WO2003018839A1 - Identification of a common promoter module in disease associated genes - Google Patents

Identification of a common promoter module in disease associated genes Download PDF

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WO2003018839A1
WO2003018839A1 PCT/IE2001/000108 IE0100108W WO03018839A1 WO 2003018839 A1 WO2003018839 A1 WO 2003018839A1 IE 0100108 W IE0100108 W IE 0100108W WO 03018839 A1 WO03018839 A1 WO 03018839A1
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genes
disease
transcription factor
binding sites
factor binding
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French (fr)
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Hugh Redmond Brady
Peter Philip Doran
Geraldine Mary Mccarthy
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University College Dublin, National University Of Ireland, Dublin
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    • C—CHEMISTRY; METALLURGY
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    • 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
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    • 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
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • This invention relates to a method of identifying common markers in cohorts of genes, the expression of which is altered in disease states and to the use of such markers in diagnosis and therapy.
  • a gene is typically defined as a functional unit composed of the set of sequences necessary to encode a protein (the open reading frame) and to regulate its transcription (the promoter and terminator).
  • the open reading frame which encodes the protein is preceded by the promoter region. This regulatory sequence determines whether the gene is transcribed or not.
  • binding of specialized effector molecules known as transcription factors to specific regions within the gene promoter is required. These factors bind to sequence specific binding sites in the promoter sequence of the gene and facilitate recruitment of the RNA Polymerase II Holoenzyme. Binding of this enzyme to the gene permits transcription to proceed.
  • Transcription factor binding sites These represent the lowest level of promoter organisation and are relatively short stretches of DNA (10 - 20 nucleotides), sufficiently conserved in sequence to allow specific recognition by the corresponding transcription factor (TF). The function of these mediators is recruitment of transcription factors by DNA binding.
  • Promoter Modules The next highest level of promoter organization is the one of promoter modules which are composed of two or more TF-sites in a defined distance range. In contrast to isolated binding sites these regions allow both synergistic or antagonistic effects of individual transcription factors.
  • Promoter Models The highest and most complex level of organization of a promoter is the complete promoter itself. Functionally related promoters often exhibit a clearly defined core organization of binding sites conserved both in orientation as well as in distances (with some variability).
  • Osteoarthritis is the most common form of arthritis. Over 50% of all people aged 65 and older present with radiographic changes of OA, with the prevalence rising to almost 100% in the over 75 population, making it one of the most expensive and debilitating diseases in terms of costs of diagnosis, therapy, complications of therapy and lost productivity. To date, there has been little success in altering the natural course of the disease and typically prescribed therapies have the potential for significant toxicity.
  • the etiology of OA is multifactorial. OA involves the entire joint, encompassing the cartilage, synovium and underlying bone. The cells in each of these tissues have independent capacities to initiate and respond to injury in the joint, ultimately resulting in degeneration.
  • Synovial fluid basic calcium phosphate (BCP) (hydroxyapatite, octacalcium phosphate and tricalcium phosphate) crystals are commonly found in osteoarthritis and are associated with severe degenerative disease (Halverson, P.B. and McCarthy, DJ. (1997) in Arthritis and Allied Conditions: A Textbook of Rheumatology (Koopman, W.J., ed) pp. 2127- 2146,). The incidence of BCP crystals in synovial fluid from patients with knee OA has been suggested, by several studies to be between 30-60 % (Gibilsco, P. A. et al ( 1985) Arthritis Rheum, 28: 5 1 1 -515).
  • Crystal deposition correlates strongly with radiographic evidence of cartilage degeneration, (Halverson, P. B., and McCarty, D. J. (1986) Ann. Rheum. Dis. 45, 603-605) and is associated with larger joint effusions when compared with joint fluid from OA knees without crystals (Carroll, G. J. et al ( 1991 ) J. Rheumatol. 18, 861 -866).
  • DN diabetic nephropathy
  • the pathological hallmark of diabetic nephropathy is glomerulosclerosis due to accumulation of extracellular matrix proteins in the glomerular mesangium.
  • Mesangial matrix accumulation reflects both increased synthesis and decreased degradation of extracellular matrix components.
  • High ambient glucose is a major stimulus for mesangial cell matrix production in DN.
  • the molecular basis for hyperglycaemia induced changes in mesangial cell function are still being appreciated.
  • OA and DN are characterized by gene expression patterns that are not easily segregated into functional categories.
  • the invention provides a method for identifying a cohort of genes the- expression of which is altered in a disease state, and the expression products of which are involved in the pathogenesis of said disease, which method comprises identifying in said genes a common promoter module which governs the transcriptomic response to disease associated stimuli.
  • the common promoter module has a number of common transcription factor binding sites within 750 base pairs (bp) of the start site of transcription.
  • the disease is osteoarthritis.
  • the genes are basic calcium phosphate (BCP) crystal responsive genes.
  • the common promoter module includes the transcription factor binding sites LM02COM, AP-1 and the sequence TTCCTGTCT.
  • the disease is diabetic nephropathy.
  • the genes are responsive to high ambient glucose.
  • high ambient glucose here is meant a concentration of glucose greater than 5 mM.
  • the common promoter module includes the transcription factor binding sites GATA- 1 , MZF, CDXA and SRY.
  • the transcription factor binding sites are located at a conserved distance from the start of the coding region of the genes and occur within a range of 10-300 bp of one another.
  • the common promoter module is elucidated by bioinformatics as hereinafter exemplified.
  • the underlying method involves building a profile of the consensus sequences for transcription factor binding sites with the promoter modules of interest.
  • a suitable method involves use of the statistical algorithm for the identification of TF binding sites in human genomic DNA developed by Schweizert, K et al ((1996) Comput. Appl. Biosci. 72, 405-413).
  • the transcription factor binding sites are predicted by a comparison of putative sequences against a vertebrate transcription factor database yielding both a core and a matrix similarity value and selecting those sequences where a similarity score of greater than 0.9 is recorded.
  • diagnostic assays could be based on the measurement of either the mRNA or protein levels of the gene of interest.
  • Various assay methods known per se could be utilised in this regard.
  • An advantage of the method according to the invention is that it would permit the design of a diagnostic assay based on the measurement of the expression and/or activity of a number of genes, possessing this common promoter structure, that are implicated in a given disease.
  • the invention further provides a promoter module common to a cohort of genes the expression of which is altered in a disease state, and the expression products of which are involved in the pathogenesis of said disease, said promoter module comprising two or more common transcription factor binding sites which act in concert to regulate the expression of said genes.
  • Fig 1 is a graphical representation of the transcription factor binding complement of crystal responsive genes according to Example 1 ;
  • Fig. 2 is a graphical representation of the most common transcription factor binding sites in crystal responsive genes according to Example 1 ;
  • Fig. 3 is a series of ethidium stained panels of agarose gel containing PCR reaction after electrophoresis according to Example 3.
  • Example 1 The invention will be further illustrated by the following Examples.
  • Example 1 The invention will be further illustrated by the following Examples.
  • Example 1 The invention will be further illustrated by the following Examples.
  • the first stage of the analysis was to obtain the putative promoter sequences of the genes of interest. Initially the human nucleotide databases were searched for actual promoter sequences. This analysis led to the identification of fourteen promoter sequences from the genes of interest as described in further detail in Example 4. These searches allowed fourteen full-length promoter sequences corresponding to BCP crystal responsive genes to be identified and utilised in subsequent analysis.
  • a large library was used consisting of more than 300 transcription factor binding site matrices that was compiled on the basis of published matrices as well as entries from the TRANSFAC database, with emphasis on sequences with experimentally verified binding capacity.
  • the advantages of the Stant K et al (( 1996) supra) method include position weighting of the matrices based on the information content of individual positions and calculation of a relative matrix similarity.
  • the promoter sequences from the transcripts of interest were used as input to this application in searches against the vertebrate transcription factor database and the data collated. When scoring a consensus sequence match this application provides both a core and a matrix similarity value.
