EP1232261A2

EP1232261A2 - Cloning and characterization of the cap gene promoter

Info

Publication number: EP1232261A2
Application number: EP00973600A
Authority: EP
Inventors: Christian A. Baumann; Vered Ribon; Alan Robert Saltiel
Original assignee: Warner Lambert Co LLC
Current assignee: Warner Lambert Co LLC
Priority date: 1999-11-12
Filing date: 2000-10-17
Publication date: 2002-08-21
Also published as: WO2001036619A3; WO2001036619A2; BR0015542A; CA2385090A1; AU1209601A; TR200201302T2; JP2003517302A

Abstract

The present invention relates to novel polynucleotides that contain peroxisome proliferator response elements (PPREs). The invention also describes vectors and host cells comprising the novel polynucleotides. The invention further describes methods for using the novel polynucleotides, gene therapy applications, diagnostics for syndromes involving insulin resistance, development of proprietary screening strategies for inhibitors of ligands or proteins that bind to, associate with, or interact with the CAP promoter.

Description

CLONING AND CHARACTERIZATION OF THE CAP GENE PROMOTER

BACKGROUND OF THE INVENTION

The present invention relates to novel polynucleotides that encompass the promoter region of the CAP gene containing peroxisome prohferator response elements (PPREs). The invention also describes vectors and host cells comprising the novel polynucleotides. The invention further describes methods for using the novel polynucleotides, in the detection of genetic deletions of the polynucleotides, subcellular localization of the polynucleotides, diagnostics for syndromes involving abnormal activity (including the presence or absence of) of elements within the promoter, development of proprietary screening strategies for assessing regulation/binding of ligands or proteins to the promoter.

SUMMARY OF THE RELATED ART

Although the tyrosine kinase activity of the insulin receptor is essential for the full expression of insulin action, the precise role of its different cellular substrates remains uncertain. Insulin stimulates the tyrosine phosphorylation of the c-Cbl proto-oncogene product ( Ribon, V., and Saltiel, A. R. (1997) Biochem J 324(Pt 3), 839-45). This phosphorylation requires the expression of a novel protein called CAP, which recruits c-Cbl to the insulin receptor (Ribon, V., Printen, J. A., Hoffman, N. G., Kay, B. K., and Saltiel, A. R. (1998) Mol Cell Biol 18(2), 872-9). CAP is a multifunctional protein with three adjacent SH3 domains in the C-terminus and a sorbin homology domain in the N-terminus. CAP associates with both c-Cbl and the insulin receptor in the basal state. Insulin stimulation causes the disassociation of CAP from the insulin receptor. However, CAP remains associated with c-Cbl after insulin stimulation. Additionally, overexpression of CAP causes the formation of focal adhesions and stress fibers due to its association with pl25FAK and actin stress fibers (Ribon V., Herrera R., Kay B.K., and Saltiel A.R. (1998) J Biol Chem 273(7), 4073-80). While the role of CAP in insulin action has not been definitively proven, several lines of evidence suggest an important function. It is expressed predominantly in insulin sensitive tissues. In the 3T3-L1 adipocyte cell line, its CAP expression correlates well with insulin sensitivity. Moreover, stimulation of the nuclear receptor PPARγ with thiazolidinediones (TZDs) in 3T3-L1 adipocytes or in diabetic rodents, leads to increased CAP expression and increased insulin- stimulated c-Cbl phosphorylation (Ribon V., Johnson J.H., Camp H.S., and Saltiel A.R. (1998) Proc Natl Acad Sci USA 95(25), 14751-6). The effects of TZDs on CAP expression is a direct result of increased transcription of the CAP gene. This TZD-induced increase expression of CAP correlates well with increased insulin sensitivity both in vitro and in vivo.

