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    • 专利摘要:
    The present invention provides two embodiments. One aspect of the invention is a method for testing a compound that induces transcription of STF-1. To isolate pancreatic islets expressing the STF-1 / lacZ fusion gene and determine the effect of various compounds on the expression of STF-1, compounds of interest are added to the cells expressing STF-1 / lacZ. . Then, colorimetric analysis measures LacZ activity in the control and treated cell populations. Using this method, numerous compounds can be screened and the compounds that induce STF-1 can be easily identified. Another aspect of the invention is a method of labeling insulin-producing pancreatic islet cells in vivo using the STF-1 promoter. At this time, green fluorescent protein (GFP) was used as a label. Transgenes of STF-1-green fluorescent protein can be introduced into swine to efficiently and rapidly recover insulin-producing cells from the pancreas.
    公开号:KR19990081853A
    申请号:KR1019980705564
    申请日:1997-01-22
    公开日:1999-11-15
    发明作者:마크 알. 몬트미니；시마 샤르마
    申请人:와일러 제임스 에프.；리서치 디벨럽먼트 파운데이션；
    IPC主号:
    专利说明:

    Novel methods describing the properties of compounds that promote the expression of STF-1 in pancreatic islet cells
    Many neuroendocrine systems work together to maintain glucose homeostasis. In mammals, the pancreatic islet is considered the primary "glucose sensor." The pancreatic islet has four major cell populations characterized by the insulin cell population, glucagon cell population, somatostatin cell population, and pancreatic polypeptide-producing cell population. Among them, the β cell group producing insulin has an overwhelming majority. Increased serum glucose facilitates the secretion and production of insulin, which in turn absorbs glucose from certain tissues. Thus, when β cells cause or destroy dysfunction, serum glucose levels increase and eventually develop diabetes mellitus.
    Genetic association analysis shows that genetic factors have a significant effect on diabetes. For example, more than 18 genetic locus have some association with insulin dependent diabetes mellitus (IDDM). The disease-sensitive locus called IDDM2 contains the human insulin gene, which is associated with altered transcriptional regulation of insulin promoter function. Therefore, it can be said that diabetes can occur by destroying the expression regulation action of the insulin gene. It is consistent with the assumption that impairment of β cells is a very common feature in diabetes.
    Non-insulin dependent diabetes mellitus (NIDDM) is believed to be due to external and complex genetic effects. Interestingly, allelic variants at the insulin locus are associated with this disease. These variants appear to contain normal insulin genes, but show altered properties in regulation at the transcriptional stage.
    It is estimated that 20 million Americans suffer from non-insulin dependent diabetes mellitus (type II). The progression of this disease, in which tissues do not properly absorb glucose in response to insulin signals, seems to require specific diabetes-sensitive genes that can cause environmental factors and insulin resistance in the peripheral, which is not yet sufficiently identified. Alternatively, a decrease in the susceptibility to glucose of the β cells of the insulin-producing pancreas seen in people with this disease may be due to genetic factors. These two physiological states eventually lead to marked hyperglycemia, the major marker of diabetes validation.
    Regulation at the transcriptional stage of the insulin gene is via short sides of the DNA that interact with cell-specific and glucose-sensitive signal molecules. It is generally known that basic helix-loop-helix (BHLH) and homeodomain-containing factors are key components of transcriptional devices that regulate β cell-specific insulin expression, but the exact nature of these regulators There are many things that I do not understand yet. Pancreatic islet-specific base helix-loop-helix complexes interact with proximal E-boxes, which have been variously called Nir, IEB1 or ICE; This E-box appears twice in the insulin I gene of mice and only once in the insulin II gene of mice and human insulin genes.
    The results of transient assays of insulin-producing cell lines revealed β cell-specific proteins and E-binding to adjacent AT-rich sequences (called FLAT) containing the validation markers of homeodomain recognition sequences. It is assumed that the factors that bind to the box are synergistic together. Several characteristic homeodomain proteins, such as ISl-1, lmx-1, cdx-3, STF-1, have been found to bind to FLAT elements. In addition, STF-1 corresponds to major binding activity in evolutionarily conserved AT-rich sequences (called P elements). Isl-1 binds weakly to FLAT elements and is unlikely to be present in the FLAT-binding complexes detected in extracts of insulin-producing cells. Recently known evidence suggests Isl-1 plays a more important role in neurogenesis. Although there are interesting facts that the homeodomain factors lmx-1 and cdx-3 interact with each other in insulin promoter function in heterologous cells, their cell dispersion and FLAT-binding capacity in β cells are not known. . In addition, there is little direct data on the function of these factors in the β cell line.
    Of the factors with insulin promoter-binding activity, STF-1 is the most potent as a substantial regulator of insulin promoter function. In mice, STF-1 is first detected in the nucleus of primordial cells prior to pancreas at day 8.5, which is slightly earlier than the earliest time insulin expression was detected at this site. Throughout the ongoing development of the endocrine pancreas, STF-1 and insulin are expressed in large quantities together. In addition, in extracts of insulin-producing cell lines, STF-1 appears to be a component with endogenous DNA-binding activity in the FLAT and P elements in the insulin promoter. STF-1 strongly synergizes with Pan-1, an E-box binding factor, as expected from the FLAT-binding factor. On the other hand, DNA binding assay results indicate that other unknown factors in β cell extracts also contribute significantly to FLAT binding activity. It is not yet known whether FLAT-mediated insulin promoting activity requires all or only some of these species to be detected.
    STF-1 is expressed in both the exocrine and endocrine cells of the pancreas in the early stages of development, but gradually production of STF-1 is limited to insulin-producing pancreatic islet cells and somatostatin-producing pancreatic islet cells. STF-1 seems to play an important role in the continued expression of high levels of somatostatin and insulin genes in these cells.
    STF-1 recognizes two kinds of pancreatic islet-specific elements, the FLAT and P elements, on the insulin promoter. When STF-1 binds to this site, STF-1 promotes the transcription of insulin along with E47, a helix-loop-helix protein that recognizes two E-box elements called Far and Nir. Similarly, STF-1 regulates the expression of somatostatin in pancreatic islet cells through two kinds of pancreatic islet-specific elements called TSEI and TSEII.
    It has been found that homeodomain proteins such as STF-1 play an important role in development by establishing cellular or partial identity. In contrast to the unusual and pronounced effects in vivo, in vitro, most homeodomain proteins are weak and have overlapping DNA binding specificities. On the other hand, recent studies suggest that certain protein cofactors are factors that determine homeodomain DNA binding specificity in vivo. For example, in Drosophila, it has been observed that extradendryl (exd) modulates the activity of homeodomain proteins without changing the expression pattern. However, extradentricles seem to promote the selection of target genes by enhancing the DNA binding specificity of certain homeodomain proteins. Indeed, extradenticles are highly conserved in vertebrates, with significant sequence similarity (71%) to human proto-oncogene Pbx-1.
    The differentiation of cells into specific lineages during development is largely determined by the relative expression of various homeodomain (HOX) select proteins that mediate the activity of specific genetic programs. However, little is known about the mechanism by which individual HOX genes themselves are targeted for expression in other types of cells.
    The pancreas of vertebrates is composed of endocrine and exocrine components derived from primitive cells common in duodenal primordial (see Citation 1). Within the endocrine component of the pancreas, pluripotent progenitor cells, which initially express several types of pancreatic hormones, are increasingly defined to form four cell subpopulations, including the islet of adult Langerhans: glucagon, an insulin-producing cell subpopulation. -Producing cell population, somatostatin-producing cell population and pancreatic polypeptide-producing cell population (see citations 2 and 3). Although the mechanism for activating this developmental pathway is not clear, it is known that the homeobox factor STF-1 (IPF-1 / IDX-1) is an important determinant in this process. In fact, the STF-1 gene is essential for the development process, which is supported by homologous recombination studies that show that the destruction of the STF-1 / IPF-1 gene innately results in pancreatic insufficiency (see citation 4).
    Expression of STF-1 (also referred to as Pdx1) can be identified initially at day 8.5 of embryonic development in pancreatic progenitor cells and pluripotent progenitor cells. STF-1 production, which is transiently expressed in both the endocrine and exocrine components of the developing pancreas, is increasingly limited to insulin-producing pancreatic islet cells and somatostatin-producing pancreatic cells (see citations 5 and 6). In these cells, STF-1 is likely to bind to functional elements in each promoter to regulate insulin genes and somatostatin genes (see citations 5, 7-16). There is no conventional method for effectively controlling the expression of STF-1, a homeodomain protein in pancreatic cells. The present invention will satisfy what has been needed and required in this field for a long time.
    Summary of the Invention
    Although STF-1 is a major regulator of pancreatic genes, it is not known by which mechanism the expression of STF-1 is targeted to pancreatic cells. It is shown in the present invention that 6.5 kb of STF-1 promoter is sufficient to direct pancreatic-specific expression of the β-galactosidase reporter gene in cultured duodenal cells and transformed mice. Of particular importance for STF-1 promoter activity is the E-box element within the 6.5 kb fragment located at −104 relative to the main transcriptional start. This element is recognized by an active factor upstream that is essential for pancreatic islet-specific expression of STF-1. It is true that such STF-1 promoters have 20 to 100 fold activity in enhancing targeted expression of STF-1 in pancreatic islet cells. Removing the E-box sequence of the base located at -104 results in no STF-1 expression in HIT cells. In addition, anti-USF (parent factor) antiserum interfered with the formation of C1, C2 and C3, indicating that these proteins are formed by USF proteins.
    In addition, an STF-1 enhancer element was found at the 530 bp site at the upper 600 bp from the start of transcription. Promoter constructs containing this 530 bp fragment were completely inhibited by dexamethasone treatment, but not in trace STF-1 promoter constructs containing widely distributed USF-1 recognition sites. The activity of the 530 bp region is 5 to 10 times greater in HIT-T15 cells than in COS-7 cells, demonstrating that the 530 bp fragment has pancreatic cell-specific activity. Therefore, the present invention uses the STF-1-lacZ fusion gene to screen compounds that enhance expression of STF-1 in pancreatic islet cells. Compounds that promote the production of STF-1 enhance the production of insulin, a function of pancreatic B cells. Thus, the compounds identified by the methods described herein are particularly useful in patients with type II diabetes mellitus who have glucose non- tolerance due to unclear pancreatic islet cell function.
    In one aspect of the invention, a method is provided for testing a compound that induces transcription of STF-1.
    Using CaPO 4 co-precipitation, pancreatic islet cell lines expressing the STF-1 / lacZ fusion gene are isolated. To determine the effects of various compounds on the expression of STF-1, compounds of interest are added to cells expressing STF-1 / lacZ. Then, colorimetric analysis measures LacZ activity in the control and treated cell populations. Using this method, many compounds can be screened and STF-1 derived compounds easily identified.
    In one aspect of the invention, a method is provided for labeling insulin-producing pancreatic cells in vivo using an STF-1 promoter. At this time, green fluorescent protein (GFP) was used as a label. As described in the literature, green fluorescent proteins can be detected without disruption (Ogawa et al., Proc. Natl. Acad. Sci., 1995, 92: 11899-11903). In order to easily recover β cells from the pancreas of an animal, the STF-1 promoter was fused to a gene encoding GFP. By introducing a transgene of STF-1-green fluorescent protein into pigs, insulin-producing cells can be efficiently and quickly recovered from the pancreas. The pancreas is briefly recovered from pigs, and then the cells are dispersed by treatment with collagenase. Insulin-producing pancreatic ducts are also effectively recovered through fluorescence-activated cell sorting (FACS) based on the expression of the transgene of STF-1-green fluorescent protein. This purified β cell population can be used for cell treatment of diabetic patients.
    In one aspect of the invention,
    (1) a STF-1 enhancer, a promoter having a sequence selected from the group consisting of SEQ ID NO: 1 or SEQ ID NO: 2 or fragments thereof, and a reporter gene under the transcriptional control of the STF-1 enhancer and the promoter, wherein the reporter gene is Providing a vector containing a detectable signal to the pancreatic cell, which is also a host cell);
    (2) transferring this vector into said host cell;
    (3) culturing said host cell in the presence of a test compound to determine if the test agent can stimulate said host cell to generate said detectable signal; And
    (4) analyzing said signal to determine if the test agent can stimulate this host cell to generate said detectable signal, wherein if said signal is present, the test compound is capable of Stimulating to induce transcription of STF-1, and the absence of said signal means that the test compound does not stimulate pancreatic islet cells to induce the transcription of STF-1). It provides a method of measuring the ability of a test compound to stimulate the transcription of STF-1 by stimulating it.
    In a preferred embodiment for the purposes of the present invention, an enzyme is used as the reporter gene. In a more preferred embodiment, this enzyme is selected from the group consisting of luciferase and β-galactosidase.
    In another embodiment of the invention, said metastasis step is carried out by introduction of the trans gene into the animal via transfection or micro injection.
    In another object of the present invention, there is provided a method comprising the steps of labeling insulin-producing pancreatic cells in vivo: a vector containing a reporter gene under the transcriptional control of the STF-1 promoter and STF-1 promoter Providing a gene wherein the reporter gene can deliver detectable signals to pancreatic islet cells; Introducing the vector into an animal embryo using the vector as a trans gene; Growing this embryo into an animal having pancreatic islet cells; Analyzing said detectable signal to determine whether said pancreatic islet cells of said animal express said reporter gene (wherein a signal indicates that insulin-producing pancreatic islet cells are present, and Is not present, insulin-producing pancreas also means no cells).
    In a preferred embodiment for the purposes of the present invention, the reporter gene produces a fluorescent protein and further comprises the step of sorting insulin-producing pancreatic islet and non-insulin-producing pancreatic islet cells by fluorescence-activated cell sorting (FACS). It provides a method to include.
    Through the preferred embodiments of the present invention described below, further aspects, features and advantages of the present invention will become apparent.
    The present invention relates generally to the fields of biochemical endocrinology, molecular biology and protein chemistry. More specifically, the present invention relates to a new method of characterizing a compound that promotes the expression of STF-1 in pancreatic islet cells.
    The invention, briefly summarized above, is described in more detail by the specific embodiments shown in the accompanying drawings, in order to achieve the features, advantages and objects of the invention described above and to understand the invention in detail. These drawings form part of the specification. The accompanying drawings, however, are illustrative of the preferred embodiments of the invention and are not to be considered as limiting the scope of the invention.
    Figure 1 shows the location on the chromosome of the STF-1 gene. 1A shows an orphan homeo box gene located at the distal end of chromosome 5 in a diagram showing the relative position of STF-1 (called Pdx1) relative to other markers on chromosome 5 in rats. It is shown that the STF-1 gene is encoded by. The centimorgan unit is shown on the right. 1B is a table showing the recombination frequency between STF-1 and various markers on chromosome 5. The left column shows the markers used for chromosome studies. STF-1 is referred to herein as Pdx1.
    Figure 2 shows that the STF-1 promoter does not have TATA and utilizes several transcriptional initiation points. In FIG. 2A kb units are indicated above and a 15 kb genomic clone of STF-1 is shown schematically. The 5 'side of 6.5 kb, the intron of 4 kb and the 3' side of 3 kb are indicated. IVS refers to a 4 kb intron inserted between exon I (dark areas labeled I) and exon II (dark areas labeled II). 2B shows the nucleotide sequence of the 5 'side of the STF-1 gene. Transcription initiation points (blacked arrowheads) and primer elongation (empty arrowheads) mapped through protection from RNase are indicated as S1 (main starting point), S2 and S3. BHLH / BHLH-ZIP protein (E-box), CTF / NF-1 (CAAT) and C / EBP are underlined where possible as factor binding sites and indicate relative positions relative to the major transcription initiation point Doing. 2C shows the RNase protection assay of Tu6 RNA (Tu6, lane 2) or control yeast tRNA (yeast, lane 3) using an anti-sense STF-1 RNA probe. The uncut probe is on the left (lane 1). 2D shows primer extension analysis in yeast (lane 4), Tu6 mRNA (lane 5) or RIN mRNA (lane 6) using anti-sense STF-1 primers. The sequence ladder is at the right end (GATC, lanes 7-10). The corresponding sites between the product protected from RNase and the primer extension product are indicated and three starting points S1, S2 and S3 are shown (see FIG. 2B).
    3 shows that 6.5 kb of the STF-1 promoter targets the expression of the β-galactosidase reporter gene in transgenic pancreatic islet cells. Using Xgal as a pigment source material, the lacZ activity of representative frozen cut sections of mature rat pancreas extracted from transformed littermate and control littermate is measured. Arrows point to pancreatic islet cells.
    4 shows that the terminal and base elements within the STF-1 promoter induce STF-1 expression in pancreatic islet cells. Figure 4A shows -6500 STF-1 luciferase after transfection of -6500 STF-1 luciferase reporter plasmid into pancreatic islet cell line (βTC3, HIT) and non-pancreatic islet cell line (PC12, COS, HeLa). The activity of reporter plasmids is compared. After normalizing with the transfected RSV-CAT control plasmid, STF-1 promoter activity in HIT cells (100%) relative to other cell lines is shown in a representative assay. Repeat this analysis three more times. 4B shows a representative analysis of the STF-1 luciferase (STF luc) promoter construct after transfection of the STF-1 luciferase (STF luc) promoter construct into HIT cells. The construct was named along the 5 'boundary of the promoter based on the major transcription start point (see S1, Figures 2A and 2B). The possible locations as binding sites for nuclear factors are shown graphically: the blacked arrows indicate the major transcription initiation points. Asterisks indicate uncharacterized binding activity at the terminal 3 kb. For each construct, normalize transfection efficiency using RSV-CAT for internal control, then calculate relative activity against -6500 STF Luc (100%). Repeat this analysis four more times.
    5 shows that the E-box element of the base in the STF-1 promoter binds to the upper factor USF. 5A shows the results of a DNase I protection assay using STP-1 promoter fragments labeled with 32 P ranging from -182 to -37 (145 bp). Cleavage patterns without extracts in radiographs (lanes 1 and 4), cleavage patterns of nuclear extracts in HIT to HeLa cells (lanes 2 and 3, respectively) or cleavage patterns of recombinant USF-1 (lanes 5) Shows.
    5B shows the electrophoretic mobility change of HIT nuclear extracts incubated with 32 P labeled double stranded oligonucleotides containing STF-1 E-box (-118 / -95) (lane 1). 50 mole times of unlabeled competing oligonucleotide is added to the binding reaction (lanes 2-5): STF E is a wild type STF-1 E-box oligo; STF-E MUTs are mutated STF-E oligos with substitutions at -106 (C / A) and -102 (T / G) in the E-box motif; Ins-1 (P) is the P element of the insulin I promoter; And Gal4 represents a GAL4 recognition site.
    Figure 5C shows the results of gel transfer analysis of HIT nuclear extract using the STF-E box as a probe (lane 1). Add USF or TFE-3 antibody to the reaction (lanes 2 and 3). Complexes C1, C2 and C3 are as indicated.
    5D shows the gel transfer analysis of the HIT extract using the STF-E probe (lanes 1 to 3). Add USF-1 and USF-2 specific antiserum to the binding reaction.
    6 shows that binding of USF to the -104 E-box is important for STF-1 promoter activity. 6A shows the effect of E-box mutations on USF-binding activity. Gel transfer analysis of HIT nuclear extract using wild type STF-1 E box probe (E-WT) and mutant STF-1 E box probe (E-MUT). The sequence from -106 to -102 is also at the bottom. C1-3 means complexes C1, C2 and C3. 6B shows the effect of E-box mutations on STF-1 promoter activity in HIT cells. Representative assay of HIT cells transfected with wild type STF-1 E box motif, mutant STF-1 E box motif or truncated STF-1 E box motif (-118 / -95) at 6500bp or 190bp of STF-1 promoter Is showing. After normalizing transfection efficiency with the co-transfected RSV-CAT control plasmid, the reporter activity was shown to be relative to the wild type -6500 STF-1 Luc construct (100%). Repeat this analysis three more times.
    Figure 7 shows that glucocorticoids inhibit the expression of STF-1 via the pancreatic islet-specific enhancer on the 5 'side of the STF-1 gene.
    7A shows the activity of the STF-1 promoter construct after treatment of HIT-T15 cells with dexamethasone (10 −7 M) or a control ethanol vehicle for 18-24 hours. -6.2 / -5.67 Set the activity of the STF Luc reporter to 100%. All constructs are evaluated in STF-1 luciferase vector (STF-1 Luc) containing 120 flanking bases at the base of 5 '. STF-1 sequences inserted into STF-1 Luc reporters are represented by nucleotide numbers. For example, -6.5 / -6.2 STF Luc contains an STF-1 sequence from -6500 to -6200 fused to 120 bp of the 5 'base of STF-1. Terminal sites containing the active element are shown as ellipses and squares, and small amounts of the STF-1 promoter are shown as rectangles. Standard error lines are shown. Repeat this analysis three more times. STF-1 reporter activity is normalized to transfection efficiency using a CMV-β gal control plasmid infected together.
    7B (top) shows the results of transient transfection assay of STF-1 reporter constructs in HIT T15 cells. The activity of -6500 STF Luc containing 6500 bases of the 5 'flanking sequence is set to 100%. Repeat this analysis four more times. Standard error is as shown. (Bottom) shows the activity of the STF-1 luciferase reporter plasmid after transient infection of the STF-1 luciferase reporter plasmid into COS-7 cells. Normalizing the activity of the promoter to CMV-β gal control activity allows direct comparison with STF-reporter activity in HIT cells.
    7C shows the nucleotide sequence of small amounts of pancreatic islet-specific enhancers in the STF-1 gene ranging from -6.2 to -5.67 kb. HNF-3 and E box-binding motifs are written in bold and underlined.
    8 shows that the pancreatic islet-specific enhancer in the STF-1 gene contains binding sites for HNF-3β and beta-2.
    FIG. 8A shows DNase I protection of nuclear extracts of HIT T15 pancreatic islet cells, HeLa or COS-7 cells using a 32 P labeled STF-1 probe ranging from -5870 to -6100 of a mouse STF-1 promoter. The results of the analysis are shown. NONE; Control reaction without added extract; HNF-3α; Reactants using recombinant protein.
    8B shows the results of gel mobility change analysis for nuclear extracts of HeLa, HepG2, glucagon-producing αTC or insulin-producing HIT and RIN cell lines. In analysis, a 32 P labeled STF-1 oligonucleotide probe containing H elements is used. Nucleotide sequences of the wild type H element (WT) and the mutant H element (MT) are shown below. As indicated above lanes, a 100-fold excess of unlabeled wild type competitor DNA or mutant competitor DNA is added.
    8C from HIT insulinoma (HIT NE) and pancreatic islet cells (ISLET NE) of primary cultured adult mice using a 32 P labeled STF-1 H element probe ranging from -5907 to -5927. Results of gel mobility change analysis for the nuclear extract are shown. As indicated above each lane, HNF-3 α, β or γ antiserum is added to the reaction. -Means no antiserum added. I and II mean protein-DNA and antibody hypershift complex, respectively.
    8D shows gel mobility change analysis results for nuclear extracts from HIT T15 cells using B-element probes labeled 32 P ranging from -5963 to -5981 of the STF-1 promoter. As indicated above lanes, beta-2, E2A or STF-1 antiserum is added to the binding reaction. As indicated, a 100-fold excess of unlabeled wild type competition DNA (WT: 5'-TCAGTGACAGATGGAGTCCT-3 ') or mutant competition DNA (MT: 5'-TCAGTGAAAGACGGAGTCCT-3') was added. Lane 7 shows binding activity from reticulocyte lysates programmed with beta-2 and E-47 cDNA. The uppermost band of lane 7 is unique to reticulocyte lysate.
    9 shows that glucocorticoids inhibit the expression of STF-1 by interfering with the activity of HNF-3 against pancreatic islet-specific enhancers.
    9A shows the results of transient transfection of the STF-1 reporter plasmid in HIT T15 insulinoma cells. The activity of the -6.2 / -5.7 STF Luc constructs containing a small amount of pancreatic islet-specific enhancers (-6200 to -5700) is set to 100%. Point mutation-containing constructs to H and B elements that interfere with the binding of HNF-3β and beta 2 / E2A are indicated by X in ellipses or squares. Point mutations correspond to mutations in the E box and H elements used in gel mobility shift analysis (see FIG. 8).
    9B shows wild type STF-1 reporter activity (black bar) and H element mutant STF-1 reporter activity (white bar) in control HIT T15 cells (−) and dexamethazone treated HIT T15 cells (+). The effect of HNF-3β over-expression on) is shown. The activity of wild type -6.2 / -5.7 STF Luc constructs containing a small amount of pancreatic islet-specific enhancers (-6200 to -5700) in control cells is set to 100%. Standard error lines are as indicated. Repeat the experiment four times or more.
    9C shows the effect of increasing levels of HNF-3β effector plasmid on wild type STF-1 (-6500 STF-1 LUC) reporter activity in HIT T15 cells. The amount (μg) of HNF-3β effector plasmid is indicated below each bar. In each assay, the total amount of effector plasmid is kept constant by balancing with an empty CMV expression vector. Control cells (ethanol) and dexamethasone treated cells (DEX) are as indicated.
    The present invention shows that the homeodomain protein STF-1, which acts on pancreatic morphogenesis and glucose homeostasis, is encoded by the "orphan" homeobox gene on the chromosome 5 of the mouse. When fused to the β-galactosidase reporter gene, pancreatic islet-specific activity was shown in the transfected mice with the 5 'flanking sequence of 6.5 kb of the STF-1 gene. For pancreatic ischemia limited expression, two elements are required in the STF-1 promoter: a distal enhancer sequence located between -3 kb and -6.5 kb and a basic helix loop helix (bHLH) / leucine zipper (ZIP). ) A proximal E box sequence located at -104 which is mainly recognized by nuclear factor USF. As point mutations in the E box located at -104 destroy USF binding, the activity of the STF-1 promoter decreases, and according to the present invention, USF controls the expression of STF-1 in pancreatic islet cells. Shows the main ingredient. In addition, it has been found that glucocorticoids effectively inhibit the expression of the STF-1 gene by interfering with the activity of terminal pancreatic-specific enhancers that recognize endoderm factors HNF-3β and beta-2 / E47. Mutations in the STF-1 enhancer disrupted the binding of HNF-3β or beta-2 / E47, disrupting the activity of the STF-1 promoter. The fact that HNF-3β expression vector can restore the activity of STF-1 enhancer in cells treated with glucocorticoids suggests that HNF-3β is actually a major regulatory factor in STF-1 expression.
    One of the objectives of the present invention is to provide a method for examining a compound that induces transcription of STF-1. Pancreatic islet cell lines expressing STF-1 / lacZ fusion genes are isolated using calcium phosphate co-precipitation. To test the effect of various compounds on the expression of STF-1, the compound in question is added to cells expressing the STF-1 / lacZ fusion gene. The activity of lacZ in control and treated cells is quantified through colorimetric analysis. Using this method, numerous compounds can be screