bootstrapping analysis tool POBO (Kankainen and Holm, 2004). Promoter-proximal ES show a significantly lower number of HNF4α motifs, b) ES (300bp surround the peak position) were sorted by their Pvalue (as calculated by the MAT algorithm) and divided into bins of 1000 ES. For each bin the number of HNF4α motif occurrences and the percentage of promoter-proximal ES was calculated. As can be clearly seen, ES with a high Pvalue (weak ES) are more likely to be located promoter-proximal, and to contain no HNF4α binding motif.
Tables:
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Further evidence of the applicability ot the invention is given by the disclosure of the articles Borlak J, Niehof M. HNF4alpha and HNF1 alpha dysfunction as a molecular rational for cyclosporine induced posttransplantation diabetes mellitus. PLoS One. 2009;4(3):e4662 and
Niehof M, Borlak J. Expression of HNF4alpha in the human and rat choroid plexus: implications for drug transport across the blood-cerebrospinal-fluid (CSF) barrier. BMC MoI Biol. 2009; 10:68, which have been published after the priority date, and which are incorporated herein by reference.
The characteristics of the invention being disclosed in the preceding description, the subsequent tables, drawings, and claims can be of importance both singularly and in arbitrary combination for the implementation of the invention in its different embodiments.
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Claims

What is claimed is:
1. Use of a human gene, in particular the coding region thereof, or a gene product encoded thereby or of RNA or DNA sequences, which hybridize to said gene and which code for a polypeptide having the function of said gene product, or of an antibody directed against said gene product, for the therapy and/or diagnosis of metabolic and/or cancerous diseases and/or to screen for and to identify drugs against metabolic and/or cancerous diseases, such as diabetes mellitus and/or colorectal cancer may be, wherein the gene is selected from the group of the human chromosomal genes having at least one expression regulatory sequence according to matrix 1 ("de novo" HNF4α matrix) in the range of 100000 nucleotides upstream or downstream of their transcription start site in the human genome, and wherein the at least one expression regulatory sequence according to matrix 1 is located within the chromosomal position specified by the start and end sites according to Tables 9-32 (chromosomes 1-22, X-, Y- chromosome).
2. Use as claimed in claim 1 , wherein genes having an expression regulatory sequence according to matrix 2 (CTCF matrix) between the transcription start site and the at least one expression regulatory sequence (matrix 1 ("de novo" HNF4α matrix)) in the human genome are excluded from the group of genes.
3. Use as claimed in one the claims 1-2, wherein the gene is selected from the group of genes further having at least one expression regulatory sequence according to matrices 3-6 (AP1 , GATA2, ER, HNF1 matrices), preferably according to matrix 3, in the range of 20-60 nucleotides upstream or downstream of the at least one expression regulatory sequence according to matrix 1 ("de novo" HNF4α matrix)) in the human genome.
4. Use as claimed in one of the claims 1-3, wherein genes having an expression regulatory sequence according to matrix 7 (CART1 matrix) in the range of 20-60 nucleotides upstream or downstream of the transcription start site in the human genome are excluded from the group of genes.
5. Use as claimed in one the claims 1-4, wherein the gene is selected from the group of genes located on chromosomes 6 or 10.
6. Use as claimed in one of the claims 1-5, wherein the gene is selected from the group of genes coded at least partially in the genomic regions according to table 33.
7. Use as claimed in one of the claims 1-6, wherein the gene is selected from the group of genes according to Table 34.
8. Use as claimed in one of the claims 1-7, wherein the gene is selected from the group of genes according to Table 35.
9. Use as claimed in one of the claims 1-8, wherein the group of genes is identified by the method according to one of the claims 10-16.
10. Method for a genomewide identification of functional binding sites at targeted DNA sequences bound to DNA complexes in cells, healthy and diseased tissues and/or organs comprising the steps of a. chromatin immunoprecipitation and b. DNA-DNA hybridisation for the c. de novo identification of gene targets (RefSeq genes).
11. Method as claimed in claim 10, in particular for the identification of the functional binding sites of a protein of interest, preferably of a transcription factor, wherein a. the chromatin immunoprecipitation comprises or consists of two rounds of sequential chromatin immunoprecipitation, b. a genome wide tiling array is used for the DNA-DNA hybridisation, and/or c. the de novo identification of gene targets comprises reducing the number of false enhancer-gene associations.
12. Method as claimed in one of the claims 10-11 , in particular for determining a region of ChIP enrichment in the immunoprecipitated sample (enrichment site), with respect to a control or to genomic DNA, preferably a HNF4α binding site, wherein a. an anti HNF4α antibody is used for the immunoprecipiation b. a human or murine array with a genome-wide 20-50 bp resolution is used, and/or c. all genes, which are separated from their associated enhancer by a CTCF- binding site, are removed from the group of the de novo identified genes (target list).
13. Method as claimed in one of the claims 10-12, in particular for mapping the in vivo enrichment sites of a specific protein of interest, wherein a. Caco-2 cells are used, b. human tiling arrays with a genome-wide 35 bp resolution are used, and/or c. all genes, which are separated from their associated enhancer by the CTCF- binding site according to matrix 2, are removed from the target list.
