Assay for the detection of factors that modulate the expression of INGAP

Abstract

A reporter construct contains mammalian INGAP 5′-regulatory region or a fragment thereof, a minimal promoter element from mammalian INGAP or a heterologous promoter, and a reporter gene. The reporter construct can be used to screen for agents which alone or in combination up-regulate or down-regulate reporter gene expression. Alternatively, the reporter construct can be used to screen for agents that bind to the hamster INGAP 5′-regulatory region or a fragment thereof.

Description

FIELD OF THE INVENTION

[0002] The invention relates to the field of assays for the detection of factors that modulate gene expression. Specifically, the invention relates to reporter constructs and methods for identifying agents that modulate the expression of the INGAP gene.

BACKGROUND OF THE INVENTION

[0003] Islet neogenesis gene associated protein (INGAP protein) has been identified as a pancreatic acinar cell protein that can induce islet cell neogenesis from progenitor cells resident in the pancreas in a manner that recapitulates islet development during normal embryogenesis. INGAP is unique in its ability to stimulate growth and differentiation of islets of Langerhans from precursor cells associated with pancreas. These islets evolve a mature insulin secretory profile capable of responding to perturbations in blood glucose in a physiologic manner. This potential anti-diabetic therapeutic has been shown to demonstrate homology across several species and to exert a biological response.

[0004] Pancreatic islet cell mass is lost in type 1 diabetes mellitus, a disease in which a progressive autoimmune reaction results in the selective destruction of insulin-producing β-cells. In type 2 diabetes mellitus, so-called adult-onset disease, but also increasingly a condition in young overweight people, the β-cell mass may be reduced by as much as 60% of normal. The number of functioning β-cells in the pancreas is of critical significance for the development, course, and outcome of diabetes. In type I diabetes, there is a reduction of β-cell mass to less than 2% of normal. Even in the face of severe insulin resistance as occurs in type II diabetes, the development of diabetes only occurs if there is inadequate compensatory increase in β-cell mass. Thus, the development of either of the major forms of diabetes can be regarded as a failure of adaptive β-cell growth and a subsequent deficiency in insulin secretion. Stimulating the growth of islets and β-cells from precursor cells, known as islet neogenesis, is an attractive approach to the amelioration of diabetes. There is need in the art for methods to identify agents that can modulate the expression of INGAP, whether in animals or in cultured cells.

BRIEF SUMMARY OF THE INVENTION

[0005] It is an object of the invention to provide a reporter construct containing the 5′-regulatory region from mammalian INGAP gene.

[0006] It is another object of the invention to provide methods for identifying agents which modulate INGAP expression.

[0007] It is another object of the invention to provide a nucleic acid or fragment of INGAP 5′-regulatory region.

[0008] It is another object of the invention to provide methods for increasing INGAP expression.

[0009] It is another object of the invention to provide a kit for modulating INGAP expression.

[0010] These and other objects of the invention are provided by one or more of the embodiments described below.

[0011] In one aspect of the invention a reporter construct is provided. The reporter construct comprises a regulatory region nucleotide sequence and a nucleotide sequence encoding a detectable product. In one aspect of the invention, the reporter construct is provided in a vector. The regulatory region nucleotide sequence is linked to the nucleotide sequence encoding a detectable product. The regulatory region nucleotide sequence may comprise one or more fragments of 5′ regulatory region of the INGAP genomic sequence, SEQ ID NO:23, or it may comprise the entire length of the 5′ regulatory region. In one embodiment of the reporter construct, a promoter element is interposed between the regulatory region nucleotide sequence and the nucleotide sequence encoding a detectable product. The promoter element may be selected from the promoter elements present in the INGAP regulatory sequence. Alternatively, the promoter element present in the vector comprising the reporter construct may be used. The detectable product encoded by the said nucleotide sequence encoding a detectable product could be either a nucleic acid or a protein. The detectable product need not be the INGAP gene nucleic acid or protein.

[0012] In another embodiment of the invention, a method identifying agents that modulate INGAP expression is provided. The method comprises contacting a cell with a test agent, wherein the cell comprises a reporter construct of the present invention. Expression of the detectable nucleic acid or protein product in the cell is determined. A test agent is identified as a modulator of INGAP expression if the test agent modulates expression of the detectable product in the cell.

[0013] In another embodiment of the invention, an isolated nucleic acid comprising the genomic sequence of the hamster INGAP gene (SEQ ID NO: 2), or a fragment thereof is provided.

[0014] According to another embodiment of the invention, an in vitro method for identifying agents that modulate INGAP expression is provided. The method comprises contacting a test agent with a reporter construct of the present invention in a cell-free system that allows for transcription and translation of a nucleotide sequence. Expression of the detectable product is determined. The substance is identified as a modulator of INGAP expression if the test substance modulates expression of the detectable product.

[0015] According to another embodiment of the invention, an in vitro method for identifying an agent that modulate INGAP expression is provided. The method comprises contacting a test agent with a nucleic acid of the invention. Binding of the test agent to the nucleic acid is determined. The test agent is identified as a modulator of INGAP expression if the test agent binds to the nucleic acid.

[0016] According to another embodiment of the invention a method for increasing INGAP expression is provided. An effective amount of a factor that stimulates INGAP expression directly or indirectly, for example cytokines, chemokines, growth factors, or pharmacological agents, is administered to a mammal in need of increased INGAP expression.

[0017] According to another embodiment of the invention a kit for modulating INGAP expression is provided. The kit comprises a modulator of INGAP expression and instructions for using the modulator of INGAP expression to modulate INGAP expression.

[0018] According to another embodiment of the invention a method for modulating INGAP expression in a mammal to treat a disease state related to reduced islet cell function is provided. The method comprises the step of administering to the mammal an effective amount of a modulator of INGAP expression whereby the level of INGAP expression in the mammal is modified.

[0019] All documents cited are, in relevant part, incorporated herein by reference; the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 shows the annotation of the hamster INGAP gene structure. The boundaries of introns 1-5 are listed in Table 1.

[0021] FIG. 2 shows an overview of the 5′-regulatory region of the hamster INGAP gene (nucleotides 1-3137 of SEQ ID NO: 2) showing many well known and well-characterized transcription factor binding sites. The minimal promoter element contains the regions noted with an underline (CAAT-box, TATA-box, and GC-box).

[0022] FIG. 3 shows a schematic of many well known and well-characterized transcription factor-binding sites for nucleotides 1-3123 of the 5′-regulatory region (SEQ ID NO: 1) of the hamster INGAP gene. Table 3 further describes these transcription factor-binding sites.

[0023] FIG. 4 shows the predicted transcription start sites within the 5′-regulatory region (SEQ ID NO: 1) of the hamster INGAP gene (SEQ ID NO: 2). The predicted start site is indicated by a boldface nucleotide. The start and end nucleotide numbers are indicated for the promoter sequence. The numbers refer to nucleotide numbers of the hamster INGAP gene (SEQ ID NO: 2)

[0024] FIG. 5 shows the adapter primer structure and sequence used in gene walking. Adapter primer 1 (API) and adapter primer 2 (AP2) are shown.

[0025] FIGS. 6 and 7 show the strategy for reconstructing the hamster INGAP gene. The hamster INGAP gene was reconstructed using the technique of gene walking. Shown are the fragments and the gene specific primers (GSP1 and GSP2) used in PCR amplification for gene walking. Fragments were joined together using unique restriction enzyme sites within each fragment. The nucleotide sequences of the individual primers are listed in Table 2.

[0026] FIG. 8 shows the fragments of INGAP 5′-regulatory region, which were cloned into pβGal-basic upstream of a P-galactosidase reporter gene. The labels on the left refer to the nucleotide fragments of SEQ ID NO: 23 which were cloned upstream of pβGal-basic.

[0027] FIG. 9A shows reporter activity in human embryonic kidney cells (293T) transfected with a reporter construct that contains various fragments of the 5′-regulatory region (SEQ ID NO: 23) of hamster INGAP DNA cloned upstream of a β-galactosidase reporter gene (pβGal-basic), or in a reporter construct which contains no INGAP DNA. The cells are stimulated with phorbol myristate acetate. Promoter activity is assessed by determining the level of β-galactosidase present in the cell using a β-galactosidase luminescent assay.

[0028] FIG. 9B shows reporter activity in human embryonic kidney cells (293T) transfected with a reporter construct that contains nucleotides 2030 to 3137 of the 5′-regulatory region (SEQ ID NO: 23) of hamster INGAP cloned upstream of a β-galactosidase reporter gene, or in a reporter construct which contains no INGAP DNA. The cells are stimulated with leukemia inhibitory factor. Promoter activity is assessed by determining the level of P-galactosidase present in the cell using a β-galactosidase luminescent assay.

[0029] FIG. 10 shows the reporter activity in human embryonic kidney cells (293T) transfected with a reporter construct that contains different fragments (see FIG. 8) of the 5′-regulatory region of hamster INGAP cloned upstream of a β-galactosidase reporter gene. The cells are stimulated with phorbol myristate acetate. Concentrations of PMA used are 6 ng/ml, 17 ng/ml, 50 ng/ml, 100 ng/ml, or 300 ng/ml. Promoter activity is assessed by determining the level of β-galactosidase present in the cell using a β-galactosidase luminescent assay.

[0030] FIG. 11 shows reporter activity in human embryonic kidney cells (293T) transfected with a reporter construct that contains different fragments (see FIG. 8) of the 5′-regulatory region of hamster INGAP cloned upstream of a β-galactosidase reporter gene. The cells are stimulated with human leukemia inhibitory factor (hLIF). Concentrations of hLIF used are 1 ng/ml, 10 ng/ml, or 30 ng/ml. Promoter activity was assessed by determining the level of β-galactosidase present in the cell using a galactosidase luminescent assay.

[0031] FIG. 12 shows RNA analysis for INGAP gene upregulation in rat amphicrine pancreatic cells, AR42J, treated with cytokine IL-6 or untreated. Total RNA is probed by Northern analysis for INGAP gene.

DETAILED DESCRIPTION OF THE INVENTION

[0032] Definitions

[0033] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.

[0034] The term “promoter” is used to define the region of a gene at which initiation and rate of transcription are controlled. It contains the site at which RNA polymerase binds and also sites for the binding of regulatory proteins, e.g. transcription factors, repressors, etc. In order to differentiate between the transcription initiation site and other sites that modulate rate of transcription, promoter region is generally subdivided into “minimal promoter element” and “regulatory region”. The term “minimal promoter element” or sometimes simply referred to as “promoter” therefore may include TATA box, GC-rich sequence and CAAT box; while “regulatory region” is usually a long stretch of nucleotide sequence where transcription factors and other factors bind. Most eukaryotic genes have long regulatory regions where many different transcription factors bind. The expression or the lack of expression of a given gene in a given cell type, tissue, organ, or an organism is governed by the interactions that take place on its regulatory region.

[0035] The term “transcription factor” is used to describe the proteins that bind short stretches of DNA in the regulatory regions of a gene. Transcription factors may interact with each other as well as RNA polymerase. Thus, transcription factors may bind hormones or second messengers, DNA, RNA, other transcription factors, or other proteins. They may activate or inhibit transcription of a given gene. Transcription factors are also sometimes referred to as “enhancers” or “repressors”. Transcription factor binding sites can be used to identify agents that bind to the 5′-regulatory region of the gene and modulate the gene's expression.

[0036] The term “reporter” is used to describe a coding sequence attached to a heterologous promoter or enhancer elements and whose product, either nucleic acid or protein, is easly detected and is quantifiable. Some common reporter genes include βgalactosidase (lacZ), chloramphenicol acetyltransferase (cat), β-glucuronidase (GUS), and green fluorescent protein (GFP).

[0037] A “reporter construct” is a piece of nucleic acid that includes a promoter element and a reporter gene housed in a suitable vector plasmid DNA. Regulatory region nucleotide sequences may be cloned 5′ of the promoter element to determine if they contain transcription factor binding sites. The reporter construct-containing vector is introduced into a cell that contains many transcription factors. Activation of the reporter gene by transcription factors may be monitored by detection and quantification of the product of the reporter gene.

[0038] The term “agent” is used here to essentially describe any means to modulate INGAP expression. Agent may be a chemical compound, a biological agent, or a physical force, a mechanical contraption, or any combinations thereof.

[0039] INGAP Promoter and Regulatory Region

[0040] It is a discovery of the present inventors that INGAP gene is regulated by a 5′-regulatory region that is susceptible to modulation by many known transcription factors, including PMA and LIF.

[0041] It is a further discovery of the present invention that the 5′-regulatory region nucleotide sequence of the INGAP gene may be used in screening assays to identify agents capable of modulating the INGAP gene expression. These modulating agents have potential as therapeutic agents for treating pathological conditions including, but not limited to, diabetes mellitus, both type 1 and type 2, endocrine and non-endocrine hypoplasia, hypertrophy, adenoma, neoplasia, and nesidioblastosis.

[0042] Mammalian INGAP, like most genes, has a 5′-regulatory region followed by introns and exons. The sequence of a mammalian (Hamster sp.) INGAP gene is provided as SEQ ID NO: 2. FIG. 1 details the relative location of the 5′-regulatory region, the introns and the exons of the hamster INGAP gene. The boundaries of introns 1-5 and the location of the TATA-box and the poly-A signal are listed in Table 1.

TABLE 1
Description   Position In INGAP Gene (SEQ ID NO: 2)
TATA-Box      3094
INTRON 1      3150-3426
INTRON 2      3508-4442
INTRON 3      4562-4735
INTRON 4      4874-5459
INTRON 5      5587-5843
Poly-A Signal 6098-6103

[0043] The nucleotide sequence of the 5′-regulatory region including the promoter elements of mammalian INGAP, is shown partially in SEQ ID NO: 1, and completely in SEQ ID NO: 2 and 23 (nucleotides 1-3137 of SEQ ID NO: 2). Nucleotides 1-3120 of SEQ ID NO: 1 are identical to nucleotides 1-3120 of SEQ ID NO: 2 and SEQ ID NO: 23. An overview of the 5′-regulatory region is shown in FIG. 2. Representative transcription enhancer/repressor binding sites are shown also in FIG. 2. Predicted transcription enhancer/repressor binding sites for nucleotides 1-3123 of the 5′-regulatory region are shown in FIG. 3. Table 3 at the end of the specification details these transcription factors and their binding sites, and their locations in the regulatory region. Potential transcription factor binding analysis was done using MatInspector professional™, which is a bioinformatics software that utilizes a library of matrix descriptions for transcription factor binding sites to locate matches in sequences of unlimited length (Quandt, K., Frech, K., Karas, H., Wingender, E., Werner, T. (1995) Nucleic Acids Res. 23, 4878-4884).