Despite the growing body of evidence suggesting an important function, the role of CAP in insulin action has not been definitively proven. It would be beneficial if such a role were validated. By gaining an understanding of the biochemical mechanisms behind these insulin-stimulated reactions, new opportunities for treating and diagnosing diseases related to abnormal (high or low) storage and/or utilization of insulin or glucose, may be achieved. Stated another way, a better understanding of the molecular mechanisms of insulin action will allow improved design of therapeutic drugs that treat diseases related to abnormal storage and/or utilization of insulin or glucose. Such disease states include diabetes, glycogen storage diseases, obesity, polycystic ovarian syndrome, hypertension, atherosclerosis and other diseases of insulin-resistance.

SUMMARY OF THE INVENTION

The invention relates to the discovery of the promoter of the CAP gene, and the identification of a novel target peroxisome proliferator-response element

(PPRE) and the isolation of polynucleotide sequences encoding the 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. Sequence analysis of 2.5 kB of the 5' flanking region of the CAP gene reveals a predicted peroxisome prohferator 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 (RXR α) caused an additional 2-fold stimulation of promoter activity. The TZD rosiglitizone produced an additional 2-3 fold stimulation of the promoter. Deletion of the predicted PPRE from the CAP promoter abolished its ability to respond to rosiglitizone. Gel shift analysis of the putative PPARγ site demonstrates direct binding of PPAR/RXR heterodimers to the PPRE in the CAP gene. These data demonstrate that TZDs directly stimulate transcription of the CAP gene through activation of PPARγ. The results provided herein demonstrate the first line of evidence linking the anti-diabetic effects of PPARγ activation to improvements in insulin signaling. The properties of this PPRE in the CAP promoter has profound physiological effects. Thus, by regulating the activity levels of the subject promoter, desirable physiological effects may be obtained. Such effects may be used to design or discover treatment for a variety of diseases involving abnormal levels of insulin or glucose (ie, disease states include, but are not limited to diabetes, glycogen storage diseases, obesity, polycystic ovarian syndrome, hypertension, atherosclerosis and other diseases of insulin-resistance).

One aspect of the invention is to polynucleotide sequences encoding the promoter of the invention (including elements within the promoter such as the

PPREs) and to provide polynucleotides complementary to the polynucleotide coding strand. The polynucleotides of the invention may be used in recombinant DNA technology (cloning, subcloning, etc.). The polynucleotides of the invention may also be used for in the detection of genetic deletions of the polynucleotide. The invention also provides polynucleotides for use as hybridization probes and amplification primers for the detection of naturally occurring polynucleotides encoding PPREs.

Another aspect of the invention is to provide assays for the detection or screening of therapeutic compounds that interfere with or increase the interaction between PPREs and PPARγ and/or RXR (or other transcription factors that bind to the CAP promoter). The assays of the invention comprise the step of measuring the effect of a compound of interest on binding between the PPREs and PPARγ or RXR (or other transcription factors that bind to any part of the promoter). Binding may be measured in a variety of ways, including the use of labeled PPREs labeled transcription factors, or reporter assays contains part or all of the CAP promoter. Another aspect of the invention is to provide assays for the discovery of proteins that interact directly or indirectly with the promoter. The assays of the invention comprise a method for detecting such interactions in cells, or in biochemical assays. These interactions may be detected in a variety of ways, including the use of the CAP cDNA.

The foregoing is not intended and should not be construed as limiting the invention in any way. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All U.S. patents and all publications mentioned herein are incorporated in their entirety by reference thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Structure of the 5' Flanking Region of the Mouse CAP Gene. A)

Schematic of the genomic organization and restriction map of the CAP promoter and 5' coding exons. The black represents the promoter. The hatched box is the location of the PPRE. The gray boxes represent exons and the open boxes represents intronic sequences. B) Sequence and putative transcription factor binding sites in the CAP promoter. Binding sites for Spl, AP-2, C/EBP and

PPAR are highlighted. The start of transcription, as determine by SI nuclease protection, is indicated by the arrow. The cDNA encoding exonl of the CAP gene (Genebank Ascesion #U58883). The single stranded probe used for the SI nuclease protection assays is italicized.