14. Method as claimed in one of the claims 10-13, further comprising the use of DNA- sequences and/or genes identified by the method according to one of the claims 9-12 to screen for and to identify novel drug targets for the treatment of metabolic and cancerous diseases, preferably comprising the use according to one of the claims 1- 7.
15. Method as claimed in one of the claims 10-14, wherein novel identified sequences and genes encoding DNA are used.
16. Method as claimed in one of the claims 10-15, wherein polypeptides encoded by novel identified sequences and genes are used.
17. Use or method as claimed in one of the preceding claims to develop new medications for the treatment of disease as a result of HNF4α dysfunction.
18. Use or method as claimed in one of the preceding claims to develop new drug candidates for treatment of metabolic and tumorous diseases by interfering with the activity of polypeptides according to claim 16 for the purpose of normalizing its activity and to restore a healthy condition.
19. Use or method as claimed in one of the preceding claims to optimize novel chemical entities for the purpose of drug development.
20. Use or method as claimed in one of the preceding claims to identify drugs targeting chromatin and its regulation by interfering with protein-DNA, protein-protein and multiprotein-DNA complexes for the purpose of normalizing gene activities in metabolic and cancerous diseases.
21. Use or method as claimed in one of the preceding claims to identify drugs targeting carbohydrate metabolism in metabolic and tumorous disease for the purpose of its normalization as exemplified by but not limited to the following genes and gene products: ACLY1 ACO1, ACO2, ADPGK1 AKR1A1, ALDH2, ALDOB, AMDHD2, APOA2, ARPP-19, ARSB, B3GAT1, B4GALT1, B4GALT4, B4GALT5, B4GALT6, B4GALT7, C9orf103, CARKL, ChGn, CHST12, CHST13, CHST3, CHST9, CMAS, COG2, DLST, EXT1, FBP1, FBP2, FLJ10986, FN3K, FUCA2, FUT4, FUT8, G6PC, GALK1, GALM, GALNT3, GALNT4, GALNT5, GALT, GANAB, GANC, GBA3, GBGT1, GCNT2, GCNT3, GENX-3414, GK, GLCE, GMDS, GNPDA1, GNS, GPD1, GPD2, GSK3B, GUSB, GYS2, H6PD, HECA, HEXB, HIBADH, HK1, HK2, HKDC1, HS3ST4, HS3ST5, IDH1, IDH3A, IDS, IMPA1, INSR, IRS2, ITIH1, ITIH2, ITIH3, ITIH5, KHK, KIAA0100, KL, KLB1 LARGE, LCT, LCTL, LDHA, LDHAL6B, MAN2A1, MAN2C1, MANBA, MDH2, ME1, ME2, ME3, MGAM, MGAT1, MGAT2, MGC40579, MMP15, MMP16, MMP17, MMP2, MMP20, NAALADL2, NAGK, NANS, NDST1, OGDH1 PC, PCK1, PCK2, PDK1, PDK3, PDK4, PFKFB1, PFKFB2, PFKFB3, PFKM, PFKP1 PGAM4, PGD, PGK2, PGLYRP2, PGM1, PGM2, PGM2L1, PGM3, PHKB, PKM2, PMM1, POFUT1, PPARD, PPP1CB, PPP1R2, PRKAA1, PRKAB1, PTEN, PYGB, PYGL, RBKS, RPIA, SDHB, Sl, SLC2A2, SLC2A3, SLC2A8, SLC35A2, SLC35A3, SLC35D1, SLC37A4, SLC3A1, SMA4, SORD, SPAM1, ST3GAL2, ST3GAL6, ST6GAL1, ST8SIA2, ST8SIA4, SUCLA2, SUCLG1, SUCLG2, SULF2, TFF1, UAP1 and UGP2.