[0044] Table 3 lists predicted binding proteins (Further Information) based upon their classification into functionally similar matrix families (Family/matrix). The DNA sequence predicted to bind the protein (Sequence), whether sense or antisense DNA (Str) and location of the sequence in SEQ ID NO: 2, (Position) are listed. Further the similarity to the consecutive highest conserved nucleotides of a matrix (Core sim.) and similarity to all nucleotides in that matrix (Matrix sim.) along with the optimized value (Opt) defined in a way that a minimum number of matches is found in non-regulatory test sequences are also listed. Details to the algorithms used in MatInspector professional™ is referenced:

[0045] OPT: This matrix similarity is the optimized value defined in a way that a minimum number of matches are found in non-regulatory test sequences (i.e. with this matrix similarity the number of false positive matches is minimized). This matrix similarity is used when the user checks “Optimized” as the matrix similarity threshold for MatInspector professional™.

[0046] Family: Each matrix belongs to a so-called matrix family, where functionally similar matrices are grouped together, eliminating redundant matches by MatInspector professional™ professional (if the family option was selected). E.g. the matrix family V$NFKB includes 5 similar matrices for NFkappaB (V$NFKAPPAB.01, V$NFKAPPAB.02, V$NFKAPPAB.03, V$NFKAPPAB50.01, V$NFKAPPAB65.01) as well as 1 matrix for the NFkappaB related factor c-Rel (V$CREL.01).

[0047] Matrix: The MatInspector professional™ matrices have an identifier that indicates one of the following seven groups: vertebrates (V$), insects (I$), plants (P$), fungi (F$), nematodes (N$), bacteria (B$), and other functional elements (O$); followed by an acronym for the factor the matrix refers to, and a consecutive number discriminating between different matrices for the same factor. Thus, V$OCT1.02 indicates the second matrix for vertebral Oct-1 factor.

[0048] Core Sim: The “core sequence” of a matrix is defined as the (usually 4) consecutive highest conserved positions of the matrix. The core similarity is calculated as described here. The maximum core similarity of 1.0 is only reached when the highest conserved bases of a matrix match exactly in the sequence. More important than the core similarity is the matrix similarity which takes into account all bases over the whole matrix length.

[0049] Matrix Sim: The matrix similarity is calculated as described here. A perfect match to the matrix gets a score of 1.00 (each sequence position corresponds to the highest conserved nucleotide at that position in the matrix), a “good” match to the matrix usually has a similarity of >0.80. Mismatches in highly conserved positions of the matrix decrease the matrix similarity more than mismatches in less conserved regions.

[0050] Another aspect of the invention provides for a reporter construct. Reporter constructs contain a 5′ regulatory region nucleotide sequence fragment of SEQ ID NO: 23 (e.g., an enhancer and/or repressor binding site containing region), a promoter element (which may or may not be from INGAP regulatory region nucleotide sequence, SEQ ID NO: 23), and a reporter gene. The 5′-regulatory region nucleotide sequence is positioned upstream of the reporter gene. In order to determine the identity of various transcription factors that bind the 5′ regulatory region nucleotide sequence and to elucidate their binding locations within the 5′ regulatory nucleotide sequence of the INGAP gene, the region may be mapped using deletion analysis. One or more fragments of the regulatory region nucleotide sequence may be initially analyzed for their responses to various transcription factor activators. Once, a region of interest is determined, further fine mapping may be carried out where DNA from different locations within the regulatory region could be combined to make a more robust, and responsive reporter construct. DNA sequences, such as INGAP 5′-regulatory region DNA or a fragment thereof, can be manipulated by methods well known in the art. Examples of such techniques include, but are not limited to, polymerase chain reaction (PCR), restriction enzyme endonuclease digestion, ligation, and gene walking. Cloning fragments of DNA, such as 5′-regulatory regions is well known in the art.

[0051] Another approach to quantify the expression levels of a gene is to measure transcription of the gene. PCR-ELISA may be used to capture transcripts onto a solid phase using biotin or digoxigenin-labelled primers, oligonucleotide probes (oligoprobes) or directly after incorporation of the digoxigenin into the transcripts (Watzinger, F. and Lion, T. (2001) Nucleic Acids Res., 29, e52). Once captured, the transcripts can be detected using an enzyme-labeled avidin or anti-digoxigenin reporter molecule similar to a standard ELISA format. Another approach is to employ real-time PCR to detect the transcript of the reporter gene (Mackay, I. M. and Nitsche, A., Nucleic Acids Res. Mar. 15, 2002; 30(6), 1292-305). In real-time PCR fluorogenic nucleotides are used and progress of the transcript is monitored in real-time as the polymerase transcribes the reporter gene.

[0052] The promoter element in the reporter construct may or may not be from the same gene as the 5′-regulatory region. As an example, the enhancer/repressor region from the INGAP 5′-regulatory region, or a fragment of the enhancer/repressor region from the INGAP 5′-regulatory region, may be cloned upstream of a heterologous minimal promoter element, e.g., the minimal CMV promoter (Boshart et al., 1985) and the promoters for TK (Nordeen, 1988), IL-2, and MMTV.

[0053] Transcription of a gene begins around the minimal promoter. FIG. 4 shows the predicted transcription start sites for mammalian INGAP gene (SEQ ID NO: 2). SEQ ID NO: 2 was analyzed using “Neural Network Promoter Prediction” program designed by Martin Reese to identify eukaryotic promoter recognition elements such as TATA-box, GC-box, CAAT-box, and the transcription start site. These promoter elements are present in various combinations separated by various distances in sequence. The program is available on the Internet and is located at http://www.fruitfly.org/seqtools/promoter.html.

[0054] The reporter construct can be used to identify agents that modulate, either alone or in combination, the expression of INGAP. Some such agents may modulate expression of INGAP by binding to the regulatory region directly while others may regulate expression of transcription factors that bind to the INGAP regulatory region.

[0055] The reporter construct can be transfected into a host cell in vitro, or in vivo through the pancreatic duct, either transiently or stably, and a test agent introduced to the assay system. Examples of test agents include, but are not limited to organic and inorganic chemical agents, carbohydrates, proteins, oligonucleotides, cholecystokinin, mechanically induced pressure, and agents which cause a pancreatic duct obstruction. Expression of the reporter gene product can be determined by an assay appropriate for the reporter gene employed. Examples of such assays include, but are not limited to a luminescent assay for β-galactosidase or luciferase, an enzymatic assay for chloramphenicol acetyl transferase, and fluorescence detection for fluorescent proteins. Such assays are well known in the art, and a skilled artisan will be able to select an appropriate assay for the chosen reporter. A test agent is identified as a modulator of INGAP expression if the test agent modulates expression of the reporter gene product. Preferably the level of increase or decrease is at least 50%, 100%, 200%, 500%, or 1000%, but any statistically significant change can be an indicator of modulatory activity. A skilled artisan may also determine reporter gene product expression in untreated cells, and in treated and untreated cells transfected with a promoter-less reporter gene only. Such determinations can be used to determine background levels of expression.

[0056] Test agents can also be obtained by fractionating pancreatic secretion fluids. A pancreatic duct obstruction can be used as an exemplary method of harvesting pancreatic secretion fluids. The pancreatic secretion fluids can be fractionated by methods well known in the art. Examples include high-pressure liquid chromatography (HPLC), size exclusion chromatography, hydrophobic interacting columns, and density gradient centrifugation. Individual fractions can be tested for agents that modulate reporter gene expression using a method described herein. The individual fractions can be further fractionated to identify agents that modulate reporter gene expression. The identified test agents can be used to modulate the expression of INGAP.

[0057] A host cell can be any cell suitable for transfection and maintenance in a suitable assay system. Examples of suitable cells include, but are not limited to, mammalian cells, human cells, mouse cells, rat cells, monkey cells, dog cells, bovine cells, and porcine cells. Preferably the cells used will be human cells. The cells could be either transformed cells line or primary cells. Whole organ explants may also be used where the regulation may be monitored over many different cell types. Many methods exist in the art for transfecting or infecting cells with reporter construct DNA. Such methods include, but are not limited to, lipofection, electroporation, calcium phosphate precipitation, DEAE dextran, gene guns, and modified viral techniques (e.g., recombinant adenovirus or recombinant retrovirus). The skilled artisan can readily choose a method suitable for use with a given cell type and assay system.

[0058] The reporter construct can also be introduced in vivo directly into cells of the pancreas. Examples of methods to introduce the reporter construct into pancreatic cells in vivo include pancreatic duct retrograde perfusion and in vivo electroporation (Mir, 2001). The reporter construct encodes a reporter gene product that is readily measured in vivo. A test agent can be administered systemically or locally, and expression of the reporter gene in vivo can be determined by an assay appropriate for the particular reporter employed. Examples of such include a fluorescence assay for green fluorescent protein.

[0059] Methods for identifying agents that modulate INGAP expression can also be accomplished in vitro. The reporter construct can be contacted with a test agent in vitro under conditions sufficient for transcription and/or translation of the reporter gene. Components such as rabbit reticulocyte lysates or wheat germ extracts can be utilized for such a method. Subsequently, the expression level of the reporter gene can be determined as described above utilizing an appropriate assay for a given reporter gene. A test agent is identified as a modulator of INGAP expression if the test agent modulates expression of the reporter gene. Threshold levels of change can be set by the practitioner as discussed above.

[0060] A test agent can alternatively be contacted with an isolated and purified INGAP 5′-regulatory DNA molecule and one can determine if the test agent binds to the DNA molecule. Test agents can be a chemical agent, a protein, or a nucleic acid. Appropriate INGAP 5′-regulatory DNA molecules would include nucleotides 1-6586 of SEQ ID NO: 2, the 5′-regulatory region DNA (SEQ ID NO: 1, or SEQ ID NO: 23), or any fragment of the 5′-regulatory region, preferably a fragment which contains one or more enhancer/repressor binding sites. Methods to determine binding of the test agent to the fragment of DNA are well known in the art, e.g., electrophoretic mobility shift assay (EMSA). See for example Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51. Fragments of the 5′-regulatory region can be obtained by methods well known in the art using the disclosed sequence (SEQ ID NO: 2). Examples of such methods include, PCR, restriction enzyme digestion, and chemical synthesis. Any fragment of DNA within the 5′-regulatory region (SEQ ID NO: 1, or 23) can be used. The exact location that an agent binds can be determined for example by utilizing smaller fragments to map precisely the binding site for the test agent. Test agents that bind in the assay can be further tested in other assays that require modulatory activity.

[0061] An agent that causes an increase or decrease in reporter gene expression can be used as a modulator of INGAP expression. The modulator can be administered to a mammal in need of such modulation. Examples of mammals that may need INGAP expression modulation are those with reduced pancreatic function, in particular reduced islet cell function. Such mammals include those who have diabetes mellitus, impaired glucose tolerance, impaired fasting glucose, hyperglycemia, obesity, and pancreatic insufficiency.

[0062] An agent that is identified as a modulator of INGAP expression can be supplied in a kit to treat diseases associated with reduced islet cell function. The kit would comprise in single or divided containers, in single or divided doses a modulator of INGAP expression. Written instructions may be included for using the modulator of INGAP expression. The instructions may simply refer a reader to another location such as a website or other information source.

[0063] Agents that cause an increase in reporter gene expression can be used to increase INGAP expression to treat a disease state related to reduced islet cell function.

[0064] Agents that cause a decrease in reporter gene expression can be used to decrease INGAP expression to treat a disease state related to hyperactivity of islet cells or a disease where reduced INGAP expression is desirable. Examples of such agents include, but are not limited to, PMA, LIF, interleukin-6, Oncostatin M, and ciliary neurotropic factor. Agents can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, rectal, or pancreatic duct retrograde perfusion. Agents for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the mammal. Agents for intravenous, intramuscular, intra-arterial, transdermal, and subcutaneous injections can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for injection into the mammal. Agents for intranasal, topical, and rectal administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for surface administration to the mammal. Mammals in need of an increase in INGAP expression include for example, mammals with diabetes mellitus, impaired glucose tolerance, impaired fasting glucose, hyperglycemia, obesity, and pancreatic insufficiency. Mammals in need of a decrease in INGAP expression include for example, mammals with hypoglycemia.

[0065] The following examples are offered by way of illustration and do not limit the invention disclosed herein.

EXAMPLES

Example 1

[0066] Hamster INGAP Genomic Sequence and Structure

[0067] The hamster INGAP genomic sequence and structure was determined by gene walking (Clontech) and DNA sequencing. Gene walking is a method for walking upstream toward a promoter or downstream in genomic DNA from a known sequence, such as cDNA. This method utilizes four uncloned, adapter-ligated genomic fragment libraries. The manufacturer's recommended protocol is followed with one notable exception; hamster genomic DNA was used to create the uncloned, adapter-ligated genomic fragment libraries.

[0068] To create uncloned, adapter ligated genomic fragment libraries, genomic DNA was purified from hamster cells. Four separate aliquots were thoroughly digested with PvuII, StuI, DraI, or EcoRV. Following digestion, inactivation of the restriction enzymes, and dephosphorylation, each separate pool of DNA fragments was ligated to an adapter, see FIG. 5. The adapter was phosphorylated to provide the requisite phosphate group for a ligation reaction. Also note that the 3-prime side of the short adapter contains an amine group to prevent the adapters from forming concatamers.

[0069] Two gene specific primers (GSP1 and GSP2) were designed for each region of known sequence (i.e., the exons of the INGAP gene). See FIG. 6 for fragment location and GSP1 and GSP2 location. The gene specific primers were designed as reverse PCR primers for all fragments except fragments 1—2 and 14—5. The gene specific primers for fragments 1—2 and 14—5 were designed as forward primers. Adapter primer 1 (API) and adapter primer 2 (AP2) (FIG. 5) were forward PCR primers for all fragments except fragments 1—2 and 14—5, which were reverse PCR primers. The outer gene specific primer (GSP1) was used with adapter primer 1 in a PCR reaction. To increase specificity, a second, nested PCR was set up using the inner gene specific primer (GSP2) and adapter primer 2. A small aliquot of the first reaction served as template for the second reaction. Gene specific PCR primers utilized for gene walking are listed in Table 2 and the strategy used to build the INGAP genomic sequence is shown in FIGS. 6 and 7. The arrowheads in FIG. 6 represent the adapter primers (AP1 and AP2), while the circles represent the gene specific primers (GSP1 and GSP2).