Figure 2. SI Nuclease Analysis of the Transcription Initiation Site of the CAP

Gene. Total RNA (40 μg) isolated from 3T3-L1 adipocytes or mouse fat tissue were hybridized with [γ-32P]- end labeled 70 mer probe overnight at 42°C. The anti-sense 70 mer probe contains a sequence complimentary to the first 40 bases of the 5'-end of the longest CAP cDNA clone, a sequence complimentary to the 20-base genomic sequences immediately upstream of the 5 '-end of the longest clone, and a 10-base nonspecific sequence (see Fig. 3B). The samples were then digested with 250 units of SI nuclease at 37oC for 60 min, followed by ethanol precipitation. The reaction products were subjected to polyacrylamide (8%)/urea (7M) gel elecrophoresis and visualized by autoradiography. Undigested probe was loaded on lane 1. The size of the protected fragment was estimated by comparing to a DNA sequencing ladder run on the same gel.

Figure 3. The CAP Promoter is Functional and TZD Sensitive in NIH3T3 Fibroblasts. A, Aschematic representation of the various CAP promoter reporter constructs cloned into the pGL3 basic luciferase reporter plasmid. B, NIH3T3 fibroblasts were co-transfected with CMV-PPARγ and CMV-RXRα or empty vector and the various CAP promoter constructs in the pGL3 luciferase reporter system. After transfection, the cells were incubated for 48 hours in the presence of absence 20 μM rosiglitazone. All luciferase measurements are normalized to β-Gal activity and are shown as mean + S.E. (n=3). Asterisks indicate statistical difference from control-trated reporter constricts ( * = p<0.005).

Figure 4. The CAP Promoter is functional and TZD Sensitive in 3T3-L1 Adipocytes. 3T3-L1 adipocytes were electroporated with various reporter constructs and luciferase activity, in the presence and absence of 20 μM rosiglitazone, was evaluated. All luciferase activity was normalized to β-galactosidase activity and are shown as mean + S.E. (n=3). Asterisks indicate statistical difference from control-trated reporter constricts ( * = p<0.005).

Figure 5. Gel Mobility Shift Assay. This figures shows a representative autoradio graph from a gel mobility shift assay. A, Sequences of Oligo nucleotides used in gel shift studies PPRE sequences are underlined. B, In vitro translated

PPARγ and RXRα were incubated to form heterodimers prior to the addition of the radiolabeled CAP PPRE probe. To control for specificity of binding, several samples were incubated in the presence of increasing (10 and 50 fold molar excess) concentrations of cold wtPPRE, mutPPRE or 50-fold molar excess of non-specific double stranded oligo nucleotide. B, Nuclear extracts from 3T3-L1 fibroblasts or 3T3-L1 adipocytes were incubated with radiolabeled CAP PPRE probe To control for specificity of binding, several samples were incubated in the presence of increasing (10 and 50 fold molar excess) concentrations of cold wtPPRE, mutPPRE or 50-fold molar excess of non-specific double stranded oligonucleotide.

DETAILED DESCRIPTION OF THE INVENTION

Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology,

Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis et al., 1990, Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2nd ed. (R.I. Freshney, 1987, Liss, Inc. New York,

NY), and Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, NJ).

In one aspect, the present invention provides novel isolated and purified polynucleotide sequences encoding a functional PPAR response element (hereinafter referred to as "PPRE") within the CAP promoter and/or a CAP promoter comprising the PPRE. The term "PPRE" is used broadly herein. Unless noted otherwise, the term "PPRE" include, but is not limited to, any natural mammalian-derived form of PPRE and the like. It is preferred that the term PPRE include primates and humans. Also, the term "interacting" is used broadly herein. Unless noted otherwise, the term "interacting" includes, but is not limited to, binding, affecting, and regulating.

The polynucleotides provided for may encode the complete CAP promoter (SEQ ID NO.l) or portions thereof such as the PPRE (SEQ ID NO. 2). The polynucleotides of the invention may be produced by a variety of methods including in v trø chemical synthesis using well-known solid phase synthesis technique, by cloning or combinations thereof. Persons of ordinary skill in the art are familiar with the degeneracy of the genetic code and may readily design polynucleotides that encode part or all of the promoter that have either partial or polynucleotide sequence homology to naturally occurring polynucleotide sequences encoding the promoter. The polynucleotides of the invention may be single stranded or double stranded. Polynucleotide complementary to polynucleotides encoding the promoter are also provided.