22. Use or method as claimed in one of the preceding claims to identify drugs targeting lipid metabolism in metabolic and tumorous disease for the purpose of its normalization as exemplified by but not limited to the following genes and gene products: A4GALT, ABCA1, ABCD2, ABCG1, ABHD4, ACAA2, ACACA, ACADM, ACADS, ACAT2, ACBD3, ACLY, ACOT1, ACOT11, ACOT12, ACOT2, ACOT7, ACOX1, ACOX2, ACSL1, ACSL3, ACSL4, ACSL5, ACSL6, ACSS2, ADIPOR2, ADM, AGPAT1, AGPAT2, AGPAT3, AKR1B10, AKR1C1, AKR1C3, AKR1C4, AKR1D1, ALDH3A2, ALOX5AP, ALOXE3, ANGPTL3, AOAH, APOA1, APOA2, APOA4, APOB1 APOBEC1, APOC3, APOF1 APOM, ASAH1, ASAH2, ATP8B1, AYTL2, B3GALT1, B4GALNT2, B4GALT4, BAAT, BTN2A1, BTNL3, C11orf11, C14orf1, CD36, CD74, CDC91L1, CDS1, CDS2, CEL1 CERK, CHKA1 CLU, CMAS, C0Q2, CPNE3, CPNE7, CPT2, CRLS1, CROT, CUBN, CYP19A1, CYP2J2, CYP3A4, CYP3A5, CYP4A11, CYP4F3, CYP51A1, CYP7A1, DCTN6, DEGS1, DGAT1, DGKD, DHCR24, DHRS3, DHRS8, DPAGT1, EBP, EBPL, EHHADH, EL0VL2, ELOVL4, ENPP6, ENPP7, ETNK1, FABP1, FABP2, FABP5, FABP6, FADS1, FDFT1, FDX1, FLJ25084, GALC, GPAM, GPX4, HACL1, HADH, HADHA, HADHB, HAO2, HEXB, HMGCR, HMGCS1, HNF4A, HPGD, HSD17B2, HSD17B3, HSD17B4, HSD17B6, HSD3B1, HSD3B2, IHPK3, IMPA1, LARGE, LASS2, LASS5, LIPA, LIPC, LIPF, LOC340204, LPGAT1, LRP1, LRP2, LRP5, LYPLA2, MGC26963, MGLL, MGST2, MGST3, MIF, MTMR10, MTMR11, MTMR12, MTMR2, MTMR4, MTTP, MVK, NANS, NPC1, NPC2, NR1H4, NR1I2, NR2F2, NR5A1, OSBP, OSBP2, OSBPL10, OSBPL11, OSBPL1A, OSBPL3, OSBPL5, OSBPL6, OSBPL8, OSBPL9, PAFAH2, PBX1, PC, PCCA, PCCB, PCSK9, PCYT1A, PCYT1B, PCYT2, PDE3A, PDSS1, PECI, PECR, PEMT, PGDS, PHYH, PIGC, PIGF1 PIGH, PIGK, PIGL, PIGM, PIGY, PIGZ, PIK3C2A, PIK3R1, PIK4CB, PIP5K2A, PISD, PITPNA, PITPNB, PLA2G12B, PLA2G2A, PLA2G2E, PLA2G4A, PLA2G4D, PLA2G6, PLB1, PLCB1, PLCE1, PLCG1, PLCH1, PLCZ1, PLD1, PLIN, PMVK, PNLIPRP1, PNLIPRP2, PNPLA3, PNPLA8, PPAP2A, PPAP2B, PPARD, PPARG, PPP2CA, PPP2R1B, PRDX6, PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG2, PRLR, PSAP, PTDSS1, PTEN, PTGES, PTGIS, RBP3, RDH12, SAMD8, SC4MOL, SC5DL, SCAP, SCARB1, SCD, SCD5, SCP2, SEC14L2, SELI, SERINC2, SERPINA3, SGPL1, SHH, SLC27A2, SLC27A3, SMPD1, SMPD3, SOAT1, SOAT2, SORL1, SQLE, SRD5A2, SREBF1, ST3GAL6, STARD4, STARD5, SULT1A1, SULT1B1, SULT1E1, SULT2A1, TBXAS1, TMEM23, TNFRSF1A, TPP1, TRERF1, UCP3, UGCG, UGT1A1, UGT2B11, UGT2B7, UGT8, VLDLR, WWOX, YWHAH and ZNF202.
23. Use or method as claimed in one of the preceding claims to identify drugs targeting intracellular signaling in metabolic and tumorous disease for the purpose of its normalization as exemplified by but not limited to the following genes and gene products: ABL1, ABL2, ABR, ABRA, ACOT11, ACR, ADCY5, ADCY6, ADCY8, ADCY9, ADIPOR2, ADORA2A, ADORA2B, AD0RA3, ADRA1B, ADRA1D, ADRB1, AGTR1, AKAP13, AKAP7, AMBP, ANP32A, APBB2, ARF1, ARF6, ARFGEF2, ARHGAP29, ARHGAP5, ARHGAP6, ARHGEF1, ARHGEF10L, ARHGEF11, ARHGEF12, ARHGEF17, ARHGEF18, ARHGEF3, ARHGEF5, ARHGEF7, ARID1A, ARL1, ARL4A, ARL4C, ARL4D, ARL5A, ARL5B, ARL8B, ASB1, ASB13, ASB14, ASB2, ASB4, ASB7, ASB9, ASIP, ATP2C1, AVPR1A, BCAR3, BCL10, BIRC2, BLNK, BRAF, BRCA1, C20orf23, C5AR1, CALCR, CALCRL, CAP1, CARD10, CARD4, CARHSP1, CBLB, CCKAR, CCM2, CCNE1, CCR1, CDC42BPA, CDK7, CENTD3, CERK, CERKL, CFL1, CFLAR, CHN1, CHN2, CHP, CHRM1, CHRM2, CNIH, CNIH3, CNIH4, CORO2A, CRKL, CRSP2, CRSP6, CSK, CTNNB1, DAB2IP, DAPK1, DAPK2, DAPP1, DDAH1, DEPDC7, DERL1, DGKA, DGKB, DGKD, DGKH, DGKI, DIRAS3, DLG5, DNMBP, DRD2, DRD5, DSCR1, DSCR1L1, DUSP16, DUSP22, DUSP6, DUSP9, DVL3, DYNC1LI1, EDD1, EDG2, EDNRB, EGF, EGFR, ELMO1, ERBB2, ERN1, ESR1, F2, F2R, FARP2, FGD2, FGD4, FGD5, FGF2, FHL2, FLJ20184, FLJ30934, FLJ38964, FLJ41603, FRK, FYN, G3BP, GADD45B, GADD45G, GAP43, GAPVD1, GBF1, GCC2, GEM, GHRH, GJA1, GLP1R, GNAQ, GNB1L, GPR89A, GRAP1 GRB10, GRB14, GRB2, GRB7, GRK5, GRLF1, GTPBP4, GUCY2C, GUCY2D, HIPK2, HIST4H4, HMOX1, HRH2, HS1BP3, ICK, IFNAR2, IGF1, IHPK3, IL10, IL22RA2, IQGAP2, IQGAP3, IQSEC1, IQSEC3, IRAKI BP1, ITSN1, JAK1, KALRN, KIAA1804, KRAS, KSR1, KSR2, LAT, LATS 1, LATS2, LAX1, LGALS9, LHCGR, LTB4R2, LYN, MAGI3, MAP3K11, MAP3K13, MAP3K4, MAP3K7IP2, MAP3K9, MAP4K3, MAP4K4, MAP4K5, MAPK13, MAPK14, MAPK8, MAPKAPK2, MARK1, MARK2, MBIP, MC3R, MCF2L, MCTP1, MCTP2, MED4, MFHAS1, MGC39715, MINK1, MIST, NCK2, NCOA1, NCOA3, NCOA4, NDFIP1, NEK11, NEK6, NET1, NF1, NFAM1, NFKBIA, NKIRAS1, NKIRAS2, NLK1 NMUR2, NOTCH2, NPR2, NPY1 NRAS, NRIP1, NUDT4, OPRK1, OPRM1, OTUD7B, P2RY1, P2RY2, P2RY4, P2RY6, PAK1, PARD3, PARK7, PDCD11, PDK1, PDZD8, PIK3C2A, PIK3C2G, PIK3CB, PIK3R1, PIP5K3, PLCB1, PLCE1, PLCG1, PLCH1, PLCZ1, PLD1, PLEK2, PLEKHG1, PLEKHG2, PLEKHG3, PLEKHM1, PLK2, PPAP2A, PPARBP, PPARGC1B, PPM1A, PPP2CA, PPP2R1B, PRDX4, PRKAA1, PRKAR1A, PRKAR2B, PRKCA, PRKCD, PRKCE, PRKCI, PRKCSH, PRKCZ, PRLR, PSCD4, PSD3, PSD4, PSEN1, PTEN, PTPLAD1, PTPN11, RAB10, RAB11A, RAB17, RAB1A, RAB20, RAB27A, RAB30, RAB31, RAB32, RAB33A, RAB35, RAB37, RAB38, RAB3B, RAB3D, RAB43, RAB4A, RAB5A, RAB5C, RAB6C, RAB7, RAB7L1, RAB8B, RAB9A, RABIF, RABL3, RAF1, RALB, RALGPS1, RALGPS2, RAN, RANBP3, RAP1A, RAP1B, RAP2A, RAP2B, RAPGEF1, RAPGEF4, RASA1, RASA2, RASA3, RASAL2, RASGEF1C, RASGRF1, RASGRF2, RBJ, RBM9, RGL1, RGS1, RGS3, RGS9, RHEB, RHOBTB2, RHOC, RHOF, RHOH, RHOT1, RIN2, RIPK1, RND1, RND3, ROCK1, ROCK2, RP1L1, RPS6KA5, RRAGC, RREB1, S100A1, SAC, SAR1B, SCAP, SDCBP2, SELS, SGEF, SGK2, SH2D1A, SH2D3A, SH2D4A, SH2D6, SH3BP5, SH3PXD2A, SHANK2, SHC1, SHE, SHF, SHOC2, SIAH2, SLA, SLC20A1, SMAD2, SNF1LK2, SNX1, SNX10, SNX11, SNX12, SNX13, SNX14, SNX19, SNX24, SNX27, SNX3, SNX4, SNX5, SNX7, SNX9, SOCS5, SOCS6, SOCS7, SOS1, SPAG5, SPRED1, SPRED2, SRPK1, SRPK2, SSTR1, STAT5B, STAT6, STK17A, STK17B, STK3, STK38, STK38L, STK4, STMN4, SYNGAP1, TACR1, TAOK3, TBK1, TEC, TESK2, TFG, THRAP1, TIAM1, TIAM2, TMED4, TMEPAI, TMPRSS6, TNFAIP3, TNFRSF10B, TNFRSF19, TNFRSF1A, TNFSF10, TNFSF15, TNIK, TNK2, TNS1, TNS3, TRAF3IP2, TRAF5, TRAF6, TRIO, TRIP6, TSSK3, TULP4, UBE2V1, USH1C, VAPA1 VAV2, VAV3, VIPR1, WASF2, WNK1, WNK2, WNK4, WSB1, WSB2, YES1, YWHAH, ZAK and ZDHHC13.