TABLE 2
NAME (LOCATION)	SEQUENCE
INGEN 21_3 (1464, 1482)	5′-ACAAGCAATCTAGAGATGG-3′	(SEQ ID NO: 3)
INGEN 19_3 (1401, 1423)	5′-GTTCAGCTATGTTCATAGCAGGG-3′	(SEQ ID NO: 4)
INGEN 16_3 (1855, 1876)	5′-GTCTGTATGACTGTGTGGGAAG-3′	(SEQ ID NO: 5)
INGEN 15_3 (1929, 1948)	5′-GCACTTGAACTCAATGGCTC-3′	(SEQ ID NO: 6)
INGEN 14_3 (2147, 2168)	5′-GAACCACCTGACATGGGTGATG-3′	(SEQ ID NO: 7)
INGEN 13_3 (2177, 2200)	5′-GGGCATCGTATCATCTGGTTACAG-3′	(SEQ ID NO: 8)
INGEN 8_3 (2544, 2565)	5′-GGTTCAAAAAAGCTGCTTCAAC-3′	(SEQ ID NO: 9)
INGEN 7_3 (2666, 2689)	5′-GGAATAGCTGCAATTTATGCCCAT-3′	(SEQ ID NO: 10)
INGEN 4_3 (2833, 2858)	5′-CTTAGGAACATTCAGGCAGCCTCCTG-3′	(SEQ ID NO: 11)
INGEN 3_3 (2866, 2891)	5′-GTTGCCCTCTGCCACGTGTCAAGTTC-3′	(SEQ ID NO: 12)
INGEN 2_3 (3444, 3470)	5′-CATCCAAGACATCCTACAGAGGGTCAT-3′	(SEQ ID NO: 13)
INGEN 1_3 (3475, 3501)	5′-CCCAAGAAAGGAACATCAGGCAGGAAA-3′	(SEQ ID NO: 14)
INGEN 2_2 (3330, 3350)	5′-CCAAATGAGTGCTTCCCTGAA-3′	(SEQ ID NO: 15)
INGEN 1_2 (3241, 3266)	5′-GCAGCACTCTGAAACTCAGTAGAGTT-3′	(SEQ ID NO: 16)
INGEN 14_5 (5544, 5563)	5′-GCTGCTGACCGTGGTTATTG-3′	(SEQ ID NO: 17)
INGEN 13_5 (5463 5485)	5′-ACACTACCCAACGGAAGTGGATG-3′	(SEQ ID NO: 18)
INGAP1_1L (3475, 3492)	5′-TTTCCTGCCTGATGTTCC-3′	(SEQ ID NO: 19)
INGAP1_1R (5957, 5976)	5′-TCATACTTGCTTCCTTGTCC-3′	(SEQ ID NO: 20)
INGAP2_1L (4470, 4488)	5′-CTTCACGTATAACCTGTCC-3′	(SEQ ID NO: 21)
INGAP2_1R (5905, 5923)	5′-ATTAGAACTGCCCTAGACC-3′	(SEQ ID NO: 22)

[0070] The PCR fragments were sequenced to determine the nucleotide sequence of the INGAP 5′-regulatory region; the introns, the intron/exon junctions, and the 3-prime polyadenylation regions. The nucleotide sequence of hamster INGAP genomic DNA is shown in SEQ ID NO: 2.

Example 2

[0071] Cloning Hamster INGAP 5′-Regulatory Region Fragment into a Reporter Construct

[0072] To construct the INGAP 5′-regulatory region, individual PCR fragments were joined together at unique restriction sites located within two adjoining fragments. FIGS. 6 and 7 detail the strategy used to piece the INGAP 5′-regulatory region together. Fragments 8—3 and 2—3 were joined at a unique SphI site; 14—3 and 8—3 were joined at a unique BbsI site; 16—3 and 14—3 were joined at a unique PstI site. The nucleotide sequence of hamster INGAP 5′-regulatory region DNA is shown in SEQ ID NO: 1 and 23 in the sequence listing.

[0073] The hamster INGAP 5′-regulatory region or a fragment of the 5′-regulatory region was cloned into a reporter plasmid, pβGal-Basic (Clontech). The 5′-regulatory region or fragments were cloned utilizing the unique XmaI site from the gene walking adapter primer and a unique BglII site located at the 3-prime side of the regulatory region. FIG. 8 details the fragments cloned into pβGal-Basic. The sizes of the fragments are indicated to the right of the fragments and are expressed as the number of nucleotides of the fragment.

Example 3

[0074] Assay System to Screen for Factors that Modulate the Expression of INGAP

[0075] Promoter analysis of INGAP identified a number of potential promoter-proximal regulatory sites including the consensus transcription factor binding sites; cAMP response element (CRE), AP-1 and STAT. Promoter-fragment reporter-gene constructs were transiently transfected into 293T cells and co-transfection of secretory alkaline phosphatase was used to normalize for transfection efficiency.

[0076] Reporter constructs containing INGAP 5′-regulatory region fragments 2—3sP (SEQ ID NO: 37), 2—3dP (SEQ ID NO: 38), 2—3pP (SEQ ID NO: 36), 14—3P (SEQ ID NO: 34), 16—3P (SEQ ID NO: 31), or 19—3P (SEQ ID NO: 23) were transfected into human cells. The pβGal-Basic plasmid without the hamster INGAP DNA was also transfected into human cells as a control to measure the level of endogenous reporter activity. Two days following transfection, the cells were treated with PMA for 24 hours or were untreated. To determine the level of promoter activity, the amount of β-galactosidase gene product was determined using a luminescent assay for β-galactosidase. FIG. 9A shows that construct 14—3P activated the INGAP expression the most, followed by 2—3pP, and 16—3P.

[0077] Reporter construct containing INGAP 5′-regulatory region DNA nucleotides 2030 to 3120 was transfected into human cells. The pβGal-Basic plasmid without the hamster INGAP DNA was also transfected into human cells as a control to measure the level of endogenous reporter activity. Two days following transfection, the cells were treated with LIF for 24 hours or were untreated. To determine the level of promoter activity, the amount of β-galactosidase gene product was determined using a luminescent assay for β-galactosidase. FIG. 9B shows the results. LIF was determined to increase the activity of the 5′-regulatory region of mammalian INGAP. Forskolin (an activator of cAMP/CREB/CRE) did not modulate gene expression (data not shown).

[0078] It is important to note that when present in human cells, the hamster INGAP 5′-regulatory region is transactivated by the human transcription factors. Thus, linked to a reporter gene, the 5′-regulatory region of hamster INGAP creates a sensitive assay system to screen for factors that modulate the expression of INGAP.

Example 4

[0079] Determination of Approximate Location of PMA and LIF-Mediated Transcription Factor Binding in the 5′-Regulatory Region

[0080] To map the approximate location of PMA-initiated or LIF-initiated transcription factor binding different fragments of the hamster INGAP 5′-regulatory region were cloned into pβGal-Basic. See FIG. 8. The fragments cloned into the reporter construct were 2—3sP (SEQ ID NO: 37), 2—3dP (SEQ ID NO: 38), 2—3pP (SEQ ID NO: 36), 14—3P (SEQ ID NO: 34), 16—3P (SEQ ID NO: 31), or 19—3P (SEQ ID NO: 23). The reporter constructs were transfected into human cells. Two days following transfection, the cells were treated with different concentrations of PMA or LIF for 24 hours. The concentrations of PMA used were 6 ng/ml, 17 ng/ml, 50 ng/ml, 100 ng/ml, or 300 ng/ml. The concentrations of LIF used were 1 ng/ml, 10 ng/ml, or 30 ng/ml. To determine the level of promoter activity, the amount of β-galactosidase gene product was determined using a luminescent assay for β-galactosidase. FIGS. 10 and 11 show the results for PMA and LIF treatment, respectively. Both PMA and LIF activated the cell reporter constructs. The exact location of the DNA contact sites can be narrowed further by cloning smaller fragments of the hamster INGAP 5′-regulatory region and by site directed mutations or deletions.

Example 5

[0081] RNA Analysis of INGAP Gene Upregulation

[0082] To determine if INGAP RNA levels increase after stimulation with a cytokine that signals through STAT, rat amphocrine pancreatic cells, AR42J were treated with IL-6 (1000 U/ml) for 24 hours. Total RNA was extracted from the treated and untreated cells using techniques well known in the art, e.g., using TRIZOL® reagent.

[0083] Equal amounts of total RNA (10 kg) were loaded in 2.5% formaldehyde gel and electrophoresed for 4 hours at 70V with a constant circulation of the buffer using a circulating pump. The gel was photographed and washed with water twice at room temperature and soaked in 20×SSC. The gel was transferred to a nylon membrane (Amersham) in 20×SSC overnight following a standard procedure. The membrane was washed with 20×SSC to remove any agar that might have attached to the membrane and baked for 4 hours at −80° C.

[0084] One hundred nanograms of hamster INGAP cDNA was labeled using Random Prime Labeling kit (Roche-BMB) and alpha-P32 dCTP (ICN). Approximately 20 million counts were used for hybridization in 20 ml hybridization buffer following the standard procedure at 42° C. for overnight. The blot was washed as follows: 2-times at room temperature with 2×SSC for 10 minutes each; 2-times at 42° C. with 2×SSC for 10 minutes each; 2-times at 55° C. with 1×SSC for 10 minutes each. The membrane was exposed to the film (XOMAT-Kodak) and kept at −80° C. overnight before developing.

[0085] Treatment with IL-6 caused an increase in INGAP gene expression (FIG. 12). These data demonstrate that extracellular factors that elevate AP-1-binding transcription factors and STAT-binding transcription factors are involved in the regulation of INGAP gene expression. These studies suggest that it is feasible to enhance INGAP expression as a means of inducing islet neogenesis.