Libraries, either cDNA or genomic, are screened with probes designed to identify the gene of interest. For cDNA libraries, suitable probes include carefully selected oligonucleotide probes (usually of about 20-80 bases in length) that encode known or suspected portions of a PPRE from the same or different species, and/or complementary or homologous cDNAs or fragments thereof that encode the same or a similar gene, and/or homologous genomic DNAs or fragments thereof. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures as described in Chapters 10-12 of Sambrook et al., Molecular Cloning: A Laboratory Manual, New York, Cold Spring Harbor Laboratory Press, 1989).

The oligonucleotide must be labeled such that it can be detected upon hybridization to DNA in the library being screened. The preferred method of labeling is to use ATP (eg, T32P) and polynucleotide kinase to radiolabel the

5' end of the oligonucleotide. However, other methods may be used to label the oligonucleotide, including, but not limited to, biotinylation or enzyme labeling. DNA sequences containing the CAP promoter can also be identified and isolated by other known techniques of recombinant DNA technology, such as by direct expression cloning or by using the polymerase chain reaction (PCR) as described in US Patent No. 4,683,195, in Section 14 of Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York, 1989, or in Chapter 15 of Current Protocols in Molecular Biology, Ausubel et al., eds., Green Publishing Associates and Wiley-Interscience, 1991. This method requires the use of oligonucleotide probes that will hybridize to DNA encoding a PPRE. In a preferred embodiment, the invention comprises DNA sequences substantially similar to those shown in SEQ ID NO. 1 and 2 (mouse PPRE polynucleotides) As defined herein, "substantially similar" includes identical sequences, as well as deletions, substitutions or additions to a DNA. Preferably, the DNA sequences according to the mvention consist essentially of the DNA sequence of SEQ ID NO. 1 and 2. These novel purified and isolated DNA sequences can be used for mutational analysis of promoter function.

Mutated sequences according to the invention can be identified in a routine manner by those skilled in the art using the teachings provided herein, and techniques well-known in the art.

In a preferred embodiment, the present invention comprises a nucleotide sequence that hybridizes to the nucleotide sequence shown in SEQ ID NO. 1 and 2 under high stringency hybridization conditions. As used herein, the term "high stringency hybridization conditions" refers to hybridization at 65°C in a low salt hybridization buffer to the probe of interest at 2 x 10^ cpm/μg for between about

8 hours to 24 hours, followed by washing in 1% SDS, 20 mM phosphate buffer and 1 mM EDTA at 65°C, for between about 30 minutes to 4 hours. In a preferred embodiment, the low salt hybridization buffer comprises between, 0.5-10% SDS, and 0.05 M and 0.5 M sodium phosphate. In a most preferred embodiment, the low salt hybridization buffer comprises, 7% SDS, and 0.125 M sodium phosphate.

The polynucleotides of the invention have a variety of uses, some of which have been indicated or will be addressed in greater detail, infra. The particular uses for a given polynucleotide depend, in part, on the specific polynucleotide embodiment of interest. The polynucleotides of the invention may be used as hybridization probes to recover the promoter or a portion thereof from genetic libraries. The polynucleotides of the invention may also be used as primers for the amplification of the promoter or a portion thereof through the polymerase chain reaction (PCR) and other similar amplification procedures. The polynucleotides of the invention may also be used as probes and amplification primers to detect mutations in the promoter or a portion thereof that have been correlated with diseases, particularly diseases related to overexpression or underexpression of Hgands that bind to or associate or interact with the promoter. The invention also provides a variety of polynucleotide expression vectors, comprising the promoter or a portion thereof or a sequence substantially similar to it subcloned into an extra-chromosomal vector. As used herein, the term "extra- chromosomal vector" includes, but is not limited to, plasmids, bacteriophages, cosmids, retroviruses and artificial chromosomes. In a preferred embodiment, the extra-chromosomal vector comprises a vector that allows for PPRE cloning when the recombinant DNA molecule is inserted into a host cell. The vectors may comprise additional polynucleotide sequences for gene expression, regulation, or the convenient manipulation of the vector, such additional sequences include terminators, enhancers, selective markers, packaging sites, and the like. Detailed description of polynucleotide expression vectors and their use can be found in, among other places Gene Expression Technology: Methods in Enzymology, Volume 185, Goeddel, ed., Academic Press Inc., San Diego, CA (1991), Protein Expression in Animal Cells, Roth, ed., Academic Press, San Diego, CA (1994). In a further aspect, the present invention provides recombinant host cells that are stably transfected with a recombinant DNA molecule comprising the promoter subcloned into an extra-chromosomal 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., Cold Spring Harbor Press, 1989) and, as used herein, includes, but is not limited to calcium phosphate transfection, dextran sulfate transfection, electroporation, lipofection and viral infection..