  • the most reliable predictions of binding sites have been shown to occur when a matrix similarity score of greater than 0.9 is recorded. For this reason only sequence matches with this minimum score were considered for the pu ⁇ ose of this study.
  • To standardize the data reporting in the following description the position of the site of interest is reported as downstream from the start site of translation as set out in Tables 1-14.
  • AP 4 -382 0.980 A graphical representation of the transcription factor binding site complements of the analysed genes is depicted in Fig. 1, in which indicative locations of TF sites from start site of transcription of the gene product are shown.
  • Example 1 the apparent key transcription factor binding sites and the AP-1 / LM02COM promoter module in crystal responsive genes it is of great potential utility to use this information to identify other transcripts containing this module to enable identification of novel putative crystal responsive genes.
  • this approach is somewhat at variance with conventional gene finding methodologies, it represents a viable approach in light of developments in bioinformatics that permit accurate homology matrices to be constructed. To facilitate this work it was necessary to first of all construct a model of the two transcription factor binding site sequences and the distance between them. Having defined the module we were then in a position to use this information to search the nucleotide databases to find genomic segments containing this specific series of sites.
  • the sequences of the two binding sites of interest were obtained from the TRANSFAC TF database. As shown in Example 1 the distance between these two factors ranged from 10 to over 300 nucleotides. To facilitate model construction a matrix can be computed which essentially allows a floating sequence with 10 to 300 bases between the two sites to be constructed. This process leads to the generation of a large number of potential sequence models which are then simultaneously used as input in a BLAST (Basic Local Alignment Search Tool) search of the human nucleotide databases. The result of this analysis is a set of fragments which contain the two binding sites of interest, separated by a distance similar to that seen in the initial data analysis. A control on the approach utilised was the fact that the genes analysed during the course of this work were all represented in the final dataset. Pertinent among the resulting matches were the matches set out in Table 16.
  • Homo sapiens gene for leukotriene B4 omega-hydroxylase AF221943 Homo sapiens DNA, chromosome 21q22.2, PAC clone 25P16 AB003151 Homo sapiens DNA for human type I collagen alpha 2 chain NM_000089
  • Homo sapiens for RGS5 XM002185 Homo sapiens gene for hippocalcin ABO 15202
  • Sections of osteoarthritic hip and knee were obtained from patients undergoing total hip replacement surgery. Subchondral bone, cartilage and synovial lining tissue were retained. Normal joint tissue was obtained from patients undergoing amputation procedures for peripheral vascular disease.
  • SSH was performed with the PCR-SELECT cDNA subtraction kit (Clontech) as directed by the manufacturer.
  • Starting material consisted of 2 ⁇ g of fibroblast mRNA exposed to BCP crystals for 4 hours as “tester” and 2 ⁇ g of fibroblast mRNA cultured in the absence of BCP crystals as "driver”. Thirty primary PCR cycles and 12 secondary PCR cycles were performed.
  • PCR products generated by SSH were subcloned into the PCR 2.1 vector using the TA cloning kit (Invitrogen). Subcloned cDNAs were isolated by colony PCR amplification. Sequencing was performed using an automated ABI 370 A DNA sequencing system. Sequence reactions were carried out with the ABI prism dye terminator cycle sequencing ready reaction kit (Perkin Elmer). The sequences obtained were compared against the GenBank/EMBL nucleotide databases using BLAST.
  • Table 17 a refers to the sequence identity based on comparison with the Genbank / EMBL database
  • b refers to the fold upregulation of each gene based on Northern blot analysis of total RNA prepared from primary human foreskin fibroblasts stimulated with BCP crystals for 4 hours relative to the expression in the absence of these crystals. Values were obtained by Phosphor-Imaging and were normalized by comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A comparison with GAPDH is used to allow accurate determination of the quantity of the transcript of interest. This gene is expressed at constant levels and as such controls for the amount of RNA being analysed. This control removes the suggestion that the increased quantities of a gene is due to a larger amount of RNA sample being analysed.
  • GAPDH glyceraldehyde-3-phosphate dehydrogenase
  • RNA levels were measured in RNA isolated from synovial tissue explanted from patients with end stage osteoarthritis.
  • PCR primers for a representative set of these transcripts were synthesized. The nucleotide sequences of these primers are shown in Table 18.
  • Insulin like growth factor binding protein-3 Forward Primer GAC TCT GCT GGT GCT GCT C
  • RT-PCR was performed on total RNA extracted from synovial lining tissue from patients with end stage osteoarthritis and non osteoarthritic tissue.
  • Fig. 3 depicts ethidium stained panels of a 2 % (w/v) agarose gel containing 10 ⁇ l of PCR reaction after electrophoresis. Amplification of GAPDH was used to control for equivalence of loading. In Fig. 3 the lanes represent the following
  • Lanes 1 and 2 RT-PCR products from synovial tissue explanted from non-osteoarthritic tissue.
  • Lanes 3 and 4 RT-PCR products from synovial tissue explanted from patients with established end-stage osteoarthritis.
  • OA pathogenesis
  • genes include the insulin-like growth factor binding proteins, which may potentiate the mitogenic activities of the crystals and immediate early genes such as c-fos and c-myc may also play a role in mitogenesis.
  • the upregulation of matrix metalloproteinases is also of interest and is a previously well-defined response.
  • Cytoskeletal rearrangement is also an important aspect of OA, in that it is an effector of the gross structural changes seen in the osteoarthritic joint. Thus it was of interest that a number of cytoskeletal transcripts were found to be differentially expressed. These included ⁇ -actin 1, vimentin, tubulin ⁇ -2 and actinin ⁇ 4.
  • the induction of these mediators may represent an important mechanism in the pathogenesis of OA.
  • the destruction of the joint compartment by proteolytic digestion is the pathological hallmark of OA.
  • the upregulation of cathepsin K is thus of major interest as this gene is known to be the dominant cysteine protease involved in bone remodeling. Secretion of the mediator by synovial fibroblasts may thus be an important effector of joint degradation.
  • the genes identified as upregulated present a host of new target transcripts for BCP crystal induced damage.
  • Example 3 stimulation of human fibroblasts with BCP crystals leads to the coordinate induction of a wide variety of functionally diverse gene products. All of these transcripts may represent a key molecular event in the initiation and progression of osteoarthritis. Thus the challenge is to delineate the pathway that when perturbed by a therapeutic agent will have the greatest effect on the disease. Regardless of stimulus employed, we have found that for a gene to be upregulated, transcription promoting events centred at the promoter region of the gene must be initiated. This fact offers an excellent opportunity for exploitation in the search for novel therapeutic targets in a disease. In this experiment the regulatory elements in the promoters of a well-defined group of crystal responsive genes were analysed.
  • the first stage of this analysis was to obtain the promoter sequences of the genes of interest.
  • human nucleotide databases were searched for actual promoter sequences using the general procedures set out in Example 1. This analysis led to the identification of fourteen promoter sequences from the genes of interest. These searches allowed fourteen full-length promoter sequences corresponding to BCP crystal responsive genes to be identified and utilised in subsequent analysis, as shown in Table 19.
  • Arginosuccinate Synthetase X03258 Tissue Inhibitor of Metalloproteinases- 1 Y09720 To determine the capacity for transcription factor binding mediated initiation of transcription of BCP crystal responsive genes, it was necessary to build a profile of the consensus sequences for transcription factor binding sites within the promoters under study as described in Example 1. For the reasons set forth in Example 1 only sequence matches with a minimum score of greater than 0.9 were considered for the pu ⁇ ose of the study. To standardize the data reporting, the position of the site of interest is reported as downstream from the start site of translation of the protein for which the gene codes. This analysis led to the identification of five commonly occurring transcription factor binding sites in crystal responsive genes; IK-2, NF- B, LM02COM, AP-1 and MZF and is illustrated in Fig. 2.
  • the arrangement, as well as the types of a group of transcription factor binding sites in a promoter confers the ability to regulate the expression of a gene.