[0086] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

	TABLE 3
	Position		Core	Matrix
Family/matrix	Further Information	Opt.	from-to	anchor	Str.	sim.	sim.	Sequence
V$LEFF/LEF1.01	TCF/LEF-1, involved in the	0.86	12-28	20	(+)	1.000	0.900	ggaccatCAAAgtctgt
	Wnt signal transduction
	pathway
V$MITF/MIT.01	MIT (microphthalmia	0.81	22-40	31	(+)	1.000	0.823	agtctgtCATGtcatttgg
	transcription factor) and TFE3
V$OCT1/OCT1.05	octamer-binding factor 1	0.90	27-41	34	(+)	0.833	0.904	gTCATgtcatttggg
V$TCFF/TCF11.01	TCF11/KCR-F1/Nrf1	1.00	32-38	35	(+)	1.000	1.000	GTCAttt
	homodimers
V$MYOF/	Myogenin/nuclear factor 1 or	0.71	25-53	39	(+)	1.000	0.735	ctgtcatgtcatTTGGgggagggcctatg
MYOGNF1.01	related factors
V$ZBPF/ZBP89.01	Zinc finger transcription factor	0.93	36-48	42	(−)	1.000	0.982	gccctCCCCcaaa
	ZBP-89
V$SP1F/GC.01	GC box elements	0.88	38-52	45	(+)	0.876	0.898	tgggGGAGggcctat
V$PERO/PPARA.01	PPAR/RXR heterodimers	0.70	44-64	54	(−)	0.884	0.708	acagaggagggcATAGgccct
V$PAX5/PAX9.01	zebrafish PAX9 binding sites	0.78	43-71	57	(−)	0.800	0.811	cagataCACAgaggagggcataggccctc
V$TBPF/ATATA.01	Avian C-type LTR TATA box	0.81	68-84	76	(−)	1.000	0.987	tgctattTAAGcccaga
V$HMTB/MTBF.01	muscle-specific Mt binding	0.90	76-84	80	(−)	1.000	0.932	tgctATTTa
	site
V$OCT1/OCT1.06	octamer-binding factor 1	0.80	74-88	81	(−)	0.750	0.865	ggtatgctATTTaag
V$BRNF/BRN2.01	POU factor Brn-2 (N-Oct 3)	0.91	89-105	97	(+)	1.000	0.970	tccataggAAATgggct
V$HMTB/MTBF.01	muscle-specific Mt binding	0.90	108-116	112	(−)	1.000	0.953	tggaATTTg
	site
V$OCT1/OCT1.05	octamer-binding factor 1	0.90	106-120	113	(−)	0.944	0.917	tATATggaatttggg
V$HNF6/HNF6.01	Liver enriched Cut -	0.82	108-122	115	(+)	0.833	0.885	caaatTCCAtatatg
	Homeodomain transcription
	factor HNF6 (ONECUT)
V$SRFF/SRF.02	serum response factor	0.83	110-128	119	(+)	1.000	0.862	aattCCATatatgcactag
V$OCTP/OCT1P.01	octamer-binding factor 1,	0.86	114-126	120	(+)	1.000	0.903	ccatatATGCact
	POU-specific domain
V$MYOF/	Myogenin/nuclear factor 1 or	0.71	171-199	185	(+)	0.857	0.740	ctggtcttttagCTGGcacccatccatat
MYOGNF1.01	related factors
V$NF1F/NF1.02	Nuclear factor 1 (CTF1)	0.81	181-199	190	(+)	1.000	0.812	agcTGGCacccatccatat
V$CLOX/	cut-like homeodomain protein	0.94	187-203	195	(−)	0.929	0.940	ctgaatatgGATGggtg
CDPCR3HD.01
V$MYOF/	Myogenin/nuclear factor 1 or	0.71	181-209	195	(−)	0.785	0.767	aaccctctgaatATGGatgggtgccagct
MYOGNF1.01	related factors
V$OCTP/OCT1P.01	octamer-binding factor 1,	0.86	192-204	198	(+)	0.980	0.907	atccatATTCaga
	POU-specific domain
V$CREB/TAXCREB.02	Tax/CREB complex	0.71	202-222	212	(−)	0.750	0.721	ttgaacTGAAccaaaccctct
V$HOXF/EN1.01	Homeobox protein engrailed	0.77	210-226	218	(−)	0.782	0.823	aacaTTGAactgaacca
	(en-1)
V$BARB/BARBIE.01	barbiturate-inducible element	0.88	230-244	237	(−)	1.000	0.894	ttatAAAGctgagga
V$TBPF/TATA.01	cellular and viral TATA box	0.90	230-246	238	(−)	1.000	0.910	agttaTAAAgctgagga
	elements
V$BARB/BARBIE.01	barbiturate-inducible element	0.88	252-266	259	(−)	1.000	0.902	agtgAAAGcagagag
V$MYT1/MYT1.01	MyT1 zinc finger transcription	0.75	272-284	278	(−)	0.750	0.756	craCAGTtgacct
	factor involved in primary
	neurogenesis
V$SMAD/SMAD4.01	Smad4 transcription factor	0.94	304-312	308	(+)	1.000	0.940	GTCTtgact
	involved in TGF-beta signaling
V$HOXF/CRX.01	Cone-rod homeobox-	0.94	312-328	320	(−)	1.000	0.960	gagggATTAgaaaagga
	containing transcription factor/
	otx-like homeobox gene
V$ECAT/NFY.01	nuclear factor Y (Y-box	0.90	337-351	344	(−)	1.000	0.906	ggaatCCAAtygtag
	binding factor)
V$HOXF/PTX1.01	Pituitary Homeobox 1 (Ptx1)	0.79	337-353	345	(+)	0.789	0.802	ctacraTTGGattccat
V$FKHD/FREAC2.01	Fork head RElated ACtivator-2	0.84	362-378	370	(−)	1.000	0.897	tacagcTAAAcactgag
V$MINI/	Muscle Initiator Sequence	0.86	401-419	410	(−)	0.840	0.865	gagcctTCATccagtagct
MUSCLE_INI.02
V$MOKF/MOK2.01	Ribonucleoprotein associated	0.74	409-429	419	(−)	1.000	0.746	tgtcatcttagagCCTTcatc
	zinc finger protein MOK-2
	(mouse)
V$ZFIA/ZID.01	zinc finger with interaction	0.85	414-426	420	(+)	1.000	0.861	agGCTCtaagatg
	domain
V$CART/XVENT2.01	Xenopus homeodomain factor	0.82	418-434	426	(+)	0.750	0.837	tcTAAGatgacaattaa
	Xvent-2; early BMP signaling
	response
V$OCT1/OCT1.04	octamer-binding factor 1	0.80	421-435	428	(+)	0.807	0.840	aaGATGacaattaag
V$HOMS/S8.01	Binding site for S8 type	0.97	426-434	430	(+)	1.000	0.994	gacaATTAa
	homeodomains
V$NKXH/NKX25.02	homeo domain factor Nkx-	0.88	424-436	430	(−)	1.000	1.000	cctTAATtgtcat
	2.5/Csx, tinman homolog low
	affinity sites
V$CREB/CREBP1.01	cAMP-responsive element	0.80	425-445	435	(−)	0.766	0.808	cgacgattACCTtaattgtca
	binding protein 1
V$COMP/COMP1 .01	COMP1, cooperates with	0.76	434-454	444	(−)	0.750	0.768	aatgaggATCGacgattacct
	myogenic proteins in
	multicomponent complex
V$HOXF/HOX1-3.01	Hox-1.3, vertebrate	0.83	444-460	452	(+)	1.000	0.886	cgatcctcATTAtagtg
	homeobox protein
V$ETSF/GABP.01	GABP: GA binding protein	0.85	454-470	462	(+)	1.000	0.868	tatagtGGAAgggcttc
V$LEFF/LEF1.01	TCF/LEF-1, involved in the	0.86	463-479	471	(+)	1.000	0.904	agggcttCAAAggcagt
	Wnt signal transduction
	pathway
V$STAT/STAT6.01	STAT6: signal transducer and	0.84	464-482	473	(−)	0.758	0.867	gagacTGCCtttgaagccc
	activator of transcription 6
V$GATA/GATA1.03	GATA-binding factor 1	0.95	490-502	496	(−)	1.000	0.971	ttcaGATAggcag
V$SRFF/SRF.01	serum response factor	0.66	487-505	496	(−)	0.757	0.672	atgttcaGATAggcagtag
V$EVI1/EVI1.04	Ecotropic viral integration site	0.77	493-509	501	(−)	0.800	0.824	gGAAAtgttcagatagg
	1 encoded factor
V$AP4R/TH1E47.01	Thing1/E47 heterodimer, TH1	0.93	509-525	517	(+)	1.000	0.951	cctaatgCCAGatgtct
	bHLH member specific
	expression in a variety of
	embryonic tissues
V$AP4R/	Tal-1beta/ITF-2 heterodimer	0.85	512-528	520	(+)	1.000	0.852	aatgcCAGAtgtctctt
TAL1BETAITF2.01
V$NEUR/NEUROD1.01	DNA binding site for	0.83	514-526	520	(−)	1.000	0.851	gagaCATCtggca
	NEUROD1 (BETA-2/E47
	dimer)
V$MEF2/MEF2.05	MEF2	0.96	518-540	529	(−)	1.000	0.984	aggataggttTAAAgagacatct
V$EVI1/EVI1.04	Ecotropic viral integration site	0.77	523-539	531	(−)	1.000	0.774	gGATAggtttaaagaga
	1 encoded factor
V$MEF2/AMEF2.01	myocyte enhancer factor	0.80	521-543	532	(+)	1.000	0.813	tgtctcttTAAAcctatcctggc
V$TBPF/MTATA.01	Muscle TATA box	0.84	524-540	532	(+)	1.000	0.877	ctcttTAAAcctatcct
V$HOXF/HOX1-3.01	Hox-1.3, vertebrate	0.83	543-559	551	(+)	1.000	0.845	ctcccttcATTAaggta
	homeobox protein
V$PDX1/ISL1.01	Pancreatic and intestinal lim-	0.82	543-563	553	(−)	1.000	0.834	gagatacctTAATgaagggag
	homeodomain factor
V$OCT1/OCT1.05	octamer-binding factor 1	0.90	556-570	563	(+)	0.944	0.926	gGTATctcatttttt
V$CIZF/NMP4.01	NMP4 (nuclear matrix protein	0.97	562-572	567	(−)	1.000	0.972	gcAAAAaatga
	4)/CIZ (Cas-interacting zinc
	finger protein)
V$EVI1/EVI1.01	Ecotropic viral integration site	0.72	569-585	577	(−)	0.764	0.720	ggaaCAGAggagagcaa
	1 encoded factor
V$AP1F/AP1.01	AP1 binding site	0.95	582-602	592	(−)	0.881	0.964	aaaactgaATCAgtggnggaa
V$PIT1/PIT1.01	Pit1, GHF-1 pituitary specific	0.86	589-599	594	(+)	1.000	0.886	actgATTCagt
	pou domain transcription
	factor
V$AP1F/AP1.01	AP1 binding site	0.95	586-606	596	(+)	0.850	0.956	nccactgaTTCAgtttttctg
V$VMYB/VMYB.01	v-Myb	0.90	593-603	598	(−)	0.876	0.910	aaaAACTgaat
V$CIZF/NMP4.01	NMP4 (nuclear matrix protein	0.97	595-605	600	(−)	1.000	0.975	agAAAAactga
	4)/CIZ (Cas-interacting zinc
	finger protein)
V$GREF/PRE.01	Progesterone receptor binding	0.84	604-622	613	(+)	1.000	0.875	ctgatccctctTGTTctcc
	site
V$GKLF/GKLF.01	Gut-enriched Krueppel-like	0.91	632-646	639	(−)	1.000	0.971	gaaaaagagaAGGGa
	factor
V$CIZF/NMP4.01	NMP4 (nuclear matrix protein	0.97	637-647	642	(−)	1.000	0.987	ggAAAAagaga
	4)/CIZ (Cas-interacting zinc
	finger protein)
V$NFAT/NFAT.01	Nuclear factor of activated T-	0.97	640-650	645	(−)	1.000	0.982	ggagGAAAaag
	cells
V$MAZF/MAZ.01	Myc associated zinc finger	0.90	649-661	655	(−)	1.000	0.910	ggtgGAGGgaagg
	protein (MAZ)
V$EGRF/WT1.01	Wilms Tumor Suppressor	0.88	658-672	665	(−)	1.000	0.932	gggggTGGGagggtg
V$ZBPF/ZBP89.01	Zinc finger transcription factor	0.93	663-675	669	(+)	1.000	0.972	tcccaCCCCcatg
	ZBP-89
V$IRFF/IRF2.01	interferon regulatory factor 2	0.80	702-716	709	(−)	1.000	0.815	aggaagggGAAAggg
V$BRNF/BRN2.01	POU factor Brn-2 (N-Oct 3)	0.91	746-762	754	(−)	1.000	0.911	aaaataggAAATaagga
V$ETSF/PU1.01	Pu.1 (Pu120) Ets-like	0.86	746-762	754	(−)	1.000	0.883	aaaataGGAAataagga
	transcription factor identified
	in lymphoid B-cells
V$EVI1/EVI1.04	Ecotropic viral integration site	0.77	750-766	758	(−)	0.760	0.792	aGAGAaaataggaaata
	1 encoded factor
V$EVI1/EVI1.05	Ecotropic viral integration site	0.80	755-771	763	(−)	0.763	0.817	cccccagagaaAATAgg
	1 encoded factor
V$ZBPF/ZBP89.01	Zinc finger transcription factor	0.93	764-776	770	(−)	1.000	0.934	ccacaCCCCcaga
	ZBP-89
V$FAST/FAST1.01	FAST-1 SMAD interacting	0.81	769-783	776	(+)	0.983	0.894	gggtgtgGATTttat
	protein
V$TBPF/TATA.02	Mammalian C-type LTR TATA	0.89	771-787	779	(−)	1.000	0.942	caccaTAAAatccacac
	box
V$PAX5/PAX9.01	zebrafish PAX9 binding sites	0.78	781-809	795	(−)	0.866	0.813	aacataTGCAcagaagggcttccaccata
V$OCT1/OCT.01	Octamer binding site	0.79	793-807	800	(−)	1.000	0.790	catATGCacagaagg
	(OCT1/OCT2 consensus)
V$OCTP/OCT1P.01	octamer-binding factor 1,	0.86	798-810	804	(−)	1.000	0.910	caacatATGCaca
	POU-specific domain
V$SRFF/SRF.01	serum response factor	0.66	797-815	806	(+)	0.757	0.666	ctgtgcaTATGttgtctta
V$EVI1/EVI1.05	Ecotropic viral integration site	0.80	802-818	810	(−)	0.750	0.828	caataagacaaCATAtg
	1 encoded factor
V$CLOX/CDP.01	cut-like homeodomain protein	0.75	803-819	811	(−)	1.000	0.776	ccAATAagacaacatat
V$EVI1/EVI1.02	Ecotropic viral integration site	0.83	807-823	815	(−)	1.000	0.836	tcaaccaatAAGAcaac
	1 encoded factor
V$ECAT/NFY.02	nuclear factor Y (Y-box	0.91	810-824	817	(−)	1.000	0.960	atcaaCCAAtaagac
	binding factor)
V$HAML/AML3.01	Runt-related transcription	0.84	811-825	818	(+)	1.000	0.844	tcttatTGGTtgata
	factor 2/CBFA1 (core-
	binding factor, runt domain,
	alpha subunit 1)
V$PCAT/CAAT.01	cellular and viral CCAAT box	0.90	813-823	818	(−)	1.000	0.943	tcaaCCAAtaa
V$GATA/GATA.01	GATA binding site	0.95	818-830	824	(+)	1.000	0.956	ggttGATAaataa
	(consensus)
V$HNF1/HNF1.02	Hepatic nuclear factor 1	0.76	818-834	826	(+)	0.757	0.791	gGTTGataaataaagca
V$HOXT/	Homeobox protein MEIS1	0.79	823-835	829	(−)	0.750	0.797	gTGCTttatttat
MEIS1_HOXA9.01	binding site
V$ECAT/NFY.01	nuclear factor Y (Y-box	0.90	837-851	844	(+)	1.000	0.912	gttgtCCAAtaggga
	binding factor)
V$FKHD/FREAC2.01	Fork head RElated ACtivator-2	0.84	844-860	852	(+)	0.750	0.843	aataggGAAAcaagata
V$EVI1/EVI1.06	Ecotropic viral integration site	0.83	846-862	854	(+)	1.000	0.960	tagggaaacaAGATagg
	1 encoded factor
V$GATA/GATA1.01	GATA-binding factor 1	0.96	853-865	859	(+)	1.000	0.970	acaaGATAggtgg
V$PCAT/ACAAT.01	Avian C-type LTR CCAAT box	0.86	856-866	861	(−)	0.750	0.867	cccaCCTAtct
V$XBBF/RFX1.01	X-box binding protein RFX1	0.89	909-927	918	(−)	1.000	0.929	ggatcacatgGCAAccctc
V$EBOX/MYCMAX.02	c-Myc/Max heterodimer	0.92	912-928	920	(−)	0.895	0.936	aggatCACAtggcaacc
V$MITF/MIT.01	MIT (microphthalmia	0.81	911-929	920	(+)	1.000	0.863	gggttgcCATGtgatccta
	transcription factor) and TFE3
V$ETSF/PU1.01	Pu.1 (Pu120) Ets-like	0.86	927-943	935	(+)	1.000	0.950	ctaggaGGAAttgacac
	transcription factor identified
	in lymphoid B-cells
V$OCT1/OCT1.06	octamer-binding factor 1	0.80	932-946	939	(−)	1.000	0.800	catgtgtcAATTcct
V$TALE/TGIF.01	TG-interacting factor	1.00	936-942	939	(−)	1.000	1.000	tGTCAat
	belonging to TALE class of
	homeodomain factors
V$MITF/MIT.01	MIT (microphthalmia	0.81	935-953	944	(−)	1.000	0.835	ccattctCATGtgtcaatt
	transcription factor) and TFE3
V$OCT1/OCT1.04	octamer-binding factor 1	0.80	941-955	948	(+)	0.846	0.800	caCATGagaatgggg
V$GATA/GATA.01	GATA binding site	0.95	962-974	968	(+)	1.000	0.998	gaaaGATAagtcc
	(consensus)
V$SRFF/SRF.01	serum response factor	0.66	968-986	977	(−)	1.000	0.672	atattttTATAaggactta
V$CDXF/CDX2.01	Cdx-2 mammalian caudal	0.84	970-988	979	(−)	1.000	0.867	atatattTTTAtaaggact
	related intestinal transcr.
	factor
V$FKHD/XFD2.01	Xenopus fork head domain	0.89	972-988	980	(+)	1.000	0.894	tccttaTAAAaatatat
	factor 2
V$MEF2/MEF2.01	myogenic enhancer factor 2	0.74	970-992	981	(+)	1.000	0.740	agtccttaTAAAaatatatatta
V$TBPF/TATA.01	cellular and viral TATA box	0.90	973-989	981	(+)	1.000	0.963	ccttaTAAAaatatata
	elements
V$CART/CART1.01	Cart-1 (cartilage	0.84	978-994	986	(−)	1.000	0.870	acTAATatatattttta
	homeoprotein 1)