4. Use or method as claimed in one of the preceding claims to identify drugs targeting cell cycle and cell proliferation in metabolic and tumorous disease for the purpose of its normalization as exemplified by but not limited to the following genes and gene products: ABH, ABL1, ACHE, ACPP, ACTN4, ADRA1B, ADRA1D, AGGF1, AHR, AIF1, ALS2CR19, ANAPC4, ANKRD15, APBB1, APBB2, APC, AR, AREG, ARHGEF1, ATM, ATPIF1, AXIN1, B4GALT7, BCAR1, BCAR3, BCL10, BCL2, BCL6, BHLHB3, BIN1, BRCA1, BRCA2, BRIP1, BTC, BTG1, BTG2, BUB1, C10orf46, C10orf9, C2orf29, C9orf127, CABLES1, CCL14, CCNA2, CCND1, CCND2, CCNE1, CCNE2, CCNH, CCNJL, CCNL1, CCNT1, CCNT2, CCRK, CD160, CD164, CD28, CD3E, CD74, CD86, CDC14A, CDC2, CDC25A, CDC25B, CDC25C, CDC37L1, CDC6, CDK10, CDK3, CDK4, CDK5R1, CDK5RAP3, CDK7, CDKL1, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CDKN3, CDT1, CDV3, CENPF, CEP250, CETN2, CETN3, CHAF1A, CHES1, ChGn, CHRM1, CHRM4, CITED2, CLASP1, COL18A1, CREG1, CRTAM, CSF1, CSF1R, CSK, CTCFL, CUL1, CUL3, CUL4A, CXCL5, CYR61, DAB2, DBC1, DCC, DCTN2, DHCR24, DIP13B, DIRAS3, DLEC1, DLG1, DLG3, DLG5, DST, DUSP1, DUSP22, DUSP6, E2F3, E2F7, EDD1, EDN1, EGF, EGFR, ELF4, ELN, EML4, EMP1, ENPEP, ENPP7, EP300, EPS15, EPS8, ERBB2, ERBB2IP, ERG1 ERN1, ESCO1, ESR1, ETS1, EXT1, F2, F2R, FABP6, FGA1 FGB, FGF2, FGF8, FGF9, FGFR1OP, FGG, FHIT, FLCN, FLJ16793, FLJ40432, FLT1, FRK1 G0S2, GAB1, GADD45A, GAS2, GAS6, GMNN, GNRH1, GRLF1, HBP1, HDAC6, HDAC7A, HDAC9, HDGF, HECA, HEXIM1, HIC.1, HIPK2, HK2, HOXC10, HPGD, IGF1, IGF1R, IGFBP4, IL10, IL12RB1, IL12RB2, IL18, IL28RA, IL2RA, IL6R, IL9R, ING1, INHBA1 INSIG1, IRF1, IRF2, IRS2, ISG20, JAG1, KATNB1, KIAA0367, KIAA0376, KIF25, KITLG, KLF4, KPNA2, KRAS1 LAMB1, LAMC1, LATS1, LATS2, LIF, LIG4, LMO1, LRP1, LRP5, LTBP2, LYN1 LZTS2, MACF1, MAD2L1, MAP2K6, MAP3K11, MAPK13, MAPK6, MAPRE1, MAPRE2, MAPRE3, MCC1 MCM3, MCM8, MCRS1, MDM2, MDM4, MET, MIF1 MKI67, MLH3, MNAT1, MNT, MOS, MPL, MSH5. MTSS1, MUTYH, MXD1, MXM1 MYB, MYC, NBL1, NCK2, NDP, NEDD9, NEK11, NEK2, NEK6, NF1, NFYC, NIPBL, NME1, NOTCH2, NPY, NR6A1, NRAS, NRD1, NRP1, NUMA1, OPRM1, OSM, PAM, PAPD5, PARD3, PARD6B, PBEF1, PBK, PDCD4, PDF, PDGFB, PDGFRA, PEMT1 PFDN1, PGF, PIK3CB, PIM1, PLAGL1, PLCB1, PLG1 PMP22, POLA, POLS, POU3F2, PPAP2A, PPARD1 PPM1D, PPP1CB, PPP1R9B, PPP2CA, PPP2R1B, PPP3CB, PPP6C, PRDX1, PRKCA, PRKG2, PRKRIR1 PRL1 PRM1, PROK1, PSMD1, PTEN, PTHLH, PTK2B, PTMA, PTMS1 PTP4A1, PTPRC, PTTG1, QSCN6, RAD17, RAD50, RAD51, RAD51L1, RAD54L, RAD9B, RAF1, RAN, RAP1A, RASSF4, RB1CC1, RBL1, RBM5, RBM9, RCBTB1, RCC2, RECK, RFP, RGS2, RINT1, RPS27, RSN, RUNX3, S100A6, SASH1, SCIN, SEPT11, SEPT3, SEPT4, SEPT7, SESN1, SESN3, SGOL1, SH3BP4, SHC1, SHH, SIAH1, SIAH2, SKP2, SLAMF1, SLC12A6, SMARCB1, SMC3, SMPD3, SPAG5, SPHAR, SSR1, SSTR1, STARD13, STIM1, STRN3, SUPT3H, SYCP2, SYCP3, TACC1, TADA3L, TAL1, TBC1D8, TCF7L2, TCFL5, TERF2, TFDP1, TFDP2, TGFB3, TGFBI1 TGFBR2, THY1 , TM4SF4, TNFRSF11A, TNFRSF8, TNFSF13B, TNFSF15, TNFSF4, TOB1, TOB2, TSGA2, TSPAN2, TSPAN3, TUBG1, TXLNA, TXN, UBE2V1, UHRF1, UHRF2, UNC84B, UNG2, USP8, UTP14C, VEGF, VIPR1, WEE1, WT1, WWOX, XRN1, YWHAG, YWHAH, YWHAQ, ZAK, ZFP36L2, ZW10 and ZZEF1.