  • To facilitate this analysis the data set was simplified to only a consideration of MZF, IK-2, LM02COM and API as shown in Table 20. This analysis revealed LM02COM and AP- 1 , as the two most commonly occurring sites. The standardized location of each of these sites is as follows
  • the average distance separating these two sites was determined to be just 88 nucleotides. This distance is conducive with the two factors potentially functioning in unison in mediating the effects of BCP crystals.
  • AP-1 or activator protein 1 is a dimeric transcription factor composed of either two jun peptides or one jun and one fos peptide.
  • BCP crystal stimulated fos and jun expression has previously been demonstrated, suggesting that the early induction of these factors may lead to sustained transcriptional activation via activator protein 1. Furthermore it has been demonstrated that BCP crystal stimulation can lead to activation of AP-1 (McCarthy, G. M. et al, (1998) J Biol Chem 273; 52: 35161-35169).
  • LM02COM or rhombotin 2 is a 158 amino acid transcription factor that is essential for erythroid cell differentiation. This factor has not previously been implicated in osteoarthritis. However the frequency of its appearance and its proximity to AP- 1 in the promoter regions of crystal responsive genes suggest a role for this transcription factor in human osteoarthritis.
  • these two transcription factor binding sites are (a) present in the vast majority of genes demonstrated to be differentially expressed in the presence of BCP crystals, (b) are located at relatively similar distances from the start of the coding region of the genes and (c) occur within a narrow distance range of one another. This observation may be of use in devising novel agents to inhibit transcriptome activation in osteoarthritis.
  • the sequences of the two binding sites of interest were obtained from the TRANSFAC transcription factor database. As was previously demonstrated the distance between these two factors ranged from 10 to over 300 nucleotides. To facilitate model construction a floating sequence with 10 to 300 bases between the two sites of interest was constructed. This process led to the generation of a large number of potential sequence models that were then simultaneously used as input in a BLAST search of the human nucleotide databases. The result of this analysis is a cohort of genes that contain the two binding sites of interest, separated by a distance similar to that seen in the initial data analysis. A control on the approach utilised was the fact that the genes analysed during the course of this work were all represented in the final dataset. Other notable transcripts are listed in Table 16 above.
  • Propagation of mesangial cells under conditions of high ambient glucose has proved a useful in vitro model with which to probe the molecular basis for mesangial matrix accumulation in diabetes, attributable to high ambient glucose.
  • Exposure of cultured mesangial cells to high glucose stimulates de novo synthesis of ECM components such as type IV collagen, fibronectin and laminin (Ayo, S.H., et al (1990) Am. J. Pathol. 136, 1339-1348; Wahab, N. A., et al. (1996) Biochem J. 316, 985- 992; and Ayo, S. H., et al. (1991 ) Am J.Physiol. 260, F 185-F191 ).
  • the current transcription factor databases contain details of all the previously identified transcription factors in human promoters. We have carried out an experiment to examine the hypothesis that there may be novel transcription factor binding sites in the BCP crystal responsive gene promoters and other gene promoters that can drive the transcriptomic machinery.
  • the identification of putative novel transcription factor binding sites involves the creation of a test set of short oligonucleotide sequences, corresponding to all possible permutations. Such an approach leads to the generation of a list in which all short sequences are represented, thus providing an enabling platform for searches of homology in BCP crystal responsive promoters.
  • the initial stage of this work involved the generation of all the combinations of A, C, T, G to generate every possible sequence of DNA.
  • the sequences constructed in this manner were between 7 and 18 nucleotides long, a distance which encompasses known transcription factor binding sites.
  • the sequence combinations were created by a simple application of a random word generator programme, scripted to produce every possible word.
  • the sequences obtained in this manner were compressed into a flat sequence file, suitable for further searching.
  • To determine the reliability of this methodology a whole database comparison was performed. This comprised of comparing the putative sequence database against the TRANSFAC database of human transcription factor binding sites. This led to the identification of all vertebrate TF sites within the generated sequence file.
  • TTCCTGTCT TTCCTGTCT

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Abstract

A method for identifying a cohort of genes the expression of which is altered in a disease state, and the expression products of which are involved in the pathogenesis of said disease, comprises identifying in said genes a common promoter module which governs the transcriptomic response to disease associated stimuli. The representative diseases are osteoarthritis and diabetic nephropathy. The methods can be used: for identifying targets for the rational design of therapeutic agents for use in the treatment of an associated disease; as a basis for determining a response to therapeutic agents; for identifying targets for the rational diagnosis of disease; and as a basis for assessing disease activity.

Description

Description
IDENTIFICATION OF A COMMON PROMOTER MODULE IN DISEASE ASSOCIATED GENES
Technical Field
This invention relates to a method of identifying common markers in cohorts of genes, the expression of which is altered in disease states and to the use of such markers in diagnosis and therapy.
Background Art
Complex diseases such as osteoarthritis, rheumatoid arthritis and diabetic nephropathy are characterized by gene expression patterns that are not easily segregated into functional categories. This hampers efforts to design therapies based on perturbation of gene expression. For example, if it is known that there are twenty genes the expression of which is altered in a disease, and the gene products are directly involved in the pathogenesis thereof, choice of the gene to be targeted with a therapeutic agent becomes difficult. However if one can delineate the common factor in complex conditions then one may be in a position to intervene at an earlier stage in transcriptome activation with a therapeutic agent.
A gene is typically defined as a functional unit composed of the set of sequences necessary to encode a protein (the open reading frame) and to regulate its transcription (the promoter and terminator). The open reading frame, which encodes the protein is preceded by the promoter region. This regulatory sequence determines whether the gene is transcribed or not. For a gene to be activated, binding of specialized effector molecules known as transcription factors to specific regions within the gene promoter is required. These factors bind to sequence specific binding sites in the promoter sequence of the gene and facilitate recruitment of the RNA Polymerase II Holoenzyme. Binding of this enzyme to the gene permits transcription to proceed.
The regulation of transcription of a gene in response to a cellular stimulus is governed by its promoter sequence. Human gene promoters are hierarchically organized, with three different levels of organization with distinct functionality. The functionally relevant structures are,
• Transcription factor binding sites. These represent the lowest level of promoter organisation and are relatively short stretches of DNA (10 - 20 nucleotides), sufficiently conserved in sequence to allow specific recognition by the corresponding transcription factor (TF). The function of these mediators is recruitment of transcription factors by DNA binding.
• Promoter Modules. The next highest level of promoter organization is the one of promoter modules which are composed of two or more TF-sites in a defined distance range. In contrast to isolated binding sites these regions allow both synergistic or antagonistic effects of individual transcription factors.
• Promoter Models. The highest and most complex level of organization of a promoter is the complete promoter itself. Functionally related promoters often exhibit a clearly defined core organization of binding sites conserved both in orientation as well as in distances (with some variability).
The combination of the aforementioned factors results in a structure which contains all response elements necessary for its complete transcriptional regulation.
Osteoarthritis (OA) is the most common form of arthritis. Over 50% of all people aged 65 and older present with radiographic changes of OA, with the prevalence rising to almost 100% in the over 75 population, making it one of the most expensive and debilitating diseases in terms of costs of diagnosis, therapy, complications of therapy and lost productivity. To date, there has been little success in altering the natural course of the disease and typically prescribed therapies have the potential for significant toxicity. The etiology of OA is multifactorial. OA involves the entire joint, encompassing the cartilage, synovium and underlying bone. The cells in each of these tissues have independent capacities to initiate and respond to injury in the joint, ultimately resulting in degeneration.