V$CART/CART1.01	Cart-1 (cartilage	0.84	985-1001	993	(−)	1.000	0.855	caTAATtactaatatat
	homeoprotein 1)
V$SATB/SATB1.01	Special AT-rich sequence-	0.93	985-1001	993	(−)	1.000	0.943	cataattacTAATatat
	binding protein 1,
	predominantly expressed in
	thymocytes, binds to matrix
	attachment regions (MARs)
V$BRNF/BRN3.01	POU transcription factor Brn-3	0.78	987-1003	995	(−)	1.000	0.816	cccATAAttactaatat
V$CLOX/CDP.01	cut-like homeodomain protein	0.75	987-1003	995	(−)	0.757	0.765	ccCATAattactaatat
V$HOMS/S8.01	Binding site for S8 type	0.97	992-1000	996	(+)	1.000	0.989	agtaATTAt
	homeodomains
V$NKXH/DLX1.01	DLX-1, -2, and -5 binding	0.91	990-1002	996	(−)	1.000	0.976	ccatAATTactaa
	sites
V$HOXF/HOX1-3.01	Hox-1.3, vertebrate	0.83	989-1005	997	(−)	1.000	0.886	aacccataATTActaat
	homeobox protein
V$PDX1/PDX1.01	Pdx1 (IDX1/IPF1) pancreatic	0.74	988-1008	998	(−)	1.000	0.775	attaacccaTAATtactaata
	and intestinal homeodomain
	TF
V$FKHD/XFD3.01	Xenopus fork head domain	0.82	998-1014	1006	(+)	0.826	0.844	tatgggttAATAattaa
	factor 3
V$HNF1/HNF1.01	hepatic nuclear factor 1	0.78	1000-1016	1008	(−)	0.755	0.857	aCTTAattattaaccca
V$HNF1/HNF1.01	hepatic nuclear factor 1	0.78	1002-1018	1010	(+)	1.000	0.966	gGTTAataattaagtca
V$PAX4/PAX4.01	Pax-4 paired domain protein,	0.97	1005-1015	1010	(+)	1.000	0.972	taatAATTaag
	together with PAX-6 involved
	in pancreatic development
V$HOMS/S8.01	Binding site for S8 type	0.97	1007-1015	1011	(−)	1.000	0.995	cttaATTAt
	homeodomains
V$HOXF/HOX1-3.01	Hox-1.3, vertebrate	0.83	1003-1019	1011	(−)	1.000	0.873	ctgacttaATTAttaac
	homeobox protein
V$NKXH/DLX1.01	DLX-1, -2, and -5 binding	0.91	1005-1017	1011	(+)	1.000	0.988	taatAATTaagtc
	sites
V$RBIT/BRIGHT.01	Bright, B cell regulator of IgH	0.92	1005-1017	1011	(+)	1.000	0.931	taataATTAagtc
	transcription
V$TBPF/ATATA.01	Avian C-type LTR TATA box	0.81	1005-1021	1013	(+)	1.000	0.881	taataatTAAGtcagag
V$CREB/CREBP1.01	cAMP-responsive element	0.80	1004-1024	1014	(−)	0.766	0.819	tagctctgACTTaattattaa
	binding protein 1
V$RORA/RORA2.01	RAR-related orphan receptor	0.82	1007-1023	1015	(+)	0.750	0.874	ataattaAGTCagagct
	alpha2
V$PCAT/CAAT.01	cellular and viral CCAAT box	0.90	1022-1032	1027	(+)	0.856	0.928	ctagCCATtaa
V$NKXH/NKX25.02	homeo domain factor Nkx-	0.88	1022-1034	1028	(−)	1.000	0.903	tctTAATggctag
	2.5/Csx. tinman homolog low
	affinity sites
V$CREB/HLF.01	hepatic leukemia factor	0.84	1022-1042	1032	(−)	0.770	0.842	ctagtGTTTcttaatggctag
V$HOXF/HOX1-3.01	Hox-1.3, vertebrate	0.83	1056-1072	1064	(+)	1.000	0.891	gcttcataATTAatata
	homeobox protein
V$HOMS/S8.01	Binding site for S8 type	0.97	1061-1069	1065	(−)	1.000	0.995	attaATTAt
	homeodomains
V$NKXH/DLX1.01	DLX-1, -2, and -5 binding	0.91	1059-1071	1065	(+)	1.000	0.988	tcatAATTaatat
	sites
V$RBIT/BRIGHT.01	Bright, B cell regulator of IgH	0.92	1059-1071	1065	(+)	1.000	0.952	tcataATTAatat
	transcription
V$BRNF/BRN2.01	POU factor Brn-2 (N-Oct 3)	0.91	1058-1074	1066	(+)	1.000	0.945	ttcataatTAATatagt
V$OCT1/OCT1.06	octamer-binding factor 1	0.80	1060-1074	1067	(−)	1.000	0.885	actatattAATTatg
V$HOXF/HOX1-3.01	Hox-1.3, vertebrate	0.83	1061-1077	1069	(−)	1.000	0.854	gatactatATTAattat
	homeobox protein
V$OCT1/OCT1.06	octamer-binding factor 1	0.80	1079-1093	1086	(+)	0.750	0.875	tgtatgttCATTtgg
V$FAST/FAST1.01	FAST-1 SMAD interacting	0.81	1080-1094	1087	(+)	0.850	0.887	gtatgttCATTtggg
	protein
V$RREB/RREB1.01	Ras-responsive element	0.79	1081-1095	1088	(−)	1.000	0.816	cCCCAaatgaacata
	binding protein 1
V$E2FF/E2F.02	E2F, involved in cell cycle	0.84	1085-1099	1092	(−)	1.000	0.849	tcagcccCAAAtgaa
	regulation, interacts with Rb
	p107 protein
V$CREB/TAXCREB.01	Tax/CREB complex	0.81	1091-1111	1101	(+)	1.000	0.828	tggggcTGACacagttctggg
V$AP1F/VMAF.01	v-Maf	0.82	1092-1112	1102	(+)	1.000	0.833	ggggcTGACacagttctggga
V$MYT1/MYT1.01	MyT1 zinc finger transcription	0.75	1123-1135	1129	(+)	0.750	0.791	aggAAGAytactt
	factor involved in primary
	neurogenesis
V$CLOX/CLOX.01	Clox	0.81	1136-1152	1144	(−)	0.804	0.820	cctacaATCCatgtacc
V$HNF4/HNF4.01	Hepatic nuclear factor 4	0.82	1156-1172	1164	(−)	1.000	0.864	atagagCAAAggactac
V$LEFF/LEF1.01	TCF/LEF-1, involved in the	0.86	1157-1173	1165	(−)	1.000	0.907	catagagCAAAggacta
	Wnt signal transduction
	pathway
V$PERO/PPARA.01	PPAR/RXR heterodimers	0.70	1157-1177	1167	(−)	1.000	0.700	tagacatagagcAAAGgacta
V$CLOX/CLOX.01	Clox	0.81	1173-1189	1181	(+)	0.804	0.831	gtctaaATCCatatatg
V$HNF6/HNF6.01	Liver enriched Cut-	0.82	1175-1189	1182	(+)	0.833	0.929	ctaaaTCCAtatatg
	Homeodomain transcription
	factor HNF6 (ONECUT)
V$SRFF/SRF.02	serum response factor	0.83	1177-1195	1186	(+)	1.000	0.851	aaatCCATatatgaatgag
V$CLOX/CDPCR3.01	cut-like homeodomain protein	0.75	1180-1196	1188	(−)	1.000	0.761	actcattcatatATGGa
V$PIT1/PIT1.01	Pit1, GHF-1 pituitary specific	0.86	1186-1196	1191	(−)	1.000	0.919	actcATTCata
	pou domain transcription
	factor
V$HMTB/MTBF.01	muscle-specific Mt binding	0.90	1196-1204	1200	(−)	0.807	0.901	tggtATGTa
	site
V$FKHD/HFH8.01	HNF-3/Fkh Homolog-8	0.92	1200-1216	1208	(−)	1.000	0.922	gaaagayAAACatggta
V$E4FF/E4F.01	GLI-Krueppel-related	0.82	1223-1235	1229	(−)	0.789	0.898	gtgAGGTaacccc
	transcription factor, regulator
	of adenovirus E4 promoter
V$CREB/HLF.01	hepatic leukemia factor	0.84	1221-1241	1231	(+)	1.000	0.854	atgggGTTAcctcactcagga
V$VBPF/VBP.01	PAR-type chicken vitellogenin	0.86	1226-1236	1231	(+)	1.000	0.903	gTTACctcact
	promoter-binding protein
V$OCT1/OCT.01	Octamer binding site	0.79	1259-1273	1266	(−)	0.758	0.870	cgcAGGCaaatgaat
	(OCT1/OCT2 consensus)
V$STAT/STAT6.01	STAT6: signal transducer and	0.84	1261-1279	1270	(+)	0.758	0.850	tcattTGCCtgcgaatttt
	activator of transcription 6
V$CDXF/CDX2.01	Cdx-2 mammalian caudal	0.84	1270-1288	1279	(+)	1.000	0.869	tgcgaatTTTAagattcca
	related intestinal transcr.
	factor
V$SORY/SOX9.01	SOX (SRY-related HMG box)	0.90	1280-1296	1288	(−)	1.000	0.990	taaaaCAATggaatctt
V$FKHD/HFH2.01	HNF-3/Fkh Homolog 2	0.93	1285-1301	1293	(−)	1.000	0.931	aggaataaAACAatgga
V$CDXF/CDX2.01	Cdx-2 mammalian caudal	0.84	1286-1304	1295	(+)	1.000	0.865	ccattgtTTTAttcctctg
	related intestinal transcr.
	factor
V$OCTB/TST1.01	POU-factor Tst-1/Oct-6	0.87	1288-1302	1295	(−)	0.894	0.876	gaggAATAaaacaat
V$PDX1/ISL1.01	Pancreatic and intestinal lim-	0.82	1298-1318	1308	(+)	1.000	0.824	tcctctgagTAATactccatt
	homeodomain factor
V$SORY/SOX9.01	SOX (SRY-related HMG box)	0.90	1308-1324	1316	(−)	1.000	0.925	ttacaCAATggagtatt
V$CREB/HLF.01	hepatic leukemia factor	0.84	1310-1330	1320	(−)	0.901	0.920	ggtacATTAcacaatggagta
V$VBPF/VBP.01	PAR-type chicken vitellogenin	0.86	1315-1325	1320	(−)	1.000	0.871	aTTACacaatg
	promoter-binding protein
V$CEBP/CEBPB.01	CCAAT/enhancer binding	0.94	1313-1331	1322	(+)	0.929	0.955	tccattgtGTAAtgtacca
	protein beta
V$PDX1/ISL1.01	Pancreatic and intestinal lim-	0.82	1313-1333	1323	(+)	1.000	0.859	tccattgtgTAATgtaccaca
	homeodomain factor
V$HAML/AML1.01	runt-factor AML-1	1.00	1323-1337	1330	(−)	1.000	1.000	aaaatgTGGTacatt
V$GREF/ARE.01	Androgene receptor binding	0.80	1323-1341	1332	(+)	0.750	0.819	aatgtaccacaTTTTctcc
	site
V$TEAF/TEF1.01	TEF-1 related muscle factor	0.84	1343-1355	1349	(+)	1.000	0.896	taCATTcttcagt
V$CMYB/CMYB.01	c-Myb, important in	0.99	1352-1360	1356	(+)	1.000	0.990	caGTTGagg
	hematopoesis, cellular
	equivalent to avian
	myoblastosis virus oncogene
	v-myb
V$AP4R/TH1E47.01	Thing1/E47 heterodimer, TH1	0.93	1378-1394	1386	(−)	1.000	0.932	gcaatagCCAGaacctg
	bHLH member specific
	expression in a variety of
	embryonic tissues
V$CP2F/CP2.01	CP2	0.90	1384-1394	1389	(−)	1.000	0.945	gcaatagCCAG
V$CHOP/CHOP.01	heterodimers of CHOP and	0.90	1386-1398	1392	(−)	1.000	0.951	attTGCAatagcc
	C/EBPalpha
V$CEBP/CEBP.02	C/EBP binding site	0.85	1385-1403	1394	(+)	1.000	0.853	tggctattGCAAataaccc
V$MEF2/HMEF2.01	myocyte enhancer factor	0.76	1384-1406	1395	(+)	1.000	0.809	ctggctattgcAAATaaccctgc
V$OCT1/OCT1.03	octamer-binding factor 1	0.85	1388-1402	1395	(+)	1.000	0.889	ctattgcAAATaacc
V$HMTB/MTBF.01	muscle-specific Mt binding	0.90	1394-1402	1398	(−)	1.000	0.900	ggttATTTg
	site
V$CLOX/CDPCR3.01	cut-like homeodomain protein	0.75	1422-1438	1430	(+)	0.975	0.761	acatatgtcattATTGt
V$OCT1/OCT1.05	octamer-binding factor 1	0.90	1423-1437	1430	(+)	0.944	0.938	cATATgtcattattg
V$HOXF/HOX1-3.01	Hox-1.3, vertebrate	0.83	1423-1439	1431	(+)	1.000	0.836	catatgtcATTAttgta
	homeobox protein
V$PDX1/PDX1.01	Pdx1 (IDX1/IPF1) pancreatic	0.74	1423-1443	1433	(−)	1.000	0.889	ttcatacaaTAATgacatatg
	and intestinal homeodomain
	TF
V$SORY/SOX5.01	Sox-5	0.87	1426-1442	1434	(−)	1.000	0.870	tcataCAATaatgacat
V$OCT1/OCT1.05	octamer-binding factor 1	0.90	1444-1458	1451	(−)	0.944	0.914	aATATgtaaaacaga
V$CREB/E4BP4.01	E4BP4, bZIP domain,	0.80	1443-1463	1453	(−)	1.000	0.856	tttaaaatatGTAAaacagat
	transcriptional repressor
V$VBPF/VBP.01	PAR-type chicken vitellogenin	0.86	1449-1459	1454	(+)	1.000	0.886	tTTACatattt
	promoter-binding protein
V$TBPF/MTATA.01	Muscle TATA box	0.84	1455-1471	1463	(+)	1.000	0.841	tatttTAAAccatctct
V$PBXF/PBX1.01	homeo domain factor Pbx-1	0.78	1469-1481	1475	(−)	1.000	0.783	caagCAATctaga
V$COMP/COMP1.01	COMP1, cooperates with	0.76	1467-1487	1477	(+)	1.000	0.765	tctctagATTGcttgtaatat
	myogenic proteins in
	multicomponent complex
V$SORY/SOX5.01	Sox-5	0.87	1478-1494	1486	(−)	1.000	0.997	tttaaCAATattacaag
V$FKHD/FREAC2.01	Fork head RElated ACtivator-2	0.84	1485-1501	1493	(+)	1.000	0.885	tattgtTAAAcatagag
V$PDX1/ISL1.01	Pancreatic and intestinal lim-	0.82	1495-1515	1505	(+)	1.000	0.839	catagagagTAATaatgctat
	homeodomain factor
V$HOXF/HOX1-3.01	Hox-1.3, vertebrate	0.83	1499-1515	1507	(−)	1.000	0.872	atagcattATTActctc
	homeobox protein
V$PDX1/PDX1.01	Pdx1 (IDX1/IPF1) pancreatic	0.74	1498-1518	1508	(−)	0.826	0.843	tttatagcaTTATtactctct
	and intestinal homeodomain
	TF
V$CART/XVENT2.01	Xenopus homeodomain factor	0.82	1502-1518	1510	(+)	1.000	0.829	agTAATaatgctataaa
	Xvent-2; early BMP signaling
	response
V$CDXF/CDX2.01	Cdx-2 mammalian caudal	0.84	1507-1525	1516	(−)	1.000	0.906	tttaattTTTAtagcatta
	related intestinal transcr.
	factor
V$MEF2/MEF2.05	MEF2	0.96	1505-1527	1516	(+)	1.000	0.983	aataatgctaTAAAaattaaaaa
V$HNF1/HNF1.01	hepatic nuclear factor 1	0.78	1510-1526	1518	(−)	0.755	0.805	tTTTAatttttatagca
V$OCT1/OCT1.06	octamer-binding factor 1	0.80	1511-1525	1518	(+)	1.000	0.832	gctataaaAATTaaa
V$TBPF/TATA.02	Mammalian C-type LTR TATA	0.89	1510-1526	1518	(+)	1.000	0.991	tgctaTAAAaattaaaa
	box
V$NKXH/MSX.01	Homeodomain proteins MSX-1	0.97	1514-1526	1520	(−)	1.000	0.989	tttTAATttttat
	and MSX-2
V$RBIT/BRIGHT.01	Bright, B cell regulator of IgH	0.92	1515-1527	1521	(+)	1.000	0.944	taaaaATTAaaaa
	transcription
V$MEF2/AMEF2.01	myocyte enhancer factor	0.80	1514-1536	1525	(+)	1.000	0.807	ataaaaatTAAAaataatgataa
V$EVI1/EVI1.02	Ecotropic viral integration site	0.83	1526-1542	1534	(+)	1.000	0.872	aataatgatAAGAaaga
	1 encoded factor
V$GATA/GATA1.02	GATA-binding factor 1	0.99	1528-1540	1534	(+)	1.000	0.993	taatGATAagaaa
V$GATA/GATA3.02	GATA-binding factor 3	0.91	1537-1549	1543	(+)	1.000	0.931	gaaAGATcctata
V$GATA/GATA3.02	GATA-binding factor 3	0.91	1559-1571	1565	(+)	1.000	0.915	tacAGATgaaaat
V$OCT1/OCT1.02	octamer-binding factor 1	0.82	1561-1575	1568	(+)	0.763	0.867	cagATGAaaatttag
V$CEBP/CEBPB.01	CCAAT/enhancerbinding	0.94	1567-1585	1576	(+)	0.985	0.964	aaaatttaGAAAtacttta
	protein beta
V$PLZF/PLZF.01	Promyelocytic leukemia zink	0.86	1574-1588	1581	(−)	0.958	0.866	agcTAAAgtatttct
	finger (TF with nine Krueppel-
	like zink fingers)
V$PAX3/PAX3.01	Pax-3 paired domain protein,	0.76	1587-1599	1593	(−)	1.000	0.763	TCGTcagtggtag
	expressed in embryogenesis,
	mutations correlate to
	Waardenburg Syndrome
V$CREB/ATF.01	activating transcription factor	0.90	1588-1608	1598	(+)	1.000	0.923	taccacTGACgaaatttgtat
V$AP4R/TH1E47.01	Thinq1/E47 heterodimer, TH1	0.93	1614-1630	1622	(−)	1.000	0.959	tttaattCCAGacattc
	bHLH member specific
	expression in a variety of
	embryonic tissues
V$NKXH/MSX.01	Homeodomain proteins MSX-1	0.97	1619-1631	1625	(−)	1.000	0.977	cttTAATtccaga
	and MSX-2
V$RBIT/BRIGHT.01	Bright, B cell regulator of IgH	0.92	1620-1632	1626	(+)	1.000	0.923	ctggaATTAaaga
	transcription
V$OCTB/TST1.01	POU-factor Tst-1/Oct-6	0.87	1620-1634	1627	(+)	1.000	0.898	ctggAATTaaagaaa
V$NKXH/DLX3.01	Distal-less 3 homeodomain	0.91	1628-1640	1634	(−)	1.000	0.915	cagTAATttcttt
	transcription factor
V$GREF/PRE.01	Progesterone receptor binding	0.84	1628-1646	1637	(+)	1.000	0.922	aaagaaattacTGTTcttt
	site
