JP2003517302A - Cloning and characterization of CAP gene promoter - Google Patents

Abstract

  (57) [Summary] The present invention relates to novel polynucleotides containing a peroxisome proliferative response element (PPRE). The invention also describes vectors and host cells comprising the novel polynucleotide. The invention further relates to methods of using the novel polynucleotides, applications to gene therapy, the diagnosis of ligands involved in insulin resistance, the preferential binding of ligands or proteins that bind to, associate with or interact with the CAP promoter. The development of a screening strategy is described.

Description

Detailed Description of the Invention

[0001]

【Technical field】

The present invention relates to CAP containing a peroxisome proliferative response element (PPRE).
The present invention relates to a novel polynucleotide containing a promoter region of a gene. The invention also describes vectors and host cells containing the novel polynucleotides.
The invention further provides for the detection of genetic deletions of the polynucleotide, subcellular localization of the polynucleotide, diagnosis of conditions involving abnormal activity of elements within its promoter, including its presence or absence, Described are methods of using the novel polynucleotides in the development of unique screening strategies to assess the regulation / binding of ligands or proteins to their promoters.

[0002]

[Background technology]

The tyrosine kinase activity of the insulin receptor is essential for the full expression of insulin action, but the exact role of its various cellular substrates remains unclear. Insulin stimulates tyrosine phosphorylation of the c-Cbl proto-oncogene product (Ribon V & Saltiel AR, 1997, Biochem J 324 (Pt 3): 839-45). This phosphorylation requires the expression of a novel protein called CAP that supplies c-Cbl to the insulin receptor (Ribon V, Printen JA, Hoffman NG, Kay BK & Saltiel AR,
1998, Mol Cell Biol, 18 (2): 872-9). CAP has three flanking SH3s at the C-terminus.
It is a multifunctional protein having a domain and a solvin homology domain at the N-terminus. CAP is associated with both c-Cbl and the insulin receptor in the basal state. Insulin stimulation causes the dissociation of CAP from the insulin receptor.
However, CAP remains associated with c-Cbl after insulin stimulation.
Moreover, overexpression of CAP causes focal adhesion and formation of stress fibers by association with p125 FAK and actin stress fibers (Ri
bon V, Herrera R, Kay BK & Saltiel AR, 1998, J Biol Chem 273 (3): 4073-80
).

The role of CAP in insulin action has not yet been clearly determined, but evidence in several ways suggests an important function. It is preferentially expressed in insulin sensitive tissues. In the 3T3-L1 adipocyte line, its CAP expression correlates well with insulin sensitivity. Furthermore, stimulation of the nuclear receptor PPARγ by the thiazolidinedione (TZD) in 3T3-L1 adipocytes or diabetic rodents leads to increased CAP expression and insulin-stimulated elevation of c-Cbl phosphorylation (Ribon V, JohnsonJH). , Camp HS & Saltiel AR, 1998, Proc Natl Acad S
ci USA 95 (25): 14751-6). The effect of TZD on CAP expression is a direct result of increased CAP gene transcription. This TZD-induced increase in CAP expression correlates well with increased insulin sensitivity both in vitro and in vivo.

Despite a growing body of evidence suggesting important functions, the role of CAP in insulin action has not been clearly determined. Confirmation of such a role would be beneficial. If the biochemical mechanisms behind these insulin-stimulated responses are understood, abnormal (high or low) levels of insulin or glucose
New opportunities for treatment and diagnosis of diseases associated with storage and / or utilization are likely to open up. In other words, a better understanding of the molecular mechanism of insulin action could improve the design of therapeutics for the treatment of diseases associated with abnormal storage and / or utilization of insulin or glucose. Such disease states include diabetes, glycogenosis, obesity, polycystic ovary syndrome, hypertension, atherosclerosis and other insulin resistance diseases.

[0005]

DISCLOSURE OF THE INVENTION

The present invention relates to the discovery of the promoter of the CAP gene, the identification of a novel target peroxisome proliferative response element (PPRE), and the isolation of the polynucleotide sequence encoding PPRE in the CAP promoter. Disclosed herein is the cloning of the promoter of the CAP gene and the identification of a functional PPAR response element (PPRE) within the CAP promoter. A 2.5 kB sequence analysis of the 5'flanking region of the CAP gene reveals a predicted peroxisome proliferative response element (PPRE) from -1085 to -1097. The isolated promoter was functional in 3T3 fibroblasts and adipocytes. Cotransfection of the CAP promoter with PPARγ and retinoic acid X receptor α (RXRα) resulted in an additional 2-fold stimulation of promoter activity. The TZD rosiglitizone produced an additional 2-3 fold promoter stimulation.
The predicted deletion of PPRE from the CAP promoter abolished its ability to respond to rosiglithi zone. By gel shift analysis of the candidate site of PPARγ,
Direct binding of the PPAR / RXR heterodimer to PPRE in the CAP gene was demonstrated. From these data, TZD is mediated by CAP through activation of PPARγ.
It is apparent that it directly stimulates gene transcription.