Synovial fluid basic calcium phosphate (BCP) (hydroxyapatite, octacalcium phosphate and tricalcium phosphate) crystals are commonly found in osteoarthritis and are associated with severe degenerative disease (Halverson, P.B. and McCarthy, DJ. (1997) in Arthritis and Allied Conditions: A Textbook of Rheumatology (Koopman, W.J., ed) pp. 2127- 2146,). The incidence of BCP crystals in synovial fluid from patients with knee OA has been suggested, by several studies to be between 30-60 % (Gibilsco, P. A. et al ( 1985) Arthritis Rheum, 28: 5 1 1 -515). Crystal deposition correlates strongly with radiographic evidence of cartilage degeneration, (Halverson, P. B., and McCarty, D. J. (1986) Ann. Rheum. Dis. 45, 603-605) and is associated with larger joint effusions when compared with joint fluid from OA knees without crystals (Carroll, G. J. et al ( 1991 ) J. Rheumatol. 18, 861 -866).
Likewise diabetic nephropathy (DN) is a complex disease involving coordinated changes in mesangial cell matrix metabolism and proliferation among other functions.
The pathological hallmark of diabetic nephropathy is glomerulosclerosis due to accumulation of extracellular matrix proteins in the glomerular mesangium. Mesangial matrix accumulation reflects both increased synthesis and decreased degradation of extracellular matrix components. High ambient glucose is a major stimulus for mesangial cell matrix production in DN. The molecular basis for hyperglycaemia induced changes in mesangial cell function are still being appreciated.
Like many other complex diseases, OA and DN are characterized by gene expression patterns that are not easily segregated into functional categories.
It is becoming increasingly appreciated that the development of next generation therapeutic agents in complex diseases is dependent on discovery of the common critical disease causing pathway. Disclosure of Invention
The invention provides a method for identifying a cohort of genes the- expression of which is altered in a disease state, and the expression products of which are involved in the pathogenesis of said disease, which method comprises identifying in said genes a common promoter module which governs the transcriptomic response to disease associated stimuli.
As hereinbelow described we have demonstrated that differential expression of diverse cohorts of genes is facilitated by common promoter sequences. For example, we have shown that upregulation of a diverse cohort of genes by basic calcium phosphate (BCP) crystals is facilitated by such common promoter architecture.
Likewise we have shown that a diverse cohort of genes whose expression is altered by exposure to high extracellular glucose concentration is facilitated by a common promoter sequence.
It will be appreciated that knowledge of such common promoter modules is likely to be important in the rational design of novel compounds for intervention in many debilitating diseases.
Furthermore, knowledge of common promoter modules as described herein will offer new avenues for exploration in our efforts to understand the molecular events in the pathogenesis of disease.
The invention will be further illustrated principally with reference to osteoarthritis and diabetic nephropathy. Typically, the common promoter module has a number of common transcription factor binding sites within 750 base pairs (bp) of the start site of transcription.
In one aspect of the invention the disease is osteoarthritis.
Preferably, in the case of osteoarthritis the genes are basic calcium phosphate (BCP) crystal responsive genes.
When the genes are BCP crystal responsive genes, the common promoter module includes the transcription factor binding sites LM02COM, AP-1 and the sequence TTCCTGTCT.
In an alternative aspect of the invention the disease is diabetic nephropathy.
When the disease is diabetic nephropathy, preferably the genes are responsive to high ambient glucose.
By high ambient glucose here is meant a concentration of glucose greater than 5 mM.
Further, preferably, the common promoter module includes the transcription factor binding sites GATA- 1 , MZF, CDXA and SRY.
Eighty er cent of the promoter sequences analysed by us were found to have GATA-1, MZF, CDXA and SRY binding sites within 800 bp of the start site of transcription. Among the genes containing this fingeφrint were connective tissue growth factor and endothelin 1. These results suggest that four key transcription factor binding sites may account for a major component of the glucose-induced transcriptomic response in mesangial cells. Further analysis of the transcription factors and upstream signaling events that influence the expression of these genes should shed some light on the pathogenesis of DN.
Typically, the transcription factor binding sites are located at a conserved distance from the start of the coding region of the genes and occur within a range of 10-300 bp of one another.
Preferably, the common promoter module is elucidated by bioinformatics as hereinafter exemplified.
Preferably, the underlying method involves building a profile of the consensus sequences for transcription factor binding sites with the promoter modules of interest. A suitable method involves use of the statistical algorithm for the identification of TF binding sites in human genomic DNA developed by Quandt, K et al ((1996) Comput. Appl. Biosci. 72, 405-413).
Before this development motifs were commonly located by matches to IUPAC strings derived from consensus sequences. Although this method is simple and widely used, a major drawback of IUPAC strings is that they necessarily remove much of the information originally present in the set of sequences. Nucleotide distribution matrices retain most of the information and are thus better suited to evaluate new potential sites. The method of Quandt, K et al ((1996) supra) allows generation of new matrices and detection of potential sequence matches by automatic searches with a library of pre-compiled matrices.
Further, preferably, the transcription factor binding sites are predicted by a comparison of putative sequences against a vertebrate transcription factor database yielding both a core and a matrix similarity value and selecting those sequences where a similarity score of greater than 0.9 is recorded.
The invention also provides use of the methods hereinbefore defined:
a) For identifying targets for the rational design of therapeutic agents for use in the treatment of an associated disease;
b) As a basis for determining a response to therapeutic agents;
c) For identifying targets for the rational diagnosis of disease; and
d) As a basis for assessing disease activity.
The use of the method as hereinbefore defined under a)-d) will be apparent from the following Examples.
For example, diagnostic assays could be based on the measurement of either the mRNA or protein levels of the gene of interest. Various assay methods known per se could be utilised in this regard. An advantage of the method according to the invention is that it would permit the design of a diagnostic assay based on the measurement of the expression and/or activity of a number of genes, possessing this common promoter structure, that are implicated in a given disease.
The invention further provides a promoter module common to a cohort of genes the expression of which is altered in a disease state, and the expression products of which are involved in the pathogenesis of said disease, said promoter module comprising two or more common transcription factor binding sites which act in concert to regulate the expression of said genes.
Brief Description of Drawings
Fig 1 is a graphical representation of the transcription factor binding complement of crystal responsive genes according to Example 1 ;
Fig. 2 is a graphical representation of the most common transcription factor binding sites in crystal responsive genes according to Example 1 ; and
Fig. 3 is a series of ethidium stained panels of agarose gel containing PCR reaction after electrophoresis according to Example 3.
Modes for Carrying Out the Invention
The invention will be further illustrated by the following Examples. Example 1
Defining, promoter sequences of crystal responsive genes in osteoarthritis
The first stage of the analysis was to obtain the putative promoter sequences of the genes of interest. Initially the human nucleotide databases were searched for actual promoter sequences. This analysis led to the identification of fourteen promoter sequences from the genes of interest as described in further detail in Example 4. These searches allowed fourteen full-length promoter sequences corresponding to BCP crystal responsive genes to be identified and utilised in subsequent analysis.
Transcription factor binding sites in BCP responsive genes
To determine the capacity for transcription factor binding mediated initiation of transcription of BCP crystal responsive genes, it is necessary to build a profile of the consensus sequences for transcription factor binding sites within the promoters under study. For this puφose we used the method of Quandt, K et al ( 1996) supra.
A large library was used consisting of more than 300 transcription factor binding site matrices that was compiled on the basis of published matrices as well as entries from the TRANSFAC database, with emphasis on sequences with experimentally verified binding capacity. The advantages of the Quandt K et al (( 1996) supra) method include position weighting of the matrices based on the information content of individual positions and calculation of a relative matrix similarity. The promoter sequences from the transcripts of interest were used as input to this application in searches against the vertebrate transcription factor database and the data collated. When scoring a consensus sequence match this application provides both a core and a matrix similarity value. The most reliable predictions of binding sites have been shown to occur when a matrix similarity score of greater than 0.9 is recorded. For this reason only sequence matches with this minimum score were considered for the puφose of this study. To standardize the data reporting, in the following description the position of the site of interest is reported as downstream from the start site of translation as set out in Tables 1-14.