V$TBPF/TATA.01	cellular and viral TATA box	0.90	1636-1652	1644	(−)	1.000	0.934	ttataTAAAgaacagta
	elements
V$FKHD/XFD2.01	Xenopus fork head domain	0.89	1637-1653	1645	(−)	1.000	0.890	attataTAAAgaacagt
	factor 2
V$TBPF/TATA.01	cellular and viral TATA box	0.90	1638-1654	1646	(−)	0.891	0.923	tattaTATAaagaacag
	elements
V$CREB/E4BP4.01	E4BP4, bZIP domain,	0.80	1638-1658	1648	(−)	0.769	0.856	ctattattatATAAagaacag
	transcriptional repressor
V$PDX1/ISL1.01	Pancreatic and intestinal lim-	0.82	1644-1664	1654	(+)	1.000	0.836	tttatataaTAATagactgta
	homeodomain factor
V$COMP/COMP1.01	COMP1, cooperates with	0.76	1648-1668	1658	(+)	0.791	0.760	tataataATAGactgtaaaat
	myogenic proteins in
	multicomponent complex
V$TBPF/TATA.02	Mammalian C-type LTR TATA	0.89	1658-1674	1666	(+)	1.000	0.912	gactgTAAAatggcaac
	box
V$IRFF/ISRE.01	interferon-stimulated	0.81	1662-1676	1669	(+)	0.750	0.817	gtaaaatgGCAActt
	response element
V$XBBF/RFX1.01	X-box binding protein RFX1	0.89	1660-1678	1669	(+)	1.000	0.907	ctgtaaaatgGCAActttt
V$MYT1/MYT1.02	MyT1 zinc finger transcription	0.88	1667-1679	1673	(−)	1.000	0.882	taaAAGTtgccat
	factor involved in primary
	neurogenesis
V$OCT1/OCT1.06	octamer-binding factor 1	0.80	1683-1697	1690	(+)	1.000	0.878	tatttgctAATTcac
V$AP1F/	TCF11/MafG heterodimers,	0.81	1681-1701	1691	(−)	0.777	0.865	tcctgTGAAttagcaaatatt
TCF11MAFG.01	binding to subclass of AP1
	sites
V$NKXH/MSX2.01	Muscle segment homeo box	0.95	1687-1699	1693	(+)	1.000	0.969	tgCTAAttcacag
	2, homologue of Drosophila
	(HOX 8)
V$FAST/FAST1.01	FAST-1 SMAD interacting	0.81	1687-1701	1694	(−)	0.850	0.866	tcctgtgAATTagca
	protein
V$PBXC/	Binding site for a Pbx1/Meis1	0.76	1686-1702	1694	(+)	0.750	0788	ttgctaatTCACaggat
PBX1_MEIS1.03	heterodimer
V$CIZF/NMP4.01	NMP4 (nuclear matrix protein	0.97	1699-1709	1704	(−)	1.000	0.973	agAAAAaatcc
	4)/CIZ (Cas-interacting zinc
	finger protein)
V$STAT/STAT6.01	STAT6: signal transducer and	0.84	1702-1720	1711	(−)	1.000	0.908	agatgTTCCaaagaaaaaa
	activator of transcription 6
V$AP4R/	Tal-1beta/E47 heterodimer	0.87	1710-1726	1718	(−)	1.000	0.919	ttgttCAGAtgttccaa
TAL1BETAE47.01
V$SORY/HMGIY.01	HMGI(Y) high-mobility-group	0.92	1720-1736	1728	(+)	1.000	0.953	tgaacaAATTtccctta
	protein I (Y), architectural
	transcription factor organizing
	the framework of a nuclear
	protein-DNA transcriptional
	complex
V$MYT1/MYT1.01	MyT1 zinc finger transcription	0.75	1723-1735	1729	(+)	0.750	0.757	acaAATTtccctt
	factor involved in primary
	neurogenesis
V$SRFF/SRF.01	serum response factor	0.66	1728-1746	1737	(+)	1.000	0.771	tttccctTATAtgaatcac
V$HOXF/HOXA9.01	Member of the vertebrate	0.87	1731-1747	1739	(−)	1.000	0.908	agtGATTcatataaggg
	HOX - cluster of homeobox
	factors
V$HOXT/	Homeobox protein MEIS1	0.79	1734-1746	1740	(−)	1.000	0.797	gTGATtcatataa
MEIS1_HOXA9.01
	binding site
V$PIT1/PIT1.01	Pit1, GHF-1 pituitary specific	0.86	1737-1747	1742	(−)	1.000	0.912	agtgATTCata
	pou domain transcription
	factor
V$AP1F/AP1.01	AP1 binding site	0.95	1734-1754	1744	(+)	0.881	0.958	ttatatgaATCActtacattt
V$VBPF/VBP.01	PAR-type chicken vitellogenin	0.86	1746-1756	1751	(+)	1.000	0.860	cTTACattttt
	promoter-binding protein
V$FAST/FAST1.01	FAST-1 SMAD interacting	0.81	1757-1771	1764	(+)	0.850	0.829	gcctgttCATTtaaa
	protein
V$HOXF/EN1.01	Homeobox protein engrailed	0.77	1759-1775	1767	(−)	1.000	0.832	gtttTTTAaatgaacag
	(en-1)
V$TBPF/MTATA.01	Muscle TATA box	0.84	1763-1779	1771	(+)	1.000	0.853	tcattTAAAaaactgca
V$ETSF/ETS2.01	c-Ets-2 binding site	0.86	1774-1790	1782	(+)	1.000	0.866	actgcAGGAaagttgtg
V$MYT1/MYT1.02	MyT1 zinc finger transcription	0.88	1780-1792	1786	(+)	1.000	0.891	ggaAAGTtgtgat
	factor involved in primary
	neurogenesis
V$GFI1/GFI1.01	Growth factor independence 1	0.97	1782-1796	1789	(−)	1.000	1.000	ataAATCacaacttt
	zinc finger protein acts as
	transcriptional repressor
V$TBPF/TATA.01	cellular and viral TATA box	0.90	1784-1800	1792	(−)	1.000	0.931	cattaTAAAtcacaact
	elements
V$BRNF/BRN2.01	POU factor Brn-2 (N-Oct 3)	0.91	1786-1802	1794	(−)	1.000	0.933	tgcattatAAATcacaa
V$HOXT/	Homeobox protein MEIS1	0.79	1788-1800	1794	(+)	1.000	0.924	gTGATttataatg
MEIS1_HOXA9.01	binding site
V$MEF2/AMEF2.01	myocyte enhancer factor	0.80	1783-1805	1794	(−)	0.866	0.827	agttgcatTATAaatcacaactt
V$OCTB/TST1.01	POU-factor Tst-1/Oct-6	0.87	1787-1801	1794	(+)	0.894	0.898	tgtgATTTataatgc
V$HOXF/HOXA9.01	Member of the vertebrate	0.87	1787-1803	1795	(+)	1.000	0.971	tgtGATTtataatgcaa
	HOX - cluster of homeobox
	factors
V$BRNF/BRN2.01	POU factor Brn-2 (N-Oct 3)	0.91	1788-1804	1796	(+)	1.000	0.916	gtgatttaTAATgcaac
V$PARF/DBP.01	Albumin D-box binding	0.84	1791-1805	1798	(+)	0.884	0.891	atttaTAATgcaact
	protein
V$OCT1/OCT1.02	octamer-binding factor 1	0.82	1795-1809	1802	(+)	1.000	0.861	ataATGCaactgcac
V$FKHD/FREAC2.01	Fork head RElated ACtivator-2	0.84	1816-1832	1824	(+)	1.000	0.910	cagtctTAAAcaatgct
V$SORY/SOX5.01	Sox-5	0.87	1821-1837	1829	(+)	1.000	0.992	ttaaaCAATgctaacca
V$AREB/AREB6.04	AREB6 (Atp1a1 regulatory	0.98	1837-1849	1843	(+)	1.000	0.981	actgtGTTTcagc
	element binding factor 6)
V$MYT1/MYT1.02	MyT1 zinc finger transcription	0.88	1848-1860	1854	(−)	1.000	0.889	gggAAGTttatgc
	factor involved in primary
	neurogenesis
V$RBPF/RBPJK.01	Mammalian transcriptional	0.84	1851-1865	1858	(−)	1.000	0.878	tgtgTGGGaagttta
	repressor RBP-Jkappa/CBF1
V$OCT1/OCT1.02	octamer-binding factor 1	0.82	1875-1889	1882	(+)	0.763	0.826	actATGAaaacacat
V$FKHD/FREAC4.01	Fork head RElated ACtivator-4	0.78	1875-1891	1883	(+)	1.000	0.786	actatgaaAACAcatgc
V$EBOX/MYCMAX.02	c-Myc/Max heterodimer	0.92	1880-1896	1888	(+)	0.895	0.920	gaaaaCACAtgcttaaa
V$PAX6/PAX6.01	Pax-6 paired domain protein	0.75	1880-1898	1889	(−)	0.773	0.791	cctttAAGCatgtgttttc
V$IRFF/IRF3.01	Interferon regulatory factor 3	0.86	1891-1905	1898	(+)	1.000	0.874	cttaaaggCAAAtct
	(IRF-3)
V$HNF1/HNF1.02	Hepatic nuclear factor 1	0.76	1895-1911	1903	(−)	0.858	0.782	aGGTAaagatttgcctt
V$FKHD/FREAC2.01	Fork head RElated ACtivator-2	0.84	1898-1914	1906	(−)	1.000	0.853	ctgaggTAAAgatttgc
V$E4FF/E4F.01	GLI-Krueppel-related	0.82	1902-1914	1908	(−)	0.789	0.830	ctgAGGTaaagat
	transcription factor, regulator
	of adenovirus E4 promoter
V$CREB/CREBP1.01	cAMP-responsive element	0.80	1900-1920	1910	(+)	0.766	0.820	aaatctttACCTcagttaact
	binding protein 1
V$VBPF/VBP.01	PAR-type chicken vitellogenin	0.86	1905-1915	1910	(+)	1.000	0.862	tTTACctcagt
	promoter-binding protein
V$MYT1/MYT1.01	MyT1 zinc finger transcription	0.75	1912-1924	1918	(−)	0.750	0.775	gaaTAGTtaactg
	factor involved in primary
	neurogenesis
V$HNF1/HNF1.01	hepatic nuclear factor 1	0.78	1913-1929	1921	(+)	1.000	0.811	aGTTAactattccatag
V$PCAT/CAAT.01	cellular and viral CCAAT box	0.90	1928-1938	1933	(+)	0.856	0.925	agagCCATtga
V$HNF6/HNF6.01	Liver enriched Cut -	0.82	1929-1943	1936	(−)	1.000	0.873	tgaacTCAAtggctc
	Homeodomain transcription
	factor HNF6 (ONECUT)
V$PXRF/PXRCAR.01	Halfsite of PXR (pregnane X	0.98	1935-1945	1940	(−)	1.000	0.980	ctTGAActcaa
	receptor)/RXR resp. CAR
	(constitutive androstane
	receptor)/RXR heterodimer
	binding site
V$RARF/RTR.01	Retinoid receptor-related	0.81	1934-1952	1943	(+)	1.000	0.854	attgagtTCAAgtgcattt
	testis-associated receptor
	(GCNF/RTR)
V$HOXF/EN1.01	Homeobox protein engrailed	0.77	1936-1952	1944	(+)	0.782	0.813	tgagTTCAagtgcattt
	(en-1)
V$NKXH/NKX25.01	homeo domain factor Nkx-	1.00	1939-1951	1945	(+)	1.000	1.000	gttcAAGTgcatt
	2.5/Csx, tinman homolog,
	high affinity sites
V$GATA/GATA3.02	GATA-binding factor 3	0.91	1953-1965	1959	(+)	1.000	0.928	agaAGATataatg
V$TBPF/TATA.01	cellular and viral TATA box	0.90	1968-1984	1976	(−)	0.891	0.912	atataTATAtggccata
	elements
V$SRFF/SRF.01	serum response factor	0.66	1969-1987	1978	(+)	1.000	0.777	atggccaTATAtatatata
V$CLOX/CDPCR3.01	cut-like homeodomain protein	0.75	1972-1988	1980	(−)	1.000	0.806	atatatatatatATGGc
V$PAX1/PAX1.01	Pax1 paired domain protein,	0.61	2016-2034	2025	(−)	0.750	0.675	CTGTgctgatatatatata
	expressed in the developing
	vertebral column of mouse
	embryos
V$TBPF/ATATA.01	Avian C-type LTR TATA box	0.81	2019-2035	2027	(+)	0.750	0.827	atatataTCAGcacagt
V$GFI1/GfI1B.01	Growth factor independence 1	0.82	2021-2035	2028	(+)	1.000	0.904	ataTATCagcacagt
	zinc finger protein Gfi-1B
V$NRSF/NRSF.01	neuron-restrictive silencer	0.69	2025-2045	2035	(+)	1.000	0.704	atcAGCAcagtggaaacagtt
	factor
V$NFAT/NFAT.01	Nuclear factor of activated T-	0.97	2033-2043	2038	(+)	1.000	0.970	agtgGAAAcag
	cells
V$AREB/AREB6.04	AREB6 (Atp1a1 regulatory	0.98	2034-2046	2040	(−)	1.000	0.991	taactGTTTccac
	element binding factor 6)
V$HNF1/HNF1.01	hepatic nuclear factor 1	0.78	2036-2052	2044	(−)	1.000	0.798	tGTTAttaactgtttcc
V$FKHD/XFD3.01	Xenopus fork head domain	0.82	2038-2054	2046	(+)	0.826	0.824	aaacagttAATAacatt
	factor 3
V$PDX1/PDX1.01	Pdx1 (IDX1/IPF1) pancreatic	0.74	2036-2056	2046	(+)	1.000	0.749	ggaaacagtTAATaacatttt
	and intestinal homeodomain
	TF
V$OCT1/OCT1.01	octamer-binding factor 1	0.77	2050-2064	2057	(−)	1.000	0.863	taTATGctaaaatgt
V$TBPF/TATA.01	cellular and viral TATA box	0.90	2053-2069	2061	(−)	0.891	0.908	tagtaTATAtgctaaaa
	elements
V$ETSF/GABP.01	GABP: GA binding protein	0.85	2080-2096	2088	(+)	1.000	0.897	gaggctGGAAgggggct
V$BEL1/BEL1.01	Bel-1 similar region (defined	0.78	2083-2105	2094	(+)	1.000	0.787	gctggaagggggcTCAGcagtta
	in Lentivirus LTRs)
V$VMYB/VMYB.01	v-Myb	0.90	2097-2107	2102	(−)	0.876	0.901	attAACTgctg
V$GREF/ARE.01	Androgene receptor binding	0.80	2106-2124	2115	(+)	0.750	0.840	atagcacatacTATTcttc
	site
V$PDX1/PDX1.01	Pdx1 (IDX1/IPF1) pancreatic	0.74	2137-2157	2147	(+)	0.782	0.747	gtttggtttTCATcacccatg
	and intestinal homeodomain
	TF
V$MYOD/MYOD.02	myoblast determining factor	0.98	2154-2168	2161	(−)	1.000	0.988	gaacCACCtgacatg
V$GATA/GATA1.03	GATA-binding factor 1	0.95	2169-2181	2175	(−)	1.000	0.958	tacaGATAgaaat
V$AP4R/	Tal-1beta/E47 heterodimer	0.87	2179-2195	2187	(+)	1.000	0.924	gtaacCAGAtgatacga
TAL1BETAE47.01
V$OAZF/ROAZ.01	Rat C2H2 Zn finger protein	0.73	2204-2220	2212	(−)	0.750	0.762	agGTACccaaggggact
	involved in olfactory neuronal
	differentiation
V$GATA/GATA1.01	GATA-binding factor 1	0.96	2217-2229	2223	(−)	1.000	0.960	aggtGATAgaggt
V$MYOD/E47.02	TAL1/E47 dimers	0.93	2220-2234	2227	(−)	1.000	0.939	atagCAGGtgataga
V$LTUP/TAACC.01	Lentiviral TATA upstream	0.71	2225-2247	2236	(+)	0.759	0.710	cacctgctattctCACCcaaaga
	element
V$RREB/RREB1.01	Ras-responsive element	0.79	2239-2253	2246	(+)	1.000	0.805	aCCCAaagacacaca
	binding protein 1
V$OCT1/OCT1.05	octamer-binding factor 1	0.90	2251-2265	2258	(−)	0.944	0.904	tGTATgtgagtgtgt
V$OCT1/OCT1.02	octamer-binding factor 1	0.82	2282-2296	2289	(+)	1.000	0.854	tgcATGCacatagtt
V$COUP/COUP.01	COUP antagonizes HNF-4 by	0.81	2284-2298	2291	(−)	0.977	0.855	tGAACtatgtgcatg
	binding site competition or
	synergizes by direct
	protein-
	protein interaction with HNF-4
V$MEF2/MEF2.01	myogenic enhancer factor 2	0.74	2290-2312	2301	(+)	0.750	0.767	catagttcAAAAaataaaatttt
V$CDXF/CDX2.01	Cdx-2 mammalian caudal	0.84	2296-2314	2305	(−)	1.000	0.896	ttaaaatTTTAttttttga
	related intestinal transcr.
	factor
V$MYT1/MYT1.01	MyT1 zinc finger transcription	0.75	2301-2313	2307	(−)	0.750	0.798	taaAATTttattt
	factor involved in primary
	neurogenesis
V$NFAT/NFAT.01	Nuclear factor of activated T-	0.97	2314-2324	2319	(+)	1.000	0.991	aaagGAAAaaa
	cells
V$CIZF/NMP4.01	NMP4 (nuclear matrix protein	0.97	2317-2327	2322	(+)	1.000	0.977	ggAAAAaaagc
	4)/CIZ (Cas-interacting zinc
	finger protein)
V$GATA/GATA3.02	GATA-binding factor 3	0.91	2326-2338	2332	(−)	1.000	0.946	aaaAGATttgagc
V$HMTB/MTBF.01	muscle-specific Mt binding	0.90	2351-2359	2355	(−)	1.000	0.901	aggaATTTt
	site
V$NOLF/OLF1.01	olfactory neuron-specific	0.82	2350-2372	2361	(+)	0.806	0.820	taaaatTCCTatgagtgtgtgat
	factor
V$PDX1/PDX1.01	Pdx1 (IDX1/IPF1) pancreatic	0.74	2363-2383	2373	(−)	0.782	0.753	tactgacttTGATcacacact
	and intestinal homeodomain
	TF
V$GATA/GATA3.02	GATA-binding factor 3	0.91	2395-2407	2401	(−)	1.000	0.942	cacAGATtatacc
V$NFAT/NFAT.01	Nuclear factor of activated T-	0.97	2406-2416	2411	(+)	1.000	0.971	tgtgGAAAaca
	cells
V$OCTP/OCT1P.01	octamer-binding factor 1,	0.86	2433-2445	2439	(+)	0.980	0.879	ctcagtATTCaca
	POU-specific domain
V$MITF/MIT.01	MIT (microphthalmia	0.81	2438-2456	2447	(−)	1.000	0.827	ctactttCATGtgtgaata
	transcription factor) and TFE3
V$PAX8/PAX8.01	PAX 2/5/8 binding site	0.88	2441-2453	2447	(−)	0.850	0.952	cttTCATgtgtga
V$TBPF/ATATA.01	Avian C-type LTR TATA box	0.81	2451-2467	2459	(+)	1.000	0.838	aagtagcTAAGaataaa