25. Use or method as claimed in one of the preceding claims to identify drugs targeting programmed cell death in metabolic and tumorous disease for the purpose of its normalization as exemplified by but not limited to the following genes and gene products: ABL1, ACIN1, ACTN1, ACTN4, ADORA2A, AHR, ALB, AMID, AMIG02, ANXA4, ANXA5, APOE, APP, ASAH2, ATG5, AXIN1, BAD, BAG2, BAG3, BBC3, BCAR1, BCL10, BCL2, BCL2A1, BCL2L1, BCL2L10, BCL2L11, BCL2L14, BCLAF1, BID, BIRC2, BIRC3, BIRC4, BMF, BRAF, BRCA1, BRE, BTG1, CARD10, CARD4, CASP10, CASP3, CASP6, CBX4, CD28, CD3E, CD74, CDC2, CDK5R1, CDKN1A, CDKN2A, CEBPG, CFL1, CFLAR, CIAS1, CLU, COP1, CRADD, CROP, CRTAM, CTNNBL1, CTSB, CUL1, CUL3, CUL4A, CYCS1 DAD1, DAP, DAPK1, DAPK2, DCC, DHCR24, DIDO1, DNASE1, DNASE1L3, DUSP22, EBAG9, EDAR, EFHC1, EGLN3, ELMO1, ELMO2, EP300, ERCC3, ERN1, F2, F2R, FAIM, FAIM3, FASTKD1, FOXL2, FOXO1A, FOXO3A, GADD45A, GADD45B, GADD45G, GAS2, GPR65, GSTP1, HBXIP, HIPK2, HMGB1, ICEBERG, IER3, IGF1R, IHPK2, IHPK3, IL10, IL18, IL2RA, INHBA, KIAA0367, KNG1, MAGEH1, MAGI3, MCL1, MDM4, MIF, MOAP1, MRPS30, MTP18, NCKAP1, NEK6, NFKB1, NFKBIA1 NME1, NME6, NOTCH2, NRG2, NTF3, NUAK2, NUDT2, OPA1, PAK1, PAWR1 PAX7, PDCD10, PDCD11, PDCD2, PDCD4, PDCD6, PECR, PERP, PHLDA1, PHLPP, PIM1, PLAGL1, PLG, PPARD, PPP2CA, PPP2R1B, PRF1, PRKAA1, PRKCA1 PRKCE, PRKCZ, PRLR, PROC, PRODH, PSEN1, PTEN, PTH, PTK2B, PTPRC1 PTRH2, RAF1, RASA1, RFFL, RHOT1, RIPK1, RIPK3, RNF7, ROCK1, RP6-213H19.1, RRAGC1 RTN4, RUNX3, RYBP, SCARB1, SCIN1 SEMA4D, SEMA6A, SEPT4, SERPINB9, SGK1 SGPL1, SH3GLB1, SIAH1, SIAH2, SIRT1, SMNDC1, SNRK1 STK17A, STK17B, STK3, STK4, TAIP-2, TAX1BP1, TEGT, TESK2, THY1, TIA1, TIAL1, TIMP3, TLR2, TNFAIP3, TNFAIP8, TNFRSF10B, TNFRSF11B, TNFRSF19, TNFRSF1A, TNFRSF21, TNFRSF25, TNFSF10, TNFSF15, TNFSF18, TP53INP1, TP73L, TPT1, TRAF3, TRAF5, TRAF6, TRIB3, TXNDC5, UBE4B, U.NC5B, VDAC1, VEGF, YWHAG, YWHAH, ZAK, ZBTB16, ZDHHC16andZNF346.