Table 1
Transcription Factor Binding Si ites in α-Actinin 4
Trans. Factor Position Score
AP4 -15 0.925
IK2 - 153 0.945
AP2 -197 0.901
IK2 -207 0.914
CMYC -220 0.923
NF-kB -242 0.938
MZF-1 -252 0.917
NFY -351 0.901
Table 2 Transcription Factor Binding Sites in Arginosuccinate Synthetase
Trans. Factor Position Score
IK2 -25 0.932
AP2 -48 0.921
USF -72 0.948
CMYC -74 0.988
NF1 -90 0.915
LM02COM - 150 0.966
MZF 1 - 174 0.962
IK2 -205 0.907
AP I -260 0.965
NF-kB -591 0.918
IK2 -607 0.971 Table 3
Transcription Factor Binding Sites in IGFBP 3
Trans. Factor Position Score
AP4 -77 0.911 USF -88 0.907
AP2 -122 0.966
LM02COM -153 0.968
MYC -162 0.901
API -181 0.925 CREB -208 0.955
NF1 -273 0.940
SP1 -308 0.919
IK2 -341 0.902
Table 4
Transcription Factor Bi nding Sites in IGFBP5
Trans. Factor Position Score
AP4 -60 0.911
IK2 -77 0.953
NF1 -102 0.933
MYC -131 0.921
NF-Kb -170 0.928
MZF1 -202 0.986
IK2 -361 0.912
LM02COM -369 0.915
API -379 0.907
NF1 -419 0.928 Table 5
Transcription Factor Binding Sites in Cγclooxygenase-1
Trans. Factor Position Score
AP4 -14 0.896 MZF1 -86 0.982
AP4 -130 0.966
NF1 -174 0.921
LM02COM -271 0.949
API -316 0.932 IK2 -409 0.968
MZF1 -565 0.996
Table 6
Transcription Factor Binding Sites in Cycloxygenase-2 Trans. Factor Position Score
USF -54 0.930
TCF11 -63 0.977
NFAT -78 0.914
MZF1 -117 0.954 AP4 -194 0.925
NFY -222 0.918
MZF1 -228 1.000
IK2 -245 0.907
ELK1 -336 0.912 NF-KB -347 1.000
API -506 0.949
LM02COM -520 0.929 Table 7 Transcription Factor Binding Sites in Decay Accelerating Factor
Trans. Factor Position Score
API -22 0.990 USF -33 0.952
API -93 0.963
MYC -239 0.917
LM02COM -302 0.909
NF-KB -409 0.967 MZF1 -421 0.942
NF1 -428 0.950
API -472 0.931
Table 8
Transcription Factor Binding Sites in Latent TGFβ BPl
Trans. Factor Position Score
API - 184 0.919
USF -227 0.902
NF1 -272 0.901
MZF1 -359 0.959
API -380 0.979
LM02COM -494 0.942
CEBPB -518 0.955
NF-KB -575 0.950 Table 9
Transcripti on Factor Binding Sites in MMP-1
Trans. Factor Position Score
IK2 -73 0.935
NF-KB -84 0.917
API -138 1.000
BRN2 -169 0.960
LM02COM -228 0.932
NFAT -324 0.914
IK2 -441 0.921
MZF1 -517 0.938
Table 10
Transcr ipti on Factor Binding Sites in MMP-2
Trans. Factor Position Score
AP4 -41 0.905
AP2 -82 0.945
IK2 -173 0.942
API -225 0.932
LM02COM -235 0.946
IK2 -379 0.904
NF-KB -540 0.938 Table 1 1
Transcripti ion Factor Binding Sites in MMP-3
Trans. Factor Position Score
API -66 0.989
IK2 -168 0.927
ELK1 -215 0.921
LM02COM -372 0.913
MYC -443 0.958
BRN2 -452 0.925
Table 12
Transcripti on Factor Binding Sites in MMP-7
Trans. Factor Position Score
API -68 0.989
NFY -165 0.914
LM02COM -183 0.972
API -471 0.918
MYC -484 0.927
CREB -493 0.922
AP4 -557 0.917
MZF -605 0.942
NF-KB -641 0.975 Table 13
Transcripti on Factor Binding Sites in TIMP-1
Trans. Factor Position Score
API -50 0.917
IK2 -163 0.926
MZF 1 -214 0.944
API -225 0.909
MZF1 -280 0.920
LM02COM -312 0.918
IK2 -340 0.914
NF1 -400 0.934
USF -499 0.926
API -619 0.917
Table 14
Transcription Factor Binding Si ites in Tubulin β-2
Trans. Factor Position Score
API -144 0.933
LM02COM -158 0.949
MZF 1 -176 0.905
API -221 0.917
NFY -234 0.954
ELK 1 -369 0.954
AP 4 -382 0.980 A graphical representation of the transcription factor binding site complements of the analysed genes is depicted in Fig. 1, in which indicative locations of TF sites from start site of transcription of the gene product are shown.
To determine the most commonly occurring transcription factor binding sites across the data set as a whole the number of sequences with a particular consensus sequence was expressed as a proportion of the total number of genes in the cohort. This produced the data dispersion shown in Table 15.
Table 15
Frequency of occurrence of commor l transcription factor binding sites in promoters of BCP crystal responsive genes
Trans Fac No. of Prom. Frequency AP-4 7 50 %
IK-2 10 72 %
AP-2 4 29 %
MYC 7 50 %
NF-κB 9 64 % MZF 1 1 78 %
NFY 3 22 %
USF 7 50 %
LM02COM 13 93 %
AP- 1 12 85 % NF 1 6 43 %
As can be seen four of the transcription factors identified are present in over 70 % of the analysed promoter regions. These are IK-2, MZF, LM02COM and AP- 1. When only these mediators are considered for the next stage of the analysis, the distribution pattern becomes much clearer as shown in Fig. 2. These data suggest that the initiation of transcription of a large number of BCP crystal responsive genes is dependent on the presence of a select number of transcription factor binding sites. Example 2
Construction of promoter model
Having identified in Example 1 the apparent key transcription factor binding sites and the AP-1 / LM02COM promoter module in crystal responsive genes it is of great potential utility to use this information to identify other transcripts containing this module to enable identification of novel putative crystal responsive genes. Although this approach is somewhat at variance with conventional gene finding methodologies, it represents a viable approach in light of developments in bioinformatics that permit accurate homology matrices to be constructed. To facilitate this work it was necessary to first of all construct a model of the two transcription factor binding site sequences and the distance between them. Having defined the module we were then in a position to use this information to search the nucleotide databases to find genomic segments containing this specific series of sites.
To construct the promoter module the sequences of the two binding sites of interest were obtained from the TRANSFAC TF database. As shown in Example 1 the distance between these two factors ranged from 10 to over 300 nucleotides. To facilitate model construction a matrix can be computed which essentially allows a floating sequence with 10 to 300 bases between the two sites to be constructed. This process leads to the generation of a large number of potential sequence models which are then simultaneously used as input in a BLAST (Basic Local Alignment Search Tool) search of the human nucleotide databases. The result of this analysis is a set of fragments which contain the two binding sites of interest, separated by a distance similar to that seen in the initial data analysis. A control on the approach utilised was the fact that the genes analysed during the course of this work were all represented in the final dataset. Pertinent among the resulting matches were the matches set out in Table 16.
