# Patent

## Title
Regulatory sequences for modulation of INGAP expression and reporter constructs

## Abstract

A an isolated mammalian INGAP 5′′-regulatory region comprising at least about nucleotides 1-3137 of SEQ ID NO: 2 (SEQ ID NO: 23).

## Background
This application incorporates by reference provisional applications Ser. No. 60/388,315 filed Jun. 14, 2002, Ser. No. 60/361,073 filed Mar. 1, 2002, and Ser. No. 60/346,898 filed Jan. 11, 2002.


FIELD OF THE INVENTION
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
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.
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
It is an object of the invention to provide a reporter construct containing the 5′-regulatory region from mammalian INGAP gene.
It is another object of the invention to provide methods for identifying agents which modulate INGAP expression.
It is another object of the invention to provide a nucleic acid or fragment of INGAP 5′-regulatory region.
It is another object of the invention to provide methods for increasing INGAP expression.
It is another object of the invention to provide a kit for modulating INGAP expression.
These and other objects of the invention are provided by one or more of the embodiments described below.
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.
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.
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.
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.
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.
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.
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.
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.
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
FIG. 1 shows the annotation of the hamster INGAP gene structure. The boundaries of introns 1-5 are listed in Table 1.
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).
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.
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)
FIG. 5 shows the adapter primer structure and sequence used in gene walking. Adapter primer 1 (API) and adapter primer 2 (AP2) are shown.
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.
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.
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.
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.
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.
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 β-galactosidase luminescent assay.
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
Definitions
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.
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.
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.
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 β-galactosidase (lacZ), chloramphenicol acetyltransferase (cat), β-glucuronidase (GUS), and green fluorescent protein (GFP).
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.
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.
INGAP Promoter and Regulatory Region
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.
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.
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










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 professionalTM, 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).
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 professionalTM is referenced:
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 professionalTM.
Family: Each matrix belongs to a so-called matrix family, where functionally similar matrices are grouped together, eliminating redundant matches by MatInspector professionalTM 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$CREL.01).
Matrix: The MatInspector professionalTM matrices have an identifier that indicates one of the following seven groups: vertebrates (V$), 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.
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.
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.
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.
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.
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.
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/seq_tools/promoter.html.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The following examples are offered by way of illustration and do not limit the invention disclosed herein.
EXAMPLES
Example 1
Hamster INGAP Genomic Sequence and Structure
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.
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.
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)









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
Cloning Hamster INGAP 5′-Regulatory Region Fragment into a Reporter Construct
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.
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
Assay System to Screen for Factors that Modulate the Expression of INGAP
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.
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.
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).
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
Determination of Approximate Location of PMA and LIF-mediated Transcription Factor Binding in the 5′-Regulatory Region
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
RNA Analysis of INGAP Gene Upregulation
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.
Equal amounts of total RNA (10 μg) 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.
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° for overnight. The blot was washed as follows: 2-times at room temperature with 2×SSC for 10 minutes each; 2-times at 42° with 2×SSC for 10 minutes each; 2-times at 55° with 1×SSC for 10 minutes each. The membrane was exposed to the film (XOMAT-Kodak) and kept at −80° C. overnight before developing.
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.
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/enhancer binding
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
0.788
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



factor 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

## Claims

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