6. Use or method as claimed in one of the preceding claims to identify drugs targeting cell morphogenesis and cell / organ development in metabolic and tumorous disease for the purpose of its normalization as exemplified by but not limited to the following genes and gene products: ABLIM1, ABTB2, ACHE, ACIN1, ACTL6A, AGGF1, AHSG, ALX4, AMELX, AMIGO1, AMOT, ANGPTL3, ANKH, ANXA2, APBB1, APBB2, APOE, AR, ARF6, ARHGEF11, ARTS-1, ASH2L, ATP10A, ATPIF1, AXIN1, BCAR1, BCL11A, BHLHB3, BMP2, BMP3, BMP4, BMP6, BMP7, BMP8A, BMP8B, BRAF, BRD8, BTG1, BVES, CACNB2, CALCR, CAMK2D, CANX, CAP1, CAPN3, CART1, CASC5, CASQ2, CCL4, CCM2, CD164, CD1D, CD74, CD86, CD9, CDC42EP4, CDC42EP5, CDH11, CDK5R1, CDK5RAP2, CDK5RAP3, CDX2, CEACAM1, CEBPA, CEBPG, CENPF, CENTD3, CHRDL2, CITED2, CLASP1, CLEC3A, CNTN4, COL11A2, COL12A1, COL18A1, COL1A1, COL1A2, COL2A1, COL9A1, COVA1, CRB1, CREG1, CRIM1, CSDE1, CSF1, CSRP3, CTGF, CYR61, DAZL, DCC, DDX5, DGAT1, DGKD, DLC1, DLG1, DMAP1, DMD, DVL3, DZIP1, EBAG9, EBP, EGFR1 ELN, EMP1, EPAS1, ERBB2, ERBB2IP, ESR1, ETS1, ETS2, EVL1 EXT1, EYA2, FABP1, FARP2, FBLIM1, FBN1, FCMD, FEZ2, FGD2, FGD4, FGD5, FGF2, FGFR2, FHL1, FLNB1 FLT1, F0XL2, FOXN1, FOXO3A, FRZB, GAP43, GAS2, GAS6, GATA4, GATA6, GDF10, GHR, GJA1, GLCE, GNAO1, GRLF1, HAND1, HBEGF1 HCCS, HDAC7A, HDAC9, HECA1 HEY1, HILS1, HMGCR, HOXA13, HSD17B3, IFRD1, IGF1, IGFBP2, IGFBP4, IHPK2, IL10, IL18, ING1, ING2, INHBA, IPF1, ITCH, ITGA11, ITGA2, ITGB1BP2, JAG1, KAZALD1, KDR, KIRREL3, KITLG, KL, KLF6, KRT19, LAMB1, LARGE, LECT2, LHCGR, LHX3, LIMA1, LTBP4, LYN, MAFB, MAP7, MARK2, MATN1, MBNL1, MBP, MEF2A, MEF2C, MKKS, MKL2, MPZ, MSX1, MSX2, MTSS1, MUSK, MYF6, MYH10, MYH14, MYST3, NCOA4, NEDD9, NET1, NFAM1, NKX2-2, NOTCH2, NOTCH4, NR5A1, NRCAM, NRD1, NRP1, NRP2, NRXN3, NTNG1, OKL38, OSM, PAPPA2, PAPSS1, PAPSS2, PARD3, PARD6B, PAX1, PAX2, PBX1, PBX3, PDLIM5, PGF, PHEX, PHGDH, PITX1, PITX2, PITX3, PLEKHC1, PLG, P0U3F1, P0U4F1, P0U6F1, PPARD, PPP1R9B, PPP2CA, PPP2R1B, PRDX1, PRELP, PRKCI, PRL, PTEN, PTH, PTPRC, QSCN6, RAB3D, RASA1, RHOH1 RND1, ROBO1, ROBO2, RPS6KA3, RRAGC, RTN4, RTN4RL1, RTN4RL2, RUNX1, RUNX2, RUVBL1, S100A6, SCGB1A1, SCIN1 SEMA6A, SGCB, SGCE, SHCI, SHH, SIAH1, SIRT1, SIX1, SLIT1, SMARCA1, SNAM1 SNRK, SOCS5, SOCS6, SOCS7, SORT1, SOX6, SOX9, SPAG6, SPARC, SPINK5, SPN, SPRY2, SRD5A2, SRI, STIM2, SVIL, SYCP3, TAGLN3, TBX3, TCF12, TCOF1, TGFB3, THY1, TINAG1 TLE1, TLE3, TMEM97, TMPRSS6, TNFAIP2, TNFRSF11B, TNN, TNP1, TNR, TPD52, TRAPPC4, TSSK3, TUFT1, UBE3A, UTRN, VCL, VCX3A, VEGF, VIL2, WISP1 , WISP3, XRN2, YEATS4, YWHAH, ZBTB16, ZNF160 and ZNF22.
27. Use or method as claimed in one of the preceding claims to select drug candidates of an antisense molecule, ribozyme, triple helix molecule or other new chemical entities targeting the chromatin.
28. Use of a human gene, in particular the coding region thereof, or a gene product encoded thereby or of RNA or DNA sequences, which hybridize to said gene and which code for a polypeptide having the function of said gene product, as a biomarker, or of an antibody directed against said gene product, in the diagnosis, prognosis and/or treatment monitoring of metabolic and tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer, wherein the gene is selected from the group of genes according to one of the claims 1-9 or is selected from the genes specified in the claims 21-26.
29. Use as claimed in claim 28 for monitoring the therapeutic treatment of a patient suffering from a metabolic and/or tumorous disease, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer.
30. A method for diagnosing, prognosing and/or staging metabolic and tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer and/or monitoring the treatment of at least one of said diseases, comprising
(a) measuring the level of expression of at least one biomarker, wherein the biomarker is selected from the genes specified in the claims 1-9 and/or 21-26 in a patient or in a sample of a patient suffering from or being susceptible to a metabolic and/or tumorous disease, and
(b) comparing the level of said at least one biomarker in said patient or in said sample to a reference level of said at least one biomarker, in particular by the use according to one of the claims 28-29.
31. A composition for qualifying the HNF4α activity in a patient suffering or being susceptible to a metabolic and/or tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer or for classifying a patient suffering from or being susceptible to at least one of said diseases, comprising an effective amount of at least one biomarker selected from the genes specified in the claims 1-9 and/or 21-26 or from the group of gene products encoded by said genes, and/or comprising an effective amount of at least one antibody directed against at least one biomarker selected from said group of gene products.
32. Use of a composition as claimed in claims 31 for the production of a diagnostic agent, in particular of a diagnostic standard.