The results provided herein reveal evidence from a first direction that links the anti-diabetic effect of PPARγ activation to improved insulin signaling. The nature of PPRE in the CAP promoter has vital physiological effects. Therefore, by regulating the activity level of this promoter, the desired physiological effect can be obtained. Such effects can be exerted on a variety of disorders including, but not limited to, abnormal levels of insulin or glucose (ie, but not limited to diabetes, glycogenosis, obesity, polycystic ovary syndrome, hypertension, atheroma). It can be used to treat or detect disease states, including atherosclerosis and other insulin resistance diseases.

One aspect of the invention is to provide a polynucleotide complementary to a polynucleotide sequence encoding a promoter of the invention (including elements within the promoter, eg, PPRE), and to the strand encoding the polynucleotide. It is in. The polynucleotide of the present invention can be used in recombinant DNA technology (cloning, subcloning, etc.). The polynucleotides of the invention can also be used to detect genetic deletions in the polynucleotides. The invention also provides polynucleotides and amplification primers that are used as hybridization probes for the detection of naturally occurring polynucleotides encoding PPRE.

Another aspect of the present invention is to provide PPRE and PPARγ and / or RXR (or
Assays for the detection or screening of therapeutic compounds that interfere with or augment the interaction between other transcription factors (which bind to the CAP promoter). These assays of the invention consist of measuring the effect of the compound of interest on the binding between PPRE and PPARγ or RXR (or other transcription factors that bind to any part of the promoter). Binding can be measured by a variety of methods including the use of labeled PPRE-tagged transcription factors or reporter assays containing some or all of the CAP promoter.

Another aspect of the invention provides an assay for discovering proteins that interact directly or indirectly with a promoter. The assay of the present invention comprises methods for detecting such interactions in cells or in biochemical assays. These interactions can be detected by a variety of methods, including those involving the use of CAP cDNA.

The above description is not intended to limit the invention in any way,
It should not be construed as a limitation of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All US patents and publications mentioned herein are incorporated by reference in their entirety.

DESCRIPTION OF THE FIGURES FIG. Structure of the 5'flanking region of the mouse CAP gene. A: Schematic representation of the genomic organization and restriction map of the CAP promoter and 5'coding exons. Black represents the promoter. The shaded box is the PPRE position. Gray boxes represent exons and white boxes represent intron sequences. B: Sequence in the CAP promoter and putative transcription factor binding site. The binding sites of Sp1, AP-2, C / EBP and PPAR are highlighted. The start of transcription as measured by S1 nuclease protection is indicated by the arrow. A cDNA encoding exon 1 of the CAP gene (Genwbank # U58883). Single-stranded probes used in the S1 nuclease protection assay are italicized.

FIG. S1 nuclease analysis of the transcription start site of the CAP gene. Total RNA isolated from 3T3-L1 adipocytes or mouse obese adipose tissue (40 μg
Was hybridized with [γ- 32 P] -end-labeled 70-mer probe at 42 ° C. overnight. The antisense 70-mer probe is a sequence complementary to the first 40 bases of the 5'-end of the longest CAP cDNA clone, 2 immediately upstream of the 5'-end of the longest clone.
It contains a 0-base genomic sequence complementary sequence and a 10-base non-specific sequence (FIG. 3).
(See B). The sample is then 60 units at 37 ° C with 250 units of S1 nuclease.
Digested for minutes and then ethanol precipitated. The reaction product is polyacrylamide (
8%) / urea (7M) gel electrophoresis and visualized by autoradiography. Undigested probe was loaded in lane 1. The size of the protected fragments was assessed by comparison with a DNA sequencing ladder run on the same gel.

FIG. The CAP promoter is functional in NIH3T3 fibroblasts,
It is TZD sensitive. A: Schematic representation of the CAP promoter of various reporter constructs cloned into the pGL3 basic luciferase reporter plasmid. B: NIH3T3 fibroblasts were cotransfected with CMV-PPARγ and CMV-RXRα or empty vector and the CAP promoter construct in the pGL3 luciferase reporter system. Following transfection, cells were incubated for 48 hours in the presence or absence of 20 μM rosiglithizone. All luciferase measurements were normalized to β-Gal activity and average ± SE
(N = 3). Asterisk indicates significant difference from control treated reporter inhibition (* = p <
0.005).