Table 16
Sequences identified from database searches as containing BCP crystal responsive promoter module
Homo sapiens for DSIF p 160 AB000516
Homo sapiens extracellular matrix protein U65932
Homo sapiens gene for vitamin D receptor NM_000376
Homo sapiens gene for leukotriene B4 omega-hydroxylase AF221943 Homo sapiens DNA, chromosome 21q22.2, PAC clone 25P16 AB003151 Homo sapiens DNA for human type I collagen alpha 2 chain NM_000089
Homo sapiens chondroitin 6-sulfotransferase U65637
Homo sapiens gene for BCNT AB009285
Homo sapiens gene for Fas ligand AF044583
Homo sapiens APECED AIRE- 1 AB006682 Homo sapiens DNA for G-CSF receptor S71484
Homo sapiens hGLI2 AB007298
Homo sapiens KIAA0398 AB007858
Homo sapiens KIAA0439 AB007899
Homo sapiens for RGS5 XM002185 Homo sapiens gene for hippocalcin ABO 15202
Homo sapiens CRTH2 AB008535
Homo sapiens OCTN2 gene, ABO 16625
Homo sapiens gene for activin receptor type IIB AB008681
Homo sapiens CMAP AB029639 Homo sapiens gene for osteociastogenesis inhibitory factor AB008821
Homo sapiens gene for apobec-1 AB009422
Homo sapiens OR.CTL3 organic-cation transporter like 3 AB026898
Homo sapiens gene for AF-6 AB01 1399
Homo sapiens EGF2 ABO 1 1536 Example 3
Identification of fibroblast genes differentially induced by basic calcium phosphate crystals
a) Cell culture and human osteoarthritic tissue
Primary human foreskin fibroblasts were cultured as previously reported (Cheung, H. S., et al. (1984) Arthritis Rheum 27: 668-674). Cells (passage 4-8) were cultured to confluence in medium (Clonetics) and treated with BCP crystals (18μg/cm ) or left untreated for 4 hours.
Sections of osteoarthritic hip and knee were obtained from patients undergoing total hip replacement surgery. Subchondral bone, cartilage and synovial lining tissue were retained. Normal joint tissue was obtained from patients undergoing amputation procedures for peripheral vascular disease.
b) RNA Isolation
Polyadenylated RNA was isolated from fibroblasts using the Microfast Track (Microfast Track is a Trade Mark) kit (Invitrogen). Total RNA was isolated using TRIzol (TRJzol is a Trade Mark) solution (Life Technologies). c) Suppression subtractive hybridization (SSH)
SSH was performed with the PCR-SELECT cDNA subtraction kit (Clontech) as directed by the manufacturer. Starting material consisted of 2 μg of fibroblast mRNA exposed to BCP crystals for 4 hours as "tester" and 2 μg of fibroblast mRNA cultured in the absence of BCP crystals as "driver". Thirty primary PCR cycles and 12 secondary PCR cycles were performed.
d) Cloning and sequencing of cDNAs
PCR products generated by SSH were subcloned into the PCR 2.1 vector using the TA cloning kit (Invitrogen). Subcloned cDNAs were isolated by colony PCR amplification. Sequencing was performed using an automated ABI 370 A DNA sequencing system. Sequence reactions were carried out with the ABI prism dye terminator cycle sequencing ready reaction kit (Perkin Elmer). The sequences obtained were compared against the GenBank/EMBL nucleotide databases using BLAST.
SSH analysis suggested induction of 32 mRNAs in primary cultures of human fibroblasts exposed to 18μg/cm2 BCP crystals for 4 hours. Northern blots performed using formaldehyde denaturation according to standard protocols and quantitated using a phosphor imager (Biorad) confirmed differential expression of all of a test set of 10 of these mRNAs as indicated in Table 17. Table 17
Summary of Northern blot analysis of cDNAs identified by SSH as being induced in fibroblasts in response to BCP crystal stimulation
Figure imgf000027_0001
In Table 17 a refers to the sequence identity based on comparison with the Genbank / EMBL database;
b refers to the fold upregulation of each gene based on Northern blot analysis of total RNA prepared from primary human foreskin fibroblasts stimulated with BCP crystals for 4 hours relative to the expression in the absence of these crystals. Values were obtained by Phosphor-Imaging and were normalized by comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A comparison with GAPDH is used to allow accurate determination of the quantity of the transcript of interest. This gene is expressed at constant levels and as such controls for the amount of RNA being analysed. This control removes the suggestion that the increased quantities of a gene is due to a larger amount of RNA sample being analysed.
To assess the expression of these transcripts in osteoarthritis in vivo, mRNA levels were measured in RNA isolated from synovial tissue explanted from patients with end stage osteoarthritis. To this end, PCR primers for a representative set of these transcripts were synthesized. The nucleotide sequences of these primers are shown in Table 18.
Table 18 Sequences of oligonucleotide primers used for RT-PCR analyses
Insulin like growth factor binding protein-3 Forward Primer GAC TCT GCT GGT GCT GCT C
(SEQ ID NO: 1) Reverse Primer ACA GCG CTC GGT GTA GAT G
(SEQ ID NO: 2) Insulin like growth factor binding protein-5 Forward Primer CCC ATC CCC TTT AGA TTC GT
(SEQ ID NO: 3) Reverse Primer GTT CCA GTC TCT CTT CCT CCC
(SEQ ID NO: 4) Cathepsin K Forward Primer CCT TGA GGC TTC TCT TGG TG
(SEQ ID NO: 5) Reverse Primer AAA GGG TGT CAT TAC TGC GG
(SEQ ID NO: 6) Decay Accelerating Factor Forward Primer CAG CAC CAC CAC AAA TTG AC
(SEQ ID NO: 7) Reverse Primer CTG AAC TGT TGG TGG GAC CT
(SEQ ID NO: 8)
cont/. Table 18 Sequences of oligonucleotide primers used for RT-PCR analyses
Tubulin-β-2 Forward Primer AGC TCA CCC AGC AGA TGT TT
(SEQ ID NO: 9) Reverse Primer CAT TTG CTC ATC CAC CTC CT
(SEQ ID NO: 10) α-Actinin 4 Forward Primer ACC AGT TCA AGT CCA CCC TG
(SEQ ID NO: 1 1) Reverse Primer GCT TGA TGT GGT TGC TCT CA
(SEQ ID NO: 12) γ-Actin Forward Primer GGA CCT GAC CGA CTC CCT CA
(SEQ ID NO: 13) Reverse Primer GCA AGA TTC CAT ACC CAG GA
(SEQ ID NO: 14)
RT-PCR was performed on total RNA extracted from synovial lining tissue from patients with end stage osteoarthritis and non osteoarthritic tissue.
Fig. 3 depicts ethidium stained panels of a 2 % (w/v) agarose gel containing 10 μl of PCR reaction after electrophoresis. Amplification of GAPDH was used to control for equivalence of loading. In Fig. 3 the lanes represent the following
Lanes 1 and 2: RT-PCR products from synovial tissue explanted from non-osteoarthritic tissue.
Lanes 3 and 4: RT-PCR products from synovial tissue explanted from patients with established end-stage osteoarthritis.
These results indicated differential expression of the SSH identified transcripts in human osteoarthritis.
During these investigations a number of genes were identified, whose increased expression would likely contribute to the pathogenesis of OA. These include the insulin-like growth factor binding proteins, which may potentiate the mitogenic activities of the crystals and immediate early genes such as c-fos and c-myc may also play a role in mitogenesis. The upregulation of matrix metalloproteinases is also of interest and is a previously well-defined response. Cytoskeletal rearrangement is also an important aspect of OA, in that it is an effector of the gross structural changes seen in the osteoarthritic joint. Thus it was of interest that a number of cytoskeletal transcripts were found to be differentially expressed. These included γ-actin 1, vimentin, tubulin β-2 and actinin α 4. The induction of these mediators may represent an important mechanism in the pathogenesis of OA. The destruction of the joint compartment by proteolytic digestion is the pathological hallmark of OA. The upregulation of cathepsin K is thus of major interest as this gene is known to be the dominant cysteine protease involved in bone remodeling. Secretion of the mediator by synovial fibroblasts may thus be an important effector of joint degradation. In aggregate the genes identified as upregulated present a host of new target transcripts for BCP crystal induced damage.
Example 4
Identification of a BCP crystal responsive promoter module
As indicated above in Example 3 stimulation of human fibroblasts with BCP crystals leads to the coordinate induction of a wide variety of functionally diverse gene products. All of these transcripts may represent a key molecular event in the initiation and progression of osteoarthritis. Thus the challenge is to delineate the pathway that when perturbed by a therapeutic agent will have the greatest effect on the disease. Regardless of stimulus employed, we have found that for a gene to be upregulated, transcription promoting events centred at the promoter region of the gene must be initiated. This fact offers an excellent opportunity for exploitation in the search for novel therapeutic targets in a disease. In this experiment the regulatory elements in the promoters of a well-defined group of crystal responsive genes were analysed. The hypothesis explored and confirmed was that the upregualtion of a diverse cohort of genes by BCP crystals is facilitated by common promoter sequence architecture. Knowledge of this structure will offer exciting new avenues for exploration in efforts to understand the molecular events in the pathogenesis of osteoarthritis.