The CAP promoter may be used to discover molecules that interfere with or increase its activity. For example, molecules that increase the binding of

PPARγ and or RXR to the CAP promoter in insulin responsive tissues, thus increasing insulin action. Additionally, the promoter may be used to find other proteins that can directly interact with it, representing additional important regulators of glucose transport. The PPRE of the present invention have the biological activity of associating with PPARγ and/or RXR. The PPRE of the invention may be isolated from a variety of mammalian animal species. Preferred mammalian species for isolation are primates and humans. Another aspect of the invention is to provide assays useful for determining if a compound of interest can bind to the promoter so as to interfere with or increase the binding of PPARγ and/or RX (or other ligands) to the promoter. The assay comprises the steps of measuring the binding of a compound of interest to a the promoter. Either the promoter or the compound of interest to be assayed may be labeled with a detectable label, eg, a radioactive or fluorescent label, so as to provide for the detection of complex formation between the compound of interest and the promoter. In another embodiment of the subject assays, the assays involve measuring the interference, ie, competitive binding, of a compound of interest with the binding interaction between the promoter and PPARγ and/or RX (or another ligand already known to bind to PPRE). For example, the effect of increasing quantities of a compound of interest on the formation of complexes between radioactivity labeled PPARγ and/or RX and the promoter may be measured by quantifying the formation of labeled ligand- PPRE complex formation.

In a further aspect, the present invention provides a diagnostic assay for detecting cells containing CAP promoter polynucleotide deletions, comprising isolating total genomic DNA from the cell and subjecting the genomic DNA to PCR amplification using primers derived from the DNA sequence of SEQ ID NO. 1 and 2.

This aspect of the invention enables the detection of CAP prompter polynucleotide deletions in any type of cell, and can be used in genetic testing or as a laboratory tool. The PCR primers can be chosen in any manner that allows the amplification of a promoter polynucleotide fragment large enough to be detected by gel electrophoresis. Detection can be by any method, including, but not limited to ethidium bromide staining of agarose or polyacrylamide gels, autoradiographic detection of radio-labeled promoter gene fragments, Southern blot hybridization, and DNA sequence analysis. In a preferred embodiment, detection is accomplished by polyacrylamide gel electrophoresis, followed by DNA sequence analysis to verify the identity of the deletions. PCR conditions are routinely determined based on the length and base-content of the primers selected according to techniques well-known in the art (Sambrook et al., 1989). An additional aspect of the present invention provides a diagnostic assay for detecting cells containing promoter polynucleotide deletions, comprising isolating total cell RNA and subjecting the RNA to reverse transcription-PCR amplification using primers derived from the DNA sequence of SEQ ID NO. 1 and 2. This aspect of the invention enables the detection of promoter deletions in any type of cell, and can be used in genetic testing or as a laboratory tool.

Reverse transcription is routinely accomplished via standards techniques (Ausubel et al, in Current Protocols in Molecular Biology, ed. John Wiley and Sons, Inc., 1994) and PCR is accomplished as described above. The present invention may be better understood with reference to the accompanying examples that are intended for purposes of illustration only and should not be construed to limit the scope of the invention, as defined by the claims appended hereto.