V$GATA/GATA3.02	GATA-binding factor 3	0.91	2462-2474	2468	(−)	1.000	0.960	aatAGATtttatt
V$CLOX/CLOX.01	Clox	0.81	2462-2478	2470	(+)	0.806	0.819	aataaaATCTattcatc
V$HNF6/HNF6.01	Liver enriched Cut-	0.82	2464-2478	2471	(+)	0.785	0.846	taaaaTCTAttcatc
	Homeodomain transcription
	factor HNF6 (ONECUT)
V$PIT1/PIT1.01	Pit1, GHF-1 pituitary specific	0.86	2468-2478	2473	(+)	1.000	0.890	atctATTCatc
	pou domain transcription
	factor
V$AP4R/	Tal-1beta/ITF-2 heterodimer	0.85	2469-2485	2477	(−)	1.000	0.881	aaaaaCAGAtgaataga
TAL1BETAITF2.01
V$CIZF/NMP4.01	NMP4 (nuclear matrix protein	0.97	2477-2487	2482	(−)	1.000	0.981	ggAAAAacaga
	4)/CIZ (Cas-interacting zinc
	finger protein)
V$NFAT/NFAT.01	Nuclear factor of activated T-	0.97	2480-2490	2485	(−)	1.000	0.976	taagGAAAaac
	cells
V$STAT/STAT.01	signal transducers and	0.87	2479-2497	2488	(−)	1.000	0.872	aggattttaaGGAAaaaca
	activators of transcription
V$TBPF/TATA.02	Mammalian C-type LTR TATA	0.89	2484-2500	2492	(+)	1.000	0.897	ttcctTAAAatcctggc
	box
V$FKHD/XFD3.01	Xenopus fork head domain	0.82	2501-2517	2509	(−)	1.000	0.880	actgagtcAACActgta
	factor 3
V$AP1F/AP1.01	AP1 binding site	0.95	2500-2520	2510	(−)	1.000	0.984	accactgaGTCAacactgtag
V$AP1F/AP1.01	AP1 binding site	0.95	2504-2524	2514	(+)	0.964	0.984	agtgttgaCTCAgtggttgct
V$PCAT/CAAT.01	cellular and viral CCAAT box	0.90	2513-2523	2518	(−)	0.826	0.904	gcaaCCACtga
V$CDXF/CDX2.01	Cdx-2 mammalian caudal	0.84	2524-2542	2533	(+)	1.000	0.883	tttaaatTTTAtgctcaaa
	related intestinal transcr.
	factor
V$MYT1/MYT1.02	MyT1 zinc finger transcription	0.88	2539-2551	2545	(+)	1.000	0.891	caaAAGTtgaagc
	factor involved in primary
	neurogenesis
V$ETSF/FLI.01	ETS family member FLI	0.81	2560-2576	2568	(+)	1.000	0.829	tgaaCCGGtaattctac
V$MYT1/MYT1.01	MyT1 zinc finger transcription	0.75	2569-2581	2575	(−)	1.000	0.757	acaAAGTagaatt
	factor involved in primary
	neurogenesis
V$TBPF/ATATA.01	Avian C-type LTR TATA box	0.81	2576-2592	2584	(−)	0.750	0.816	aagtattTAATacaaag
V$SATB/SATB1.01	Special AT-rich sequence-	0.93	2578-2594	2586	(−)	1.000	0.939	acaagtattTAATacaa
	binding protein 1,
	predominantly expressed in
	thymocytes, binds to matrix
	attachment regions (MARs)
V$NKXH/NKX31.01	prostate-specific	0.84	2584-2596	2590	(−)	1.000	0.865	taacAAGTattta
	homeodomain protein NKX3.1
V$PARF/DBP.01	Albumin D-box binding	0.84	2589-2603	2596	(+)	1.000	0.882	acttgTTATgcatcg
	protein
V$PAX5/PAX5.02	B-cell-specific activating	0.75	2591-2619	2605	(−)	1.000	0.758	aacttgatttgttgAGCGatgcataacaa
	protein
V$ECAT/NFY.03	nuclear factor Y (Y-box	0.80	2604-2618	2611	(+)	0.750	0.809	ctcaaCAAAtcaagt
	binding factor)
V$GFI1/GFI1.01	Growth factor independence 1	0.97	2608-2622	2615	(+)	1.000	0.976	acaAATCaagtttta
	zinc finger protein acts as
	transcriptional repressor
V$HNF6/HNF6.01	Liver enriched Cut -	0.82	2608-2622	2615	(+)	1.000	0.830	acaaaTCAAgtttta
	Homeodomain transcription
	factor HNF6 (ONECUT)
V$MYT1/MYT1.01	MyT1 zinc finger transcription	0.75	2610-2622	2616	(−)	0.750	0.756	taaAACTtgattt
	factor involved in primary
	neurogenesis
V$PAX8/PAX8.01	PAX 2/5/8 binding site	0.88	2610-2622	2616	(+)	1.000	0.907	aaaTCAAgtttta
V$TTFF/TTF1.01	Thyroid transcription factor-1	0.92	2609-2623	2616	(+)	1.000	0.936	caaatCAAGttttaa
	(TTF1) binding site
V$MYT1/MYT1.02	MyT1 zinc finger transcription	0.88	2612-2624	2618	(+)	1.000	0.887	atcAAGTtttaac
	factor involved in primary
	neurogenesis
V$CDXF/CDX2.01	Cdx-2 mammalian caudal	0.84	2612-2630	2621	(+)	1.000	0.883	atcaagtTTTAacacacca
	related intestinal transcr.
	factor
V$SORY/HMGIY.01	HMGI(Y) high-mobility-group	0.92	2649-2665	2657	(−)	1.000	0.925	ttaaaaAATTtaagata
	protein I (Y), architectural
	transcription factor organizing
	the framework of a nuclear
	protein-DNA transcriptional
	complex
V$HOXF/EN1.01	Homeobox protein engrailed	0.77	2657-2673	2665	(+)	1.000	0.780	atttTTTAaatgggcat
	(en-1)
V$OCT1/OCT1.06	octamer-binding factor 1	0.80	2662-2676	2669	(−)	0.750	0.818	tttatgccCATTtaa
V$BCL6/BCL6.01	POZ/zinc finger protein,	0.76	2683-2699	2691	(+)	1.000	0.796	ctaTTCCtacagaagtc
	transcriptional repressor,
	translocations observed in
	diffuse large cell lymphoma
V$OCTP/OCT1P.01	octamer-binding factor 1,	0.86	2715-2727	2721	(+)	1.000	0.860	ctgaaaATGCatt
	POU-specific domain
V$TEAF/TEF1.01	TEF-1 related muscle factor	0.84	2722-2734	2728	(+)	1.000	0.898	tgCATTcctgatt
V$GFI1/GFI1.01	Growth factor independence 1	0.97	2723-2737	2730	(−)	1.000	0.981	ataAATCaggaatgc
	zinc finger protein acts as
	transcriptional repressor
V$HOXT/	Homeobox protein MEIS1	0.79	2729-2741	2735	(+)	1.000	0.929	cTGATttatgtaa
MEIS1_HOXA9.01	binding site
V$HOXF/HOXA9.01	Member of the vertebrate	0.87	2728-2744	2736	(+)	1.000	0.964	cctGATTtatgtaaata
	HOX - cluster of homeobox
	factors
V$PARF/DBP.01	Albumin D-box binding	0.84	2729-2743	2736	(+)	1.000	0.861	ctgatTTATgtaaat
	protein
V$VBPF/VBP.01	PAR-type chicken vitellogenin	0.86	2732-2742	2737	(−)	1.000	0.929	tTTACataaat
	promoter-binding protein
V$CREB/E4BP4.01	E4BP4, bZIP domain,	0.80	2728-2748	2738	(+)	1.000	0.943	cctgatttatGTAAatatatg
	transcriptional repressor
V$OCT1/OCT1.01	octamer-binding factor 1	0.77	2733-2747	2740	(+)	1.000	0.895	ttTATGtaaatatat
V$FKHD/XFD1.01	Xenopus fork head domain	0.90	2733-2749	2741	(+)	1.000	0.940	tttatgTAAAtatatgt
	factbr 1
V$SRFF/SRF.01	serum response factor	0.66	2736-2754	2745	(+)	1.000	0.691	atgtaaaTATAtgtatata
V$OCTP/OCT1P.01	octamer-binding factor 1,	0.86	2746-2758	2752	(+)	0.849	0.883	atgtatATACata
	POU-specific domain
V$CLOX/CDPCR3.01	cut-like homeodomain protein	0.75	2748-2764	2756	(+)	0.888	0.755	gtatatacatatATAGc
V$TBPF/TATA.01	cellular and viral TATA box	0.90	2749-2765	2757	(−)	0.891	0.903	ggctaTATAtgtatata
	elements
V$SRFF/SRF.01	serum response factor	0.66	2750-2768	2759	(+)	1.000	0.709	atatacaTATAtagcctta
V$TBPF/ATATA.01	Avian C-type LTR TATA box	0.81	2759-2775	2767	(−)	1.000	0.816	ttgttttTAAGgctata
V$TBPF/TATA.02	Mammalian C-type LTR TATA	0.89	2762-2778	2770	(+)	1.000	0.899	agcctTAAAaacaaaga
	box
V$CABL/CABL.01	Multifunctional c-Abl src type	0.97	2769-2779	2774	(+)	1.000	0.973	aaAACAaagat
	tyrosine kinase
V$LEFF/LEF1.01	TCF/LEF-1, involved in the	0.86	2766-2782	2774	(+)	1.000	0.863	ttaaaaaCAAAgattgt
	Wnt signal transduction
	pathway
V$OCT1/OCT1.06	octamer-binding factor 1	0.80	2775-2789	2782	(+)	1.000	0.811	aagattgtAATTttt
V$MEF2/MMEF2.01	myocyte enhancer factor	0.90	2776-2798	2787	(−)	1.000	0.900	acaatttaTAAAaattacaatct
V$OCT1/OCT1.06	octamer-binding factor 1	0.80	2780-2794	2787	(−)	1.000	0.844	tttataaaAATTaca
V$TBPF/TATA.01	cellular and viral TATA box	0.90	2779-2795	2787	(−)	1.000	0.956	atttaTAAAaattacaa
	elements
V$CART/CART1.01	Cart-1 (cartilage	0.84	2780-2796	2788	(+)	1.000	0.875	tgTAATttttataaatt
	homeoprotein 1)
V$FKHD/XFD2.01	Xenopus fork head domain	0.89	2780-2796	2788	(−)	1.000	0.903	aatttaTAAAaattaca
	factor 2
V$MEF2/MEF2.05	MEF2	0.96	2778-2800	2789	(−)	1.000	0.973	tcacaatttaTAAAaattacaat
V$BRNF/BRN3.01	POU transcription factor Brn-3	0.78	2785-2801	2793	(−)	0.750	0.798	atcACAAtttataaaaa
V$TBPF/TATA.01	cellular and viral TATA box	0.90	2786-2802	2794	(+)	1.000	0.927	ttttaTAAAttgtgatt
	elements
V$GFI1/GFI1.01	Growth factor independence 1	0.97	2791-2805	2798	(−)	1.000	0.997	aaaAATCacaattta
	zinc finger protein acts as
	transcriptional repressor
V$HOXT/	Homeobox protein MEIS1	0.79	2797-2809	2803	(+)	1.000	0.806	gTGATttttaaaa
MEIS1_HOXA9.01	binding site
V$MEF2/MMEF2.01	myocyte enhancer factor	0.90	2792-2814	2803	(−)	1.000	0.923	tattttttTAAAaatcacaattt
V$MEF2/MEF2.05	MEF2	0.96	2795-2817	2806	(+)	1.000	0.990	ttgtgattttTAAAaaaataaac
V$MEF2/MMEF2.01	myocyte enhancer factor	0.90	2797-2819	2808	(+)	1.000	0.905	gtgattttTAAAaaaataaacct
V$HNF1/HNF1.01	hepatic nuclear factor 1	0.78	2802-2818	2810	(−)	0.755	0.796	gGTTTatttttttaaaa
V$MEF2/MEF2.01	myogenic enhancer factor 2	0.74	2799-2821	2810	(+)	0.750	0.775	gatttttaAAAAaataaacctgc
V$HOXF/HOX1-3.01	Hox-1.3, vertebrate	0.83	2814-2830	2822	(+)	1.000	0.848	aaacctgcATTAtcttc
	homeobox protein
V$PARF/DBP.01	Albumin D-box binding	0.84	2816-2830	2823	(−)	0.884	0.851	gaagaTAATgcaggt
	protein
V$PDX1/ISL1.01	Pancreatic and intestinal lim-	0.82	2814-2834	2824	(−)	1.000	0.853	tgctgaagaTAATgcaggttt
	homeodomain factor
V$GATA/GATA1.02	GATA-binding factor 1	0.99	2819-2831	2825	(−)	1.000	0.993	tgaaGATAatgca
V$HEAT/HSF1.01	heat shock factor 1	0.93	2845-2855	2850	(+)	0.867	0.951	TGAAtgttcct
V$MYT1/MYT1.02	MyT1 zinc finger transcription	0.88	2853-2865	2859	(+)	1.000	0.893	cctAAGTtttgta
	factor involved in primary
	neurogenesis
V$BCL6/BCL6.02	POZ/zinc finger protein,	0.77	2857-2873	2865	(+)	1.000	0.772	agttttgTAGAacttga
	transcriptional repressor,
	translocations observed in
	diffuse large cell lymphoma
V$TTFF/TTF1.01	Thyroid transcription factor-1	0.92	2863-2877	2870	(−)	1.000	0.927	cgtgtCAAGttctac
	(TTF1) binding site
V$EBOX/USF.02	upstream stimulating factor	0.94	2868-2884	2876	(−)	1.000	0.997	tctgccaCGTGtcaagt
V$HOXF/PTX1.01	Pituitary Homeobox 1 (Ptx1)	0.79	2892-2908	2900	(+)	1.000	0.795	aggattTTAGtctacac
V$MYOD/	complex of Lmo2 bound to	0.98	2901-2915	2908	(−)	1.000	0.981	gatgCAGGtgtagac
LMO2COM.01	Tal-1, E2A proteins, and
	GATA-1, half-site 1
V$REBV/EBVR.01	Epstein-Barr virus	0.81	2904-2924	2914	(−)	1.000	0.832	ctgtcctcagatgcaGGTGta
	transcription factor R
V$ETSF/PU1.01	Pu.1 (Pu120) Ets-like	0.86	2932-2948	2940	(+)	1.000	0.873	ctaacaGGAAaggagac
	transcription factor identified
	in lymphoid B-cells
V$MITF/MIT.01	MIT (microphthalmia	0.81	2943-2961	2952	(+)	1.000	0.829	ggagacaCATGtgtggtag
	transcription factor) and TFE3
V$HAML/AML1.01	runt-factor AML-1	1.00	2950-2964	2957	(+)	1.000	1.000	catgtgTGGTagttc
V$NFKB/CREL.01	c-Rel	0.91	2954-2968	2961	(+)	1.000	0.919	tgtggtagTTCCcag
V$IKRS/IK3.01	Ikaros 3, potential regulator	0.84	2958-2970	2964	(−)	1.000	0.841	aactgGGAActac
	of lymphocyte differentiation
V$RBPF/RBPJK.01	Mammalian transcriptional	0.84	2957-2971	2964	(−)	1.000	0.842	aaacTGGGaactacc
	repressor RBP-Jkappa/CBF1
V$E2FF/E2F.01	E2F, involved in cell cycle	0.74	2966-2980	2973	(−)	0.750	0.784	ttcacgtCAAAactg
	regulation, interacts with Rb
	p107 protein
V$E4FF/E4F.01	GLI-Krueppel-related	0.82	2968-2980	2974	(−)	1.000	0.830	ttcACGTcaaaac
	transcription factor, regulator
	of adenovirus E4 promoter
V$CREB/ATF6.02	Activating transcription factor	0.85	2966-2986	2976	(+)	1.000	0.985	cagttttGACGtgaaaagtcc
	6, member of b-zip family,
	induced by ER stress
V$EBOX/ARNT.01	AhR nuclear translocator	0.89	2968-2984	2976	(+)	1.000	0.891	gttttgaCGTGaaaagt
	homodimers
V$E4FF/E4F.01	GLI-Krueppel-related	0.82	2971-2983	2977	(+)	1.000	0.909	ttgACGTgaaaag
	transcription factor, regulator
	of adenovirus E4 promoter
V$EBOR/XBP1.01	X-box-binding protein 1	0.86	2970-2984	2977	(+)	1.000	0.890	tttgACGTgaaaagt
V$E2FF/E2F.01	E2F, involved in cell cycle	0.74	2971-2985	2978	(+)	1.000	0.837	ttgacgtGAAAagtC
	regulation, interacts with Rb
	p107 protein
V$STAT/STAT.01	signal transducers and	0.87	2989-3007	2998	(+)	1.000	0.937	cattcttactGGAAacctc
	activators of transcription
V$BCL6/BCL6.02	POZ/zinc finger protein,	0.77	2991-3007	2999	(+)	0.800	0.805	ttcttacTGGAaacctc
	transcriptional repressor,
	translocations observed in
	diffuse large cell lymphoma
V$XSEC/STAF.01	Se-Cys tRNA gene	0.77	3003-3025	3014	(+)	0.782	0.791	acctCCCTgaatccatgccaagc
	transcription activating factor
V$NF1F/NF1.01	Nuclear factor 1	0.94	3007-3025	3016	(−)	1.000	0.964	gctTGGCatggattcaggg
V$OCT1/OCT1.02	octamer-binding factor 1	0.82	3014-3028	3021	(+)	1.000	0.820	tccATGCcaagcact
V$RCAT/	Mammalian C-type LTR	0.75	3019-3043	3031	(+)	1.000	0.787	gCCAAgcactacccatcaccttgac
CLTR_CAAT.01	CCAAT box
V$SF1F/SF1.01	SF1 steroidogenic factor 1	0.95	3033-3045	3039	(−)	1.000	0.954	cagtCAAGgtgat
V$OCT1/OCT1.01	octamer-binding factor 1	0.77	3038-3052	3045	(−)	1.000	0.800	ctTATGccagtcaag
V$PARF/DBP.01	Albumin D-box binding	0.84	3042-3056	3049	(−)	1.000	0.862	agtgcTTATgccagt
	protein
V$ETSF/ETS1.01	c-Ets-1 binding site	0.92	3057-3073	3065	(−)	1.000	0.920	atcaaAGGAaatgagtg
V$LEFF/LEF1.01	TCF/LEF-1, involved in the	0.86	3062-3078	3070	(−)	1.000	0.969	ggggcatCAAAggaaat
	Wnt signal transduction
	pathway
V$MAZF/MAZ.01	Myc associated zinc finger	0.90	3072-3084	3078	(−)	1.000	0.912	gaggGAGGggcat
	protein (MAZ)
V$SP1F/GC.01	GC box elements	0.88	3071-3085	3078	(−)	0.876	0.920	tgagGGAGgggcatc
V$TBPF/TATA.01	cellular and viral TATA box	0.90	3091-3107	3099	(+)	1.000	0.973	tattaTAAAagcacagt
	elements
V$SEF1/SEF1.01	SEF1 binding site	0.69	3099-3117	3108	(−)	1.000	0.700	gaaagagacgaCTGTgctt

[0087]

Claims

1. An isolated nucleic acid sequence of SEQ ID NO: 2.

2. An isolated nucleic acid sequence comprising at least about nucleotides 1-3137 of SEQ ID NO: 2 (SEQ ID NO: 23).

3. A reporter construct, comprising: a. a nucleotide sequence which encodes a detectable product; and b. a regulatory region nucleotide sequence linked to the 5′ end of nucleotide sequence that encodes a detectable product, wherein the regulatory region nucleotide sequence comprises of one or more regions chosen from nucleotides 1-3137 of SEQ ID NO: 2.

4. A reporter construct of claim 3, further comprising: a. a promoter element interposed between the regulatory region nucleotide sequence and the nucleotide sequence encoding the detectable product.

5. The reporter construct of claim 3 wherein the regulatory region nucleotide sequence is selected from the group consisting of SEQ ID NO: 1, 2, 23, 32, 35, 37, 28, 24, 25, 26, 27, 29, 30, 31, 33, 34, 38, and 36.

6. The reporter construct of claim 4 wherein the regulatory region nucleotide sequence is selected from the group consisting of SEQ ID NO: 1, 2, 23, 32, 35, 37, 28, 24, 25, 26, 27, 29, 30, 31, 33, 34, 38, and 36.

7. The reporter construct of claim 4 wherein the promoter element is selected from SEQ ID NO: 2.

8. A host cell comprising the reporter construct of claim 3.

9. A host cell comprising the reporter construct of claim 4.

10. A method for identifying agents which modulate INGAP expression, comprising: a. contacting the host cell of claim 8 with a test agent; b. determining expression of the detectable protein or nucleic acid product in the cell; and c. identifying the test agent as a modulator of INGAP expression if the test agent modulates expression of the detectable product in the cell.

11. A method for identifying agents which modulate INGAP expression, comprising: a. contacting the host cell of claim 9 with a test agent; b. determining expression of the detectable protein or nucleic acid product in the cell; and c. identifying the test agent as a modulator of INGAP expression if the test agent modulates expression of the detectable product in the cell.

12. An in vitro method for identifying agents which modulate INGAP expression, comprising: a. contacting a reporter construct according to claim 3 with a test substance under conditions sufficient for transcription and translation of said nucleotide sequence; b. determining expression of the detectable protein or nucleic acid product; and c. identifying the test substance as a modulator of INGAP expression if the test substance modulates expression of the detectable product.

13. An in vitro method for identifying agents which modulate INGAP expression, comprising: a. contacting a reporter construct according to claim 4 with a test substance under conditions sufficient for transcription and translation of said nucleotide sequence; b. determining expression of the detectable protein or nucleic acid product; and c. identifying the test substance as a modulator of INGAP expression if the test substance modulates expression of the detectable product.

14. An in vitro method for identifying agents which modulate INGAP expression, comprising: a. contacting the nucleic acid or fragments thereof of claim 2 with a test agent; b. determining binding of the test agent to the nucleic acid; and c. identifying the test agent as a potential modulator of INGAP expression if the test agent binds to the nucleic acid.

15. A method for modulating INGAP expression in a mammal in need thereof, comprising administering to the mammal an effective amount of a factor that stimulates INGAP expression in the said mammal.

16. The method of claim 15 further comprising: administering to the mammal an effective amount of hLIF.

17. The method of claim 15 further comprising: administering to the mammal an effective amount of PMA.