33. Use as claimed in claim 32 for the production of a diagnostic agent for qualifying the HNF4α activity in a patient suffering or being susceptible to a metabolic and/or tumorous disease, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer or for classifying a patient suffering from or being susceptible to at least one of said diseases.
34. Use as claimed in one of the claims 32-33 for the production of a diagnostic agent for predicting or monitoring the response of a patient suffering from a metabolic and/or tumorous disease, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer to a method of treating at least one of said diseases with a drug, in particular a drug identified by the use or by the method according to one of the claims 1-27 and/or a HNF4α activity modulator.
35. A kit for qualifying the the HNF4α activity in a patient suffering or being susceptible to metabolic and/or tumorous disease, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer or for classifying a patient suffering from or being susceptible to at least one of said diseases, in particular for predicting or monitoring the response of a patient to suffering from at least one of said diseases by a method of treating metabolic and/or tumorous diseases comprising administering a HNF4α activity modulator, comprising at least one standard indicative of the level of a biomarker selected from the genes specified in the claims 1-9 and/or 21-26 or from the group of gene products encoded by said genes in normal individuals or individuals having metabolic and/or tumorous disease associated with increased HNF4α activity, and instructions for the use of the kit.
36. The kit as claimed in claim 35, wherein the at least one standard comprises an indicative amount of at least one biomarker selected from the genes specified in the claims 1-9 and/or 21-26 or from the group of gene products encoded by said genes.
37. The kit as claimed in one of the claims 35-36, further comprising at least one primer or primer pair specifically hybridizing with the mRNA of a biomarker selected from the genes specified in the claims 1-9 and/or 21-26, preferably a primer or primer pair selected from Table M1 , and/or at least one antibody specific for a biomarker selected from the group of gene products encoded by said genes, and reagents effective to detect said biomarker(s) in a serum sample.
38. A method of qualifying the HNF4α activity in a patient suffering or being susceptible to metabolic and/or tumorous disease, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer or for classifying a patient suffering from or being susceptible to at least one said diseases, comprising determining in a sample of a subject suffering from or being susceptible to one of said diseases at least one biomarker wherein the biomarker is the gene product encoded by a gene selected from the genes specified in the claims 1-9 and/or 21-26 or and/or the biomarker is the mRNA sequence encoding the gene product of said selected gene, and wherein the sample level , of the at least one biomarker being significantly higher or lower than the level of said biomarker(s) in the sample of subjects without a disease associated with increased activity of HNF4α is indicative of induced HNF4α activity in the subject.
39. Method as claimed in claim 38 for predicting the response of a patient suffering from metabolic and/or tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer to a method of treating such diseases comprising administering a drug identified by the use or method according to one the claims 1-27 or a HNF4α activity modulator, wherein the level of the at least one biomarker being significantly higher or lower than the level of said biomarker(s) in subjects without cancer associated with increased activity of HNF4α is indicative that the subject will respond therapeutically to a method of treating cancer comprising administering a drug identified by the use or method according to one the claims 1-27 or administering a HNF4α activity modulator.
40. Method as claimed in one of the claims 38-39 for monitoring the therapeutically response of a patient suffering from metabolic and/or tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer to a method of treating at least one of said diseases comprising administering a drug identified by the use or method according to one the claims 1-27 or administering a HNF4α activity modulator, wherein the level of the at least one biomarker before and after the treatment is determined, and a significant decrease or increse of said level(s), preferably a decrease or increase to the normal level(s), of the at least one biomarker after the treatmentis indicative that the subject therapeutically responds to the administration of the drug identified by the use or method according to one the claims 1-27 to the administration of the HNF4α activity modulator.
41. The method as claimed in one of the claims 38-40, wherein a RT-PCR is performed, in particular by using the kit as claimed in one of the claims 35-37.
42. Medicament for the treatment of metabolic and tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer comprising a composition that decreases the expression or activity of a HNF4α modulated gene selected from the genes specified in the claims 1-9 and/or 21-26.
43. SiRNA composition, wherein the siRNA composition reduces the expression of a de novo identified HNF4α modulated gene selected from the genes specified in the claims 1-9 or 21-26.
44. Antisense composition, wherein the antisense composition comprises a nucleotide sequence complementary to a coding sequence of a HNF4α modulated gene selected from the genes specified in the claims 1-9 or 21-26
45. Use of an siRNA composition as claimed in claim 43 or of an antisense composition as claimed in claim 44 for the preparation of a medicament.
46. Use as claimed in claim 45 for the preparation of a medicament for preventing, treating, or ameliorating metabolic and tumorous diseases, in particular type 1 and/or type 2 diabetes mellitus, and/or diabetes-caused diseases, including diabetic nephropathy, hearing dysfunction, diabetic neuropathy, cardiovascular diseases or diseases of the retina, and/or colorectal cancer.
47. Method or use or medicament or kit as claimed in one of the preceding claims, wherein a composition according to one of the claims 44-45 is used as the drug or wherein an agent selected from the group consisting of the agonists, antagonists, drugs, agents, antiestrogens, and compounds listed in the
Tables 36-54, sulfonlyurea dehvates, and Aroclor 1254, is used and/or tested and/or screened as the drug and/or as the HNF4α activity modulator.
48. Method according to one of the claims 1-9 and 14-16 or use or method according to one of the claims 17-29, wherein Aroclor 1254 is screened and identified.
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