FIG. The CAP promoter is functional in 3T3-L1 adipocytes and
It is D-sensitive. Electroporate 3T3-L1 adipocytes with various reporter constructs and
Luciferase activity was assessed in the presence or absence of M rosiglithi zone. All luciferase activities were normalized to β-galactosidase activity and averaged ±
S.E. (n = 3). Asterisk indicates significant difference from control treated reporter inhibition (* = p <0.005).

FIG. Gel Mobility Shift Assay This figure shows a representative autoradiography of a gel mobility shift assay. A: The oligonucleotide sequence used in the gel shift test, the PPRE sequence, is underlined. B: In vitro translated PPARγ and RXRα were incubated prior to addition of radiolabeled CAP PPRE probe to form heterodimers. Some samples were cold to control binding specificity.
Incubated with increasing concentrations of wtPPRE, mutPPRE (10 and 50 fold molar excess) or in the presence of 50 fold molar excess of non-specific duplex oligonucleotides. B. Nuclear extracts from 3T3-L1 fibroblasts or 3T3-L1 adipocytes were incubated with radiolabeled CAP PPRE probe. In order to control the binding specificity, some samples used cold wtPPRE, mutPPR
Incubated with increasing concentrations of E (10 and 50 fold molar excess) or in the presence of 50 fold molar excess of non-specific duplex oligonucleotides.

[0016]

Detailed description of the invention

Within this application, unless otherwise indicated, the techniques used are described in several well-known literature, such as Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989, Cold.
Spring Harbor Laboratory Press), Gene Expression Technology (Methods in
(Enzymology, Vol. 185, D. Goeddel, 1991, Academic Press, San Diego, CA)
, “Guide to Protein Purification” in Methods in Enzymology (MPDeutshce
r, 1990, Academic Press, Inc.); PCR Protocols: A Guide to Methods and
Applicatuons (Innis et al., 1990, Academic Press, San Diego, CA), Culture of
Animal Cells: A Manual of Basic Technique, 2nd ed, (RIFreshney, 1987,
Liss, Inc. New York, NY), and Gene Transfer and Expression Protocols, p
p.109-128, edited by EJ Murray, Humana Press Inc., Clifton, NJ).

In one aspect, the invention provides a novel isolated and purified polynucleotide encoding a functional PPAR response element within the CAP promoter (hereinafter referred to as “PPRE”) and / or a CAP promoter consisting of PPRE. Provide an array. The term "PPRE" is used broadly herein. Unless specified otherwise
The term "PPRE" includes, but is not limited to, any naturally occurring mammalian-derived form of PPRE and the like. The term PPRE preferably includes primate and human PPRE. Also, the term "increase" is used extensively herein. Unless otherwise specified, the term "interaction" includes, but is not limited to, binding, influence and regulation.

The provided polynucleotide encodes the complete CAP promoter (SEQ ID NO: 1) or a portion thereof such as PPRE (SEQ ID NO: 2). The polynucleotides of the present invention are produced, for example, by various methods using well-known solid-phase synthesis techniques, cloning including chemical synthesis in vitro, or a combination thereof. One of ordinary skill in the art is well aware of the degeneracy of the genetic code and may have partial sequences that have polynucleotide sequences that are either partially or completely homologous to the naturally occurring polynucleotide sequences that encode the promoter. Polynucleotides encoding any or all promoters can be readily designed. The polynucleotide of the present invention may be single-stranded or double-stranded. Polynucleotides complementary to the polynucleotide encoding the promoter are also provided.

Libraries, either cDNA or genomic, are screened with probes designed to identify the gene of interest. In the case of a cDNA library, suitable probes include known or suspected portions of PPRE from the same or different species, and / or complementary or homologous cDNAs or fragments thereof encoding the same or similar genes. , And / or carefully selected oligonucleotide probes (generally about 20-80 bases in length) encoding homologous genomic DNA or fragments thereof. Screening a cDNA or genomic library with selected probes is done by Chapters 10-12, Sambrook et al., Molecular Cloning: A Laborato.
ry Manual, New York Cold Spring Harbor Laboratory Press, 1989.

The oligonucleotide must be detectably labeled when hybridized to the DNA of the library to be screened. A preferred method of labeling is to radiolabel the 5'end of the oligonucleotide with ATP (eg T32P) and polynucleotide kinase. However, other methods can be used to label the oligonucleotide, including, but not limited to, biotinylation or enzyme labeling.