The first stage of this analysis was to obtain the promoter sequences of the genes of interest. To achieve this the human nucleotide databases were searched for actual promoter sequences using the general procedures set out in Example 1. This analysis led to the identification of fourteen promoter sequences from the genes of interest. These searches allowed fourteen full-length promoter sequences corresponding to BCP crystal responsive genes to be identified and utilised in subsequent analysis, as shown in Table 19.
Table 19
Promoter Sequences of genes demonstrated to be induced in human fibroblasts in response to BCP crystal Stimulation
Transcript Genbank Accession Number of Promoter Sequence
Matrix Metalloproteinase 1 AF007878
Matrix Metalloproteinase 2 AJ298926
Matrix Metalloproteinase 3 U51914 Matrix Metalloproteinase 7 L22525
Latent TGF-b Binding Protein 1 AF171935
Insulin Like Growth Factor Binding Protein 3 M35878
Insulin Like Growth Factor Binding Protein 5 U20271
Decay Accelerating Factor M64356 α-Actinin-4 AF243386 β-Tubulin 2 X02344
Cyclooxygenase-1 D64068
Cyclooxygenase-2 AF276593
Arginosuccinate Synthetase X03258 Tissue Inhibitor of Metalloproteinases- 1 Y09720 To determine the capacity for transcription factor binding mediated initiation of transcription of BCP crystal responsive genes, it was necessary to build a profile of the consensus sequences for transcription factor binding sites within the promoters under study as described in Example 1. For the reasons set forth in Example 1 only sequence matches with a minimum score of greater than 0.9 were considered for the puφose of the study. To standardize the data reporting, the position of the site of interest is reported as downstream from the start site of translation of the protein for which the gene codes. This analysis led to the identification of five commonly occurring transcription factor binding sites in crystal responsive genes; IK-2, NF- B, LM02COM, AP-1 and MZF and is illustrated in Fig. 2.
The arrangement, as well as the types of a group of transcription factor binding sites in a promoter confers the ability to regulate the expression of a gene. To reconstruct the crystal responsive promoter from the transcription factor binding site data the occurrence of all the combinations of the most commonly occurring binding sites were considered. To facilitate this analysis the data set was simplified to only a consideration of MZF, IK-2, LM02COM and API as shown in Table 20. This analysis revealed LM02COM and AP- 1 , as the two most commonly occurring sites. The standardized location of each of these sites is as follows
MZF-1 -343 +/- 160
IK-2 -188 +/- 1 13
LM02COM -245 +/- 155
AP-1 286 +/- 133
As can be seen the average positions of these four binding sites varies quite substantially. However it is of interest that the closest lying sites are LM02COM and AP- 1 , which were also the two most commonly occurring sites. To probe the nature of the sharing of these sites between crystal responsive promoters it is appropriate to consider the context in which they lie. To achieve this analysis of the relative distances between these sites was performed, with a view to constructing a promoter module. The relative distances between these binding sites was assessed as shown in Table 21.
Table 20
The most frequently occurring transcription factor binding sites in BCP crystal responsive genes
Gene MZF IK-2 LM02C0MAP-1
Actinin -250 -207 **** ****
Ass -174 -25 -150 ****
IGFBP3 * *** -341 ■153 -181
IGFBP5 -202 -77 •364 -379
Cox-1 -565 -409 ■271 -316
Cox-2 -228 -245 ■520 -506
DAF -421 ##** ■302 -260
LTGF-BP1 -359 **** •494 -380
MMP-1 -517 -73 •228 -138
MMP-2 *#** -173 ■235 -225
MMP-3 **** -168 372 -66
MMP-7 -605 **** 183 -68
TIMP-1 -280 -163 312 -50
Tubulin -176 **** 158 -142
Table 21
Relative locations of AP-1 and LMQ2COM in BCP crystal responsive gene promoters
Gene LM02COM AP-1 Distance
ASS -150 N/A
IGFBP3 -153 -181 28
IGFBP5 -364 -379 15
Cox- 1 -271 -316 45
Cox-2 -520 -506 14
DAF -302 -260 42
LTGF-BP1 -494 -380 114
MMP-1 -228 -138 90
MMp-2 -235 -225 10
MMP-3 -372 -86 292
MMP-7 -183 -68 115
TIMP-1 -312 -50 262
Tubulin -158 -142 16
The average distance separating these two sites was determined to be just 88 nucleotides. This distance is conducive with the two factors potentially functioning in unison in mediating the effects of BCP crystals.
AP-1 or activator protein 1 is a dimeric transcription factor composed of either two jun peptides or one jun and one fos peptide. BCP crystal stimulated fos and jun expression has previously been demonstrated, suggesting that the early induction of these factors may lead to sustained transcriptional activation via activator protein 1. Furthermore it has been demonstrated that BCP crystal stimulation can lead to activation of AP-1 (McCarthy, G. M. et al, (1998) J Biol Chem 273; 52: 35161-35169).
LM02COM or rhombotin 2 is a 158 amino acid transcription factor that is essential for erythroid cell differentiation. This factor has not previously been implicated in osteoarthritis. However the frequency of its appearance and its proximity to AP- 1 in the promoter regions of crystal responsive genes suggest a role for this transcription factor in human osteoarthritis.
In summary these data suggest that these two transcription factor binding sites are (a) present in the vast majority of genes demonstrated to be differentially expressed in the presence of BCP crystals, (b) are located at relatively similar distances from the start of the coding region of the genes and (c) occur within a narrow distance range of one another. This observation may be of use in devising novel agents to inhibit transcriptome activation in osteoarthritis.
Having identified the putative crystal responsive AP-1 / LM02COM promoter module this information was utilised to identify other transcripts containing this module to enable identification of novel putative crystal response genes.
To construct the promoter model the sequences of the two binding sites of interest were obtained from the TRANSFAC transcription factor database. As was previously demonstrated the distance between these two factors ranged from 10 to over 300 nucleotides. To facilitate model construction a floating sequence with 10 to 300 bases between the two sites of interest was constructed. This process led to the generation of a large number of potential sequence models that were then simultaneously used as input in a BLAST search of the human nucleotide databases. The result of this analysis is a cohort of genes that contain the two binding sites of interest, separated by a distance similar to that seen in the initial data analysis. A control on the approach utilised was the fact that the genes analysed during the course of this work were all represented in the final dataset. Other notable transcripts are listed in Table 16 above.
Example 5
Identification of a high extracellular glucose responsive promoter module
Propagation of mesangial cells under conditions of high ambient glucose has proved a useful in vitro model with which to probe the molecular basis for mesangial matrix accumulation in diabetes, attributable to high ambient glucose. Exposure of cultured mesangial cells to high glucose stimulates de novo synthesis of ECM components such as type IV collagen, fibronectin and laminin (Ayo, S.H., et al (1990) Am. J. Pathol. 136, 1339-1348; Wahab, N. A., et al. (1996) Biochem J. 316, 985- 992; and Ayo, S. H., et al. (1991 ) Am J.Physiol. 260, F 185-F191 ). High extracellular glucose has previously been demonstrated to stimulate coordinate differential expression of a large number of genes using suppression subtractive hybridisation (Muφhy, M. et al (1999) J. Biol Chem). This study led to the identification of different functional categories of genes induced in the setting of mesangial cells propagated under conditions of high extracellular glucose. In the present Example the promoter structure of a cohort of high extracellular glucose responsive genes was investigated.
The human nucleotide databases were searched for actual promoter sequences corresponding to high extracellular glucose responsive genes. This analysis led to the identification of nine promoter sequences from the genes of interest. The identity and accession numbers of these promoter sequences are shown in Table 22.
Table 22
Promoter Sequences of genes demonstrated to be differentially regulated in human mesangial cells propagated under conditions of high extracellular glucose
Transcript Genbank Accession Number of Promoter Sequence
Matrix Metalloproteinase 1 AF007878
Elongation Factor 1 J04617
Connective Tissue Growth Factor X9251 1
Fibronectin M26179
FAS D31968
Plasminogen Activator Inhibitor 1 J03764
Connexin 43 U65743
Lysyl Oxidase
Ubiquitin X04803
To determine the capacity for transcription factor binding mediated initiation of transcription of these high extracellular glucose responsive genes, it was necessary to build a profile of the consensus sequences for transcription factor binding sites within the promoters under study. This analysis was performed exactly as in the case of Examples 1 , 2 and 4.
This analysis led to the identification of four commonly occurring transcription factor binding sites in high extracellular glucose responsive genes IK-2, MZF, CDXA, SRY and GAT A- 1 as illustrated in Table 23. Table 23
The most frequently occurring transcription factor binding sites in high extracellular glucose responsive genes
Gene MZF CDXA SRY GATA-1
Elongation Factor 1 -203 -191 -20 -659
CTGF -304 -450 **** -468
Fibronectin -422 -545 -433 ** **
FAS -40 -395 * *** * ** *
PAI 1 -237 **** -97 -75
Connexin 43 **** -575 -387
Lysyl Oxidase -173 -171 -24 ****
Ubiquitin -142 -1 14 -250 -654
MMP-1 -517 -193 -319 -31
These data indicated MZF, CDXA and SRY to be the most frequently occurring transcription factor binding sites in these genes. To reconstruct the high extracellular glucose promoter from the transcription factor binding site data the occurrence of all the combinations of the most commonly occurring binding sites were considered as shown in Tables 24-26.
Table 24
Relative locations of MZF and CDXA transcription factor binding sites in high extracellular glucose responsive genes
Gene MZF CDXA Distance
Elongation Factor 1 -203 -191 14
CTGF -304 -450 146
Fibronectin -422 -545 123
FAS -40 -395 355
Lysyl Oxidase - 173 -171 2
Ubiquitin -142 -1 14 28
MMP-1 -517 -193 320
Table 25 Relative locations of MZF and SRY transcription factor binding sites in high extracellular glucose responsive genes
Gene MZF SRY Distance
Elongation Factor 1 -203 -20 183
Fibronectin -422 -433 1 1
PAI 1 -237 -97 140
Lysyl Oxidase - 173 -24 149
Ubiquitin - 142 -25 1 17
MMP-1 -517 -319 198 Table 26
Relative locations of SRY and CDXA transcription factor binding sites in high extracellular glucose responsive genes
Gene CDXA SRΥ Distance
Elongation Factor 1 -191 -20 171
Fibronectin -545 -433 1 12
Connexin 43 -575 -387 192
Lysyl Oxidase -171 -24 147
Ubiquitin -114 -250 136
MMP-1 -193 -319 126
These data can be summarized as follows in Table 27
Table 27
Combination Frequency of Combination Average Distance
MZF + CDXA 78% 141 bp
MZF + SRY 67% 133 bp CDXA + SRY 67% 147 bp
This analysis revealed three combinations of transcription factor binding sites, occurring at a significant frequency in genes previously identified as being responsive to high extracellular glucose. The conserved promoter models identified herein represent potentially important sites for therapeutic intervention and may permit the regulation of a host of genes contributing to the pathogenesis of high extracellular glucose induced glomerulosclerosis with a one shot approach. These data represent an important insight into the mechanics of transcriptome activation in diabetic nephropathy.
Example 6
Identification of novel transcription factor binding sites
The current transcription factor databases contain details of all the previously identified transcription factors in human promoters. We have carried out an experiment to examine the hypothesis that there may be novel transcription factor binding sites in the BCP crystal responsive gene promoters and other gene promoters that can drive the transcriptomic machinery.
The identification of putative novel transcription factor binding sites involves the creation of a test set of short oligonucleotide sequences, corresponding to all possible permutations. Such an approach leads to the generation of a list in which all short sequences are represented, thus providing an enabling platform for searches of homology in BCP crystal responsive promoters.
The initial stage of this work involved the generation of all the combinations of A, C, T, G to generate every possible sequence of DNA. The sequences constructed in this manner were between 7 and 18 nucleotides long, a distance which encompasses known transcription factor binding sites. The sequence combinations were created by a simple application of a random word generator programme, scripted to produce every possible word. The sequences obtained in this manner were compressed into a flat sequence file, suitable for further searching. To determine the reliability of this methodology a whole database comparison was performed. This comprised of comparing the putative sequence database against the TRANSFAC database of human transcription factor binding sites. This led to the identification of all vertebrate TF sites within the generated sequence file.
A large scale alignment strategy was then used to compare all of the putative sequences to the promoter sequences of BCP crystal responsive genes. Following serial alignments a short sequence, TTCCTGTCT, was identified as being present in 86 % of the promoter sequences in question.
The location of this sequence in individual promoter sequences is shown in Table 28. This analysis revealed the location of this sequence to be on average 393 nt from the start site of transcription. The conserved nature of this sequence and its occurrence in a large proportion of BCP crystal responsive genes suggest that this sequence may be an important effector of translating BCP crystal mediated signals to gene activation. Table 28
Gene TTCCTGTCT
Actinin -306
Ass -580
IGFBP3 -540
IGFBP5 ****
Cox-1 -565 Cox-2 ****
DAF -340
LTGF-BP1 -115
MMP-1 -70
MMP-2 -540 MMP-3 -312
MMP-7 -487
TIMP-1 -440
Tubulin -430

Claims

Claims: -
1. A method for identifying a cohort of genes the expression of which is altered in a disease state, and the expression products of which are involved in the pathogenesis of said disease, which method comprises identifying in said genes a common promoter module which governs the transcriptomic response to disease associated stimuli.
2. A method according to Claim 1 , wherein the common promoter module has a number of common transcription factor binding sites within 750 base pairs (bp) of the start site of transcription.
3. A method according to Claim 1 or 2, wherein the disease is osteoarthritis.
4. A method according to Claim 3, wherein the genes are basic calcium phosphate (BCP) crystal responsive genes.
5. A method according to Claim 4, wherein the common promoter module includes the transcription factor binding sites
LM02COM, AP-1 and the sequence TTCCTGTCT.
6. A method according to Claim 1 or 2, wherein the disease is diabetic nephropathy.
7. A method according to Claim 6, wherein the genes are responsive to high ambient glucose.
8. A method according to Claim 7, wherein the common promoter module includes the transcription factor binding sites GATA-1, MZF, CDXA and SRY.
9. A method according to any one of Claims 2-8, wherein the transcription factor binding sites are located at a conserved distance from the start of the coding region of the genes and occur within a range of 10-300 bp of one another.
10. A method according to any preceding claim, wherein the common promoter module is elucidated by bioinformatics.
1 1. A method according to Claim 10, wherein the transcription factor binding sites are predicted by a comparison of putative sequences against a vertebrate transcription factor database yielding both a core and a matrix similarity value and selecting those sequences where a similarity score of greater than 0.9 is recorded.
12. Use of a method according to any one of Claims 1-1 1 for identifying targets for the rational design of therapeutic agents for use in the treatment of an associated disease.
13. Use of a method according to any one of Claims 1-1 1 as a basis for determining a response to therapeutic agents.
14. Use of a method according to any one of Claims 1-1 1 for identifying targets for the rational diagnosis of disease.
15. Use of a method according to any one of Claims 1-1 1 as a basis for assessing disease activity.
16. A promoter module common to a cohort of genes the expression of which is altered in a disease state, and the expression products of which are involved in the pathogenesis of said disease, said promoter module comprising two or more common transcription factor binding sites which act in concert to regulate the expression of said genes.
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