EXAMPLES Example 1

Materials- Cell culture reagents were purchase from GIBCO/BRL(Gaithersburg, MD). BRL 49653 (rosiglitizone) is commercially available.

Cloning of CAP Promoter and Reporter Fusion Constructs - The Bacterial artificial chromosome (BAC) library at Research Genetics was screened with the 5'end of the coding region of the CAP gene. The BAC clone was restriction digested with Hindlll or Sad and subject to sequential southern analysis with a probes corresponding to the 5"UTR and the promoter sequence of the CAP gene. The fragments were subcloned and assembled in both pBluescript (Strategene) and pGL3 basic (Promega). All sequence analysis of the promoter region and transcription factor binding sites was done with Signal Scan software (University of Minnesota). Plasmid constructs were generated by digestion using restriction sites within the CAP promoter. pCPH was generated by cloning a 550 bp Hindlll and Smal fragment of the CAP promoter into pGL3. pCPS generate by insertion of a 1070 bp Sad and Smal fragment into pGL3. pCPE was generated by insertion of the 2.6kb EcoRI/Smal fragment of the CAP promoter into pGL3. pCPEΔPPRE was generated by digestion of pCPE with Sad and removal of a 435bp fragment that contains the PPRE of the CAP promoter and ligation of the pCPE vector.

S7 Nuclease Assay- Total RNA (40 μg) isolated from 3T3-L1 adipocytes or mouse fat tissue was hybridized with [γ P]- end labeled 70 mer probe overnight at 42°C. The anti-sense 70 mer probe contains a sequence complimentary to the first 40 bases of the 5'-end of the longest CAP cDNA clone, a sequence complimentary to the 20-base genomic sequences immediately upstream of the 5'-end of the longest clone, and a 10-base nonspecific sequence (see Fig. 3B). The samples were then digested with 250 units of SI nuclease at 37°C for 60 min, followed by ethanol precipitation. The reaction products were subjected to polyacrylamide (8%)/urea (7M) gel elecrophoresis and visualized by autoradiography. Undigested probe was loaded on lane 1. The size of the protected fragment was estimated by comparing to a DNA sequencing ladder run on the same gel.

Cell Transfections and Reporter Assays- NIH3T3 and 3T3-L1 fibroblasts were maintained in DMEM, 10% calf serum. Transfections of NIH3T3 fibroblasts were done by lipofectAMINE reagent according to the manufacturers instructions (GibcoBRL). 250 ng of pGL3 basic firefly luciferase constructs (Promega) were co-transfected with 50 ng of pCMV-βGAL and/or CMV-PPARγ and CMV-

RXRα. PPARγi and RXRα were cloned into the pSG5 expression plasmid as previously described (Camp H.S. and 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 rosiglitizone. 3T3-L1 adipocytes were grown to confluence and differentiated as previously described(Lazar D.F., Wiese R.J.,

Brady M.J., Mastick C.C., Waters S.B., Yamauchi K., Pessin J.E., Cuatrecasas P., and Saltiel A.R. (1995) J Biol Chem 270(35), 20801-7). Adipocytes (day 8 post differentiation) were electroporated using a protocol previously reported by Thurmond et al. (Thurmond D.C., Ceresa B.P., Okada S., Elmendorf J.S., Coker K., and Pessin J.E. (1998) J Biol Chem 273(50), 33876-83). Briefly, adipocytes were trypsinized, washed and resuspended in PBS. 10⁷ adipocytes MISSING UPON TIME OF PUBLICATION

B. To determine the start of transcription of the CAP gene, a 70 nt single stranded DNA probe was generated that overlapped a region containing the longest known CAP cDNA sequence. The probe was hybridized with 3T3-L1 adipocyte or mouse fat tissue mRNA, and digested with SI nuclease to identified the start of transcription (figure IB, 2). The presence of one predominant transcript was confirmed by RT-PCR. The start of translation of the CAP gene was found to lie within exon 4 of the CAP gene (data not shown). A putative promoter sequence was identified by analysis of 2.6 kB of the 5'flanking region of the CAP gene using Signal Scan software (University of Minnesota). Although no TATA -box was found within the promoter, it does possess the characteristics of a TATA-less promoter (Parks C.L. and Shenk T. (1996) J Biol Chem 271(Dignam J.D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res 11(5), 1475-89), 4417-30, Blake M.C., Jambou R.C., Swick A.G., Kahn J.W., and Azizkhan J.C. (1990) Mol Cell Biol 10(12), 6632-41). The basal promoter region is GC rich and contains multiple Sp-1 binding sites. Additionally, a CAAT box was identified as well as several AP-2 and C/EBP sites. The putative PPRE was found at -1107- 1094 in the CAP promoter (SEQ ID NO. 2) (figure IB). The CAP promoter PPRE has a sequence of the direct repeats of a hexamer (DR1) type similar to PPRE's from other genes, as shown in Table I (Johnson E.F., Palmer C.N., Griffin K.J., and Hsu M.H. (1996) Faseb J 10(11), 1241-8; Frohnert B.I.,

Hui T.Y., and Bernlohr D.A. (1999) J Biol Chem 274(7), 3970-7).

TABLE I Comparison of Identified PPRE Sequences (1 1 ,12)

Gene Species Element Sequence Protein Function

Acyl CoA oxidase Rat ACOA AGGACA A AGGTCA Peroxisomal β-oxidation

ACOB AGGTAC A AGGTCA

Acyl-CoA synthase Rat ACS(CI) AGGGCA TCAGTCA Peroxisomal β-oxidation ALBP/aP2 Mouse ARE6 GGGTGA A ATGTGC Fatty acid binding protein

ARE7 GGATCA GAGTTCA

Apolipoprotein Oil Human APOCIIIB TGGGCAA AGGTCA Triglyceride clearance

Bifunctional Enzyme Rat BIF AGGTCC TAGTTCA Peroxisomal β-oxidation

Cytochrome P450 Al Rat CYP4A1 AGGGTA A AGTTCA ω-Oxidation

Cytochrome P450 A6 Rabbit CYP4A6 AGGGCA AAGTTGA ω-Oxidation

L-FABP Rat FABP AGGCCA TAGGTCA Fatty acid binding

Fatty acid transport protein Mouse FATP GGGGCAAAGGGCA Fatty acid transport

HMG-CoA synthase Rat HMG GGGCCA A AGGTCT Liver ketogenesis/sterol synthesis

Lipoprotein lipase Rat LPL GGGGGA A AGGGCA Triglyceride clearance

Malic enzyme Rat MEp GGGTCA A AGTTGA Fatty acid synthesis

Muscle-type carnitine palmitoyltransferase Human MCPT I AGGGAAA AGGTCA Fatty acid transport

PEPCK Rat PCK1 CGGCCAAAGGTCA Glycerogenesis and gluconeogenesis

PCK2 GGGTGA A ATGTGC

Uncoupling protein I Mouse URE1 TGGTCA A GGGTGA Thermogenesis c-Cbl associating protein Mouse CAPRE AGGCTA A AGGTCA Insulin signal transduction

Consensus AGGTCAAAGGTCA

Identification of a Functional PPRE in the CAP Gene- To determine the functionality of the CAP promoter as well as its putative PPRE, transient reporter assays using various CAP promoter constructs (figure 3A) were performed in NIH3T3 fibroblasts. Luciferase fusion constructs were prepared by subcloning restriction fragments from the cloned CAP promoter into the pGL3 basic luciferase reporter vector as described in "experimental procedures". Results obtained from reporter assays in NIH3T3 fibroblasts showed that the CAP promoter was functional (figure 3B). Fusion of the various CAP promoter constructs to the luciferase reporter produced a greater than 20-fold increase in luciferase activity, compared to the pGL3 basic vector alone, for all CAP promoter constructs tested (figure 3B). Co-transfection of PPARγ and RXRα with the fusion construct pCPE, containing the putative CAP PPRE, led to an additional fold stimulation of luciferase activity. Addition of rosiglitazone led to an additional 2-3 fold stimulation in luciferase activity from the pCPE fusion construct. In contrast, the addition PPARγ and RXRα, with or without rosiglitazone, was ineffective in increasing the luciferase activity of the shorter pCPH and pCPS fusion constructs. Additionally, deletion of the a region containing the PPRE of CAP promoter (fusion construct pCPEΔPPRE) abolished the response to rosiglitazone. In order to further characterize the activity of the CAP promoter and its

PPRE, fusion constructs were electroporated into 3T3-L1 adipocytes. 3T3-L1 adipocytes contain high levels of endogenous PPARγ. Moreover, CAP expression in 3T3-L1 adipocytes is responsive to TZDs. Electroporated CAP reporter constructs pCPH and pCPS exhibited a 10-fold higher level of luciferase activity compared to pGL3 basic (figure 4). The pCPE construct containing the 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 the region of the CAP promoter containing the PPRE (pCPEΔPPRE) abolished both the increase in basal luciferase activity and the response to rosiglitazone. Gel Shift Analysis ofPPARJRXR Heterodimer Binding to the CAP PPRE - To demonstrate direct binding of PPARγ/RXRα heterodimers to the CAP PPRE, gel shift analysis with the CAP PPRE was performed (figure5A). A double-stranded oligonucleotide probe for wtCAP PPRE was P end-labeled and incubated with in vitro translated proteins as well as 3T3-L1 nuclear extracts. As shown in figure

5B, neither PPARγ or RXRα alone bound to the CAP PPRE oligonucleotide. However, PPARγ/RXRα heterodimers bind to the wtCAP PPRE oligonucleotide. This binding is specific, since it could be competed with unlabeled wtCAP PPRE oligonucleotide. Incubation with either mutCAP PPRE (an oligonucleotide with a single base deletion in the DR1 motif) or non-specific double-stranded oligonucleotide did not displace the labeled wtCAP PPRE oligonucleotide. Furthermore, nuclear proteins from 3T3-L1 adipocytes were able to form protein- DNA complexes with the wtCAP PPRE with specificity similar to the experiments done with the in vitro translated PPARγ/RXRα heterodimers (figure 5C). As described above, unlabeled wtCAPPPRE competed for DNA-protein complexes that were formed with nuclear proteins. MutCAP PPRE and nonspecific double-stranded oligonucleotide probes did not compete with the radiolabeled DNA-protein interaction.

It is to be understood that the invention is not to be limited to the exact details of operation, or to the exact compounds, compositions, methods, procedures, or embodiments shown and described, as obvious modifications and equivalents will be apparent to one skilled in the art, and the invention is therefore to be limited only by the full scope of the appended claims.

Claims

CLAIMSWhat is claimed is:

1. An isolated and purified DNA sequence substantially similar to the DNA sequence shown in SEQ ID NOS 1 or 2.

2. An isolated and purified DNA sequence that hybridizes to the DNA sequence shown in SEQ ID NOS 1 or 2 under high stringency hybridization conditions.

3. An isolated and purified DNA sequence that consists essentially of the DNA sequence shown in SEQ ID NOS 1 or 2.

4. A recombinant DNA molecule comprising the isolated and purified DNA sequence of Claim 1 , 2, or 3 subcloned into an extra-chromosomal vector.

5. A recombinant host cell comprising a host cell transfected with the recombinant DNA molecule of Claim 4.

6. A diagnostic assay for detecting cells containing mutations with the CAP promoter, comprising isolating total genomic DNA from the cell and subjecting the genomic DNA to PCR amplification using primers derived from the isolated and purified DNA sequence of Claim 1 , 2, or 3 and determining whether the resulting PCR product contains a mutation.

7. A diagnostic assay for detecting or screening of therapeutic compounds that interfere with or increase the interaction between the CAP promoter and PPARγ or RXR or other transcription factors that bind to the promoter, comprising the step of measuring the interaction between the promoter and PPARγ or RXR or other molecules that bind to the promoter, while in the presence of at least one other therapeutic compound. A diagnostic assay for the discovery of proteins that interact directly or indirectly with the CAP promoter, comprising the step of detecting the interaction of a polynucleotide sequence encoding the CAP promoter with proteins in mammalian cells.