DNA sequences containing the CAP promoter may also be found in other well known techniques of recombinant DNA technology, eg, US Pat. No. 4,683,195, Section 14, Sambrook et al., Molecu.
lar Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Labora
tory Press, New York, 1989 or Chapter 15, Current Protocols in Molec
ular Biology, Ausubel et al., Green Publishing Assoiciates & Wiley-Intersci
ence, 1991 or by direct expression cloning or using the polymerase chain reaction (PCR). PP for this method
The use of oligonucleotide probes that hybridize to the DNA encoding RE is required.

In a preferred embodiment, the invention consists of a DNA sequence that is substantially similar to the sequences shown in SEQ ID NO: 1 and 2 (mouse PPRE polynucleotide). The term "substantially similar" as defined herein includes identical sequences as well as deletions, substitutions or additions of DNA. Preferably, the DNA sequences according to the invention consist essentially of the DNA sequences SEQ ID NO: 1 and 2. These novel purified and isolated DNA sequences can be used for mutational analysis of promoter function.

The mutated sequences of the present invention can be identified by those skilled in the art in a routine manner using the techniques provided herein and well known in the art.

In a preferred embodiment, the invention consists of a nucleotide sequence that hybridizes under stringent hybridization conditions with the nucleotide sequences shown in SEQ ID NOs: 1 and 2. As used herein, the term “high stringency hybridization conditions” refers to hybridization with a probe of interest in low salt hybridization buffer at 65 ° C. at 2 × 10 ⁸ cpm / μg for about 8-24 hours and then 1 % S
It means washing in DS, 20 mM phosphate buffer and 1 mM EDTA at 65 ° C. for about 30 minutes to 4 hours. In a preferred embodiment, the low salt hybridization buffer consists of 0.5-10% SDS and 0.05M and 0.5M sodium phosphate. In the most preferred embodiment, the low salt hybridization buffer consists of 7% SDS and 0.125M sodium phosphate.

The polynucleotides of the present invention have a variety of uses, some of which have already been pointed out and are described in detail below. The particular use of a given polynucleotide will depend in part on the implementation of the particular polynucleotide of interest. The polynucleotide of the present invention is used as a hybridization probe for recovering a promoter or a part thereof from a genomic library. The polynucleotides of the invention can also be used as primers for amplification of the promoter or parts thereof by the polymerase chain reaction (PCR) or other similar amplification procedures. The polynucleotides of the present invention also provide probes and amplifications for detecting mutations in the promoter or parts thereof which are associated with diseases, in particular diseases associated with overexpression or underexpression of ligands which bind to or associate with or interact with the promoter. It can also be used as a primer.

The present invention also provides various polynucleotide expression vectors in which a promoter or a part thereof or a sequence substantially similar thereto is subcloned into an extrachromosomal vector. The term "extrachromosomal vector" as used herein includes, but is not limited to, plasmids, bacteriophages, cosmids, retroviruses and artificial chromosomes. In a preferred embodiment, the extrachromosomal vector comprises a PPRE when the recombinant DNA molecule is inserted into a host cell.
It is composed of a vector that enables cloning of The vector may further consist of additional polynucleotide sequences for expression of the gene, regulation or conventional manipulation of the vector. Such additional sequences include terminators, enhancers, selectable markers, packaging sites and the like. A detailed description of polynucleotide expression vectors and their use can be found in, among others, Gene Expressio
n Technology: Methods in Enzymology, Volume 185, Goeddel, Academic Pr
ess Inc., San Diego, CA, 1991, Protein Expression in Animal Cells, Roth
Ed., Academic Press Inc., San Diego, CA, 1994.

In a further aspect, the invention provides a recombinant host cell stably transfected with a recombinant DNA molecule, the promoter of which is subcloned into an extrachromosomal vector. The host cells of the present invention may be of any type, including, but not limited to, bacterial, yeast and mammalian cells. Transfection of host cells with recombinant DNA molecules is well known in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., C
(Old Spring Harbor Laboratory Press, 1989), as used in the present invention, includes, but is not limited to, calcium phosphate transfection, dextran sulphate transfection, electroporation, lipofection and viral infection.

The CAP promoter can be used to discover molecules that interfere with or enhance its activity. Molecules that increase the binding of PPARγ and / or RXR to the CAP promoter in insulin responsive tissues, for example, increase the action of insulin. In addition, the promoter can be used to discover other proteins that can interact directly with it, ie, additional key regulators of glucose transport.

The PPRE of the present invention has a biological activity associated with PPARγ and / or RXR. The PPRE of the present invention can be isolated from various mammalian species. Preferred mammalian species for isolation are primates and humans.

Another aspect of the invention determines whether the compound of interest is capable of binding to a promoter and interfering with or increasing the binding of PPARγ and / or RXR (or other ligand) to the promoter. Useful assays are provided. This assay consists of measuring the binding of a compound of interest to a promoter. Either the promoter or the compound of interest to be assayed is labeled with a detectable label such as a radioactive or fluorescent label so that complex formation between the compound of interest and the promoter can be detected. In another embodiment of this assay, the assay involves interference or competitiveness of a compound of interest with a binding interaction between the promoter and PPARγ and / or RXR (or other ligand known to bind PPRE). Includes measurement of binding. For example, the effect of increasing amounts of the compound of interest on complex formation between radiolabeled PPARγ and / or RXR and the promoter can be measured by quantification of labeled ligand-PPRE complex formation.

In yet another aspect, the invention is a diagnostic assay for detecting cells containing a deletion of the CAP promoter polynucleotide, isolating total genomic DNA from the cells, SEQ ID NOs: 1 and 2. Provides a diagnostic assay which comprises subjecting genomic DNA to PCR amplification using primers derived from the DNA sequence of

This aspect of the invention allows for the detection of CAP promoter polynucleotide deletions in any type of cell and can be used as a genetic test or laboratory tool. PCR primers can be selected in any fashion that allows amplification of a promoter polynucleotide fragment of sufficient length for detection by gel electrophoresis. Detection includes any method, including, but not limited to, ethidium bromide staining of agarose or polyacrylamide gels, autoradiographic detection of radiolabeled promoter gene fragments, Southern blot hybridizations, and DNA sequence analysis. To be done. In a preferred embodiment, detection is by polyacrylamide electrophoresis followed by DNA sequence analysis to confirm the presence of the deletion. PCR conditions are routinely determined based on primer length and base content selected according to techniques well known in the art (Sambrook et al., 1989).

A further aspect of the invention is a diagnostic assay for detecting cells containing a deletion of a promoter polynucleotide, wherein total cellular RNA is isolated and primers derived from the DNA sequences of SEQ ID NOs: 1 and 2 are used. To provide RNA for reverse transcription PCR amplification. This aspect of the invention allows detection of promoter deletions in any type of cell and can be used as a genetic test or laboratory tool.

Reverse transcription is a standard technique (Ausubel et al., Current Protocols in Molecular Biology,
ed. John Wiley and Sons, Inc., 1994) and PCR.
Is implemented as described above.

It is believed that the present invention can be better understood with reference to the following examples. These examples are intended for purposes of illustration only and are not meant to limit the scope of the invention as defined in the claims set forth above.

[0036]

【Example】

Example 1 Materials -Cell culture reagents were purchased from GIBCO / BRL (Gaithersburg, MD). Rosiglitisone BRL (49653) is commercially available.

CAP Promoter and Reporter Fusion Construct— The bacterial artificial chromosome (BAC) library at Research Genetics was constructed with the coding region 5 ′ of the CAP gene.
Screened for ends. BAC clones were restriction digested with Hind III or Sac I and then subjected to Southern analysis with the probe corresponding to the 5 "UTR and the promoter sequence of the CAP gene. The fragment was labeled with pBluescript (Strata
gene) and pGL3 basic (Promega). All sequence analyzes of promoter region and transcription factor binding site are S
It was performed by ignal Scan software (University of Minnesota). The plasmid construct was generated by digestion using restriction sites within the CAP promoter. pCPH was generated by cloning the 550 bp HindIII-SmaI fragment of the CAP promoter into pGL3. pCPS is 10 to pGL3
It was generated by insertion of a 70 bp SacI-SmaI fragment. pCE is CA
It was generated by insertion of a 2.6 bp EcoRI / SmaI fragment of the P promoter. pCPEΔPPRE was generated by SacI digestion of pCPE and removal of the 435 bp fragment containing the PPRE of the CAP promoter and ligation of the pCPE vector.

S1 Nuclease Assay —Total RNA (40 μg) isolated from 3T3-L1 adipocytes or mouse adipose tissue was combined with [γ ³² P] -end labeled 70-mer probe and 42.
Hybridized overnight at ° C. Antisense 70mer probe is the longest CAP
Contains a sequence complementary to the first 40 bases of the 5'-end of the cDNA clone, a sequence complementary to the 20 base genomic sequence immediately upstream of the 5'-end of the longest clone, and a 10-base non-specific sequence (See FIG. 3B). The sample was then digested with 250 units of S1 nuclease for 60 minutes at 37 ° C, followed by ethanol precipitation. The reaction product was subjected to polyacrylamide (8%) / urea (7M) gel electrophoresis and visualized by autoradiography. Undigested probe was loaded in lane 1. The size of the protected fragments was assessed by comparison with a DNA sequencing ladder run on the same gel.

Cell Transfection and Reporter Assays —NIH3T3 and 3
T3-L1 fibroblasts were maintained in DMEM, 10% bovine serum. Transfection of NIH3T3 fibroblasts was performed with lipofectAMINE reagent (GibcoBRL) according to the manufacturer's instructions. 250 ng of pGL3 basic firefly luciferase construct (Promega) was added to pCMV-βGAL and / or CMV-PPAR.
Cotransfected with 50 ng of γ and CMV-RXRα. PPARγ ₁ and RXRα are based on the previously described pSG5 expression plasmid (Camp HS & Tafuri S
R, 1997, J Biol Chem 272 (16): 10811-6). Five hours after transfection, cells were incubated for 48 hours in the presence or absence of 20 μM rosiglitisone. 3T3-L1 adipocytes were grown to confluence and differentiated as previously described (Lazar DF, Wiese RJ, Brady MJ.
, Mastick CC, Waters SB, Yamauchi K, Pessin JE, Cuatrecasas P & Saltiel
AR, 1995, J Biol Chem 270 (35): 20801-7). Adipocytes (8th day after differentiation)
Protocols previously reported by urmond et al. (Thurmond DC, Ceresa BP, Okad
a S, Elmendorf JS, Coker K & Pessin JE, 1998, J Biol Chem 273 (50): 3387
6-83) and electroporated. Briefly, adipocytes were trypsinized, washed and resuspended in PBS. 10 ⁷ fat cells

B. To determine the start of transcription of the CAP gene, a 70 nt single stranded DNA probe was generated that overlapped the region containing the longest known CAP cDNA sequence. This probe was hybridized with 3T3-L1 adipocyte or mouse obesity tissue mRNA and digested with S1 nuclease to identify the initiation of transcription (Fig. 1B, 2). The presence of one predominant transcript was confirmed by RT-PCR. The initiation of translation of the CAP gene was found to be within exon 4 of the CAP gene (data not shown). Candidate sequences for promoters are signal scan software (Univers
2.6 kB of the 5'flanking region of the CAP gene was analyzed and identified using the City of Minnesota). Although no TATA box was found in the promoter, T
Has ATA-less promoter properties (Parks CL & Shenk T, 1996, J Biol Ch
em 271 (Dignam JD, Lebovitz RM & Roeder RG, 1983, Nucleic Acids Res 11 (5
) 1475-89), 4417-30, Blake MC, Jambou RC, Swick AG, Kahn JW & Azizkhan
JC, 1990, Mol Cell Biol 10 (12), 6632-41). The basal promoter region is GC-rich and contains multiple Sp-1 binding sites. In addition a CAAT box and several A's
P-2 and C / EBP sites were identified. The PPRE candidate was found at -1107-1094 (SEQ ID NO: 2) in the CAP promoter (Fig. 1B). The CAP promoter PPRE has a hexameric (DR1) type direct repeat sequence similar to PPRE from other genes, as shown in Table 1 (Johnson EF, Palmer CN, Griffin KJ.
& Hsu MH, 1996, Faseb J 10 (11), 1241-8; Frohnert BI, Hui TY & Bernlohr
DA, 1999, J Biol Chem 274 (7), 3970-7).

[0041]

[Table 1]

Identification of functional PPREs in the CAP gene— To determine the functionality of the CAP promoter as well as its candidate PPRE, a transient reporter assay (FIG. 3A) using various CAP promoter constructs was performed on NIH3T3 fibroblasts. It was carried out in. Luciferase fusion constructs were prepared by subcloning the cloned restriction fragment from the CAP promoter into the pGL3 basic luciferase reporter vector as described in the Experimental Section. NIH3T3
The results obtained from the reporter assay in fibroblasts showed that the CAP promoter was functional (Fig. 3B). Fusion of various CAP promoter constructs to the luciferase revotor resulted in 20 luciferase activities compared to pGL3 basic vector alone for all CAP promoter constructs tested.
It produced a more than doubled increase. Cotransfection of PPARγ and RXRα with the fusion construct pCPE containing CAP PPRE resulted in an additional stimulation of doubling of luciferase activity. Addition of rosiglitazone also led to a 2-3 fold additional stimulation of luciferase activity from the pCPE fusion construct. In contrast, additional PPARγ and R in the presence or absence of rosiglitazone
XRα was ineffective in increasing the luciferase activity of shorter pCPH and pCPS fusion constructs. Furthermore, deletion of the region containing the PPRE of the CAP promoter (fusion construct pCPEΔPPRE) abolished the response to rosiglitazone.

For further characterization of the CAP promoter and its PPRE activity, the fusion construct was electroporated into 3T3-L1 adipocytes. 3T3-L1 adipocytes contain high levels of endogenous PPARγ. Moreover, the expression of CAP in 3T3-L1 adipocytes is responsive to TZD. Electroporated CA
P reporter constructs pCPH and pCPS compared to pGL3 basic 10
It showed a doubling level of luciferase activity (Figure 4). The pCPE construct containing CAP PPRE produced an additional 2-fold stimulation of luciferase activity compared to pCPH. The activity of the pCPE fusion was 2-fold greater in the presence of 20 μM rosiglitazone. Deletion of CAP promoter region including PPRE (pCPEΔPPRE
) Abolished both increases in basal luciferase activity and response to rosiglitazone.

Gel shift analysis of binding of PPAR / RXR heterodimer to CAP PPRE-To demonstrate direct binding of PPARγ / RXRα heterodimer to CAP PPRE, gel shift analysis was performed on CAP PPRE (FIG. 5).
). The double-stranded oligonucleotide probe for wtCAP PPRE has ³² P-
Labeled and incubated in vitro with translated protein as well as 3T3-L1 nuclear extract. As shown in FIG. 5B, both PPARγ and RXRα alone are CA
It did not bind to the PPPRE oligonucleotide. However, PPARγ / R
The XRα heterodimer binds to the wtCAP PPRE oligonucleotide. This binding is specific because it competes with the unlabeled wtCAP PPRE oligonucleotide. Incubation with either mutCAP PPRE (an oligonucleotide with a single base deletion in the DR1 motif) or a non-specific duplex oligonucleotide did not displace the labeled wtCAP PPRE oligonucleotide.
In addition, the nuclear protein from 3T3-L1 adipocytes is in vitro translated by PPARγ.
We were able to form a protein-DNA complex with wtCAP PPRE with similar specificity to the experiments performed with the / RXRα heterodimer (FIG. 5C). As mentioned above,
Unlabeled wtCAP PPRE competed with the DNA-protein complex formed with the nuclear protein. The mutCAP PPRE and non-specific duplex oligonucleotide probes did not compete for interaction with radiolabeled DNA-protein.

The present invention provides the exact details of the operation or the precise compound, composition, method, process or treatment disclosed and described, as obvious modifications and equivalents will be apparent to those skilled in the art. It is to be understood that the invention is not limited by the embodiments, and therefore the invention is limited only by the full scope of the appended claims.

[Sequence list]

[Brief description of drawings]

FIG. 1 Structure of 5'flanking region of mouse CAP gene. A: Schematic representation of the genomic organization and restriction map of the CAP promoter and 5'coding exons. Black represents the promoter. The shaded box is the PPRE position. Gray boxes represent exons and white boxes represent intron sequences. B: Sequence in the CAP promoter and putative transcription factor binding site. The binding sites of Sp1, AP-2, C / EBP and PPAR are highlighted. The start of transcription as measured by S1 nuclease protection is indicated by the arrow. A cDNA encoding exon 1 of the CAP gene (Genwbank # U58883). Single-stranded probes used in the S1 nuclease protection assay are italicized.

FIG. 2. S1 nuclease analysis of the transcription start site of the CAP gene. Total RNA isolated from 3T3-L1 adipocytes or mouse obese adipose tissue (40 μg
Was hybridized with [γ- 32 P] -end-labeled 70-mer probe at 42 ° C. overnight. The antisense 70-mer probe is a sequence complementary to the first 40 bases of the 5'-end of the longest CAP cDNA clone, 2 immediately upstream of the 5'-end of the longest clone.
It contains a 0-base genomic sequence complementary sequence and a 10-base non-specific sequence (FIG. 3).
(See B). The sample is then 60 units at 37 ° C with 250 units of S1 nuclease.
Digested for minutes and then ethanol precipitated. The reaction product is polyacrylamide (
8%) / urea (7M) gel electrophoresis and visualized by autoradiography. Undigested probe was loaded in lane 1. The size of the protected fragments was assessed by comparison with a DNA sequencing ladder run on the same gel.

FIG. 3. The CAP promoter is functional in NIH3T3 fibroblasts, TZ
It is D-sensitive. A: Schematic representation of the CAP promoter of various reporter constructs cloned into the pGL3 basic luciferase reporter plasmid. B: NIH3T3 fibroblasts were cotransfected with CMV-PPARγ and CMV-RXRα or empty vector and the CAP promoter construct in the pGL3 luciferase reporter system. Following transfection, cells were incubated for 48 hours in the presence or absence of 20 μM rosiglithizone. All luciferase measurements were normalized to β-Gal activity and average ± SE
(N = 3). Asterisk indicates significant difference from control treated reporter inhibition (* = p <
0.005).

FIG. 4. CAP promoter is functional and TZD sensitive in 3T3-L1 adipocytes. Electroporate 3T3-L1 adipocytes with various reporter constructs and
Luciferase activity was assessed in the presence or absence of M rosiglithi zone. All luciferase activities were normalized to β-galactosidase activity and averaged ±
S.E. (n = 3). Asterisk indicates significant difference from control treated reporter inhibition (* = p <0.005).

Figure 5: Gel mobility shift assay. This figure shows a representative autoradiography of a gel mobility shift assay. A: The oligonucleotide sequence used in the gel shift test, the PPRE sequence, is underlined. B: In vitro translated PPARγ and RXRα were incubated prior to addition of radiolabeled CAP PPRE probe to form heterodimers. Some samples were cold to control binding specificity.
Incubated with increasing concentrations of wtPPRE, mutPPRE (10 and 50 fold molar excess) or in the presence of 50 fold molar excess of non-specific duplex oligonucleotides. B. Nuclear extracts from 3T3-L1 fibroblasts or 3T3-L1 adipocytes were incubated with radiolabeled CAP PPRE probe. In order to control the binding specificity, some samples used cold wtPPRE, mutPPR
Incubated with increasing concentrations of E (10 and 50 fold molar excess) or in the presence of 50 fold molar excess of non-specific duplex oligonucleotides.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12Q 1/68 C12N 5/00 A (81) Designated country EP (AT, BE, CH, CY, DE, DK) , ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR , NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AU, BA, BB, BG, BR, BZ, CA, CN, CR, CU, CZ, DM, DZ, EE, GD, E, HR, HU, ID, IL, IN, IS, JP, KP, KR, LC, LK, LR, LT, LV, MA, MG, MK, MN, MX, MZ, NO, NZ, PL, RO , SG, SI, SK, SL, TR, TT, UA, US, UZ, VN, YU, ZA (72) Inventor Viad Ribbon, Michigan, USA 48105. Unover. Timber Crest Court 2394 (72) Inventor Alan Robert Saartir 48105, Michigan, United States. Unover. Valley View Drive 2002 F-term (reference) 4B024 AA01 CA04 DA02 EA04 FA02 GA11 4B063 QA18 QQ08 QQ43 QR32 QR55 QS25 QS34 4B065 AA90X AA99Y AB01 BA02 CA44

Claims (8)

[Claims] 1. An isolated and purified DNA sequence which is substantially similar to the DNA sequence shown in SEQ ID NO: 1 or 2. 2. An isolated and purified DNA sequence which hybridizes to the DNA sequence set forth in SEQ ID NO: 1 or 2 under high stringency hybridization conditions. 3. An isolated and purified DNA sequence consisting essentially of the DNA sequence set forth in SEQ ID NO: 1 or 2. 4. A recombinant DNA molecule consisting of the isolated and purified DNA sequence according to any one of claims 1 to 3, subcloned into an extrachromosomal vector. 5. A recombinant host cell comprising a host cell transfected with the recombinant DNA molecule of claim 4. 6. In a diagnostic assay for detecting cells containing a mutation in the CAP promoter, total genomic DNA from the cells is isolated and the genomic DNA is isolated from any of the claims 1-3. An assay that is subjected to PCR amplification using primers derived from the isolated and purified DNA sequence to determine whether the resulting PCR product contains a mutation. 7. A diagnostic assay for detecting or screening a therapeutic compound that interferes with or enhances the interaction between the CAP promoter and PPARγ or RXR or other transcription factors that bind to the promoter, comprising: An assay comprising measuring the interaction between a promoter and PPARγ or RXR or other molecule that binds to the promoter in the presence of at least one other therapeutic compound. 8. Detecting the interaction of a polynucleotide sequence encoding the CAP promoter with a protein in mammalian cells in a diagnostic assay for the discovery of proteins that interact directly or indirectly with the CAP promoter. An assay comprising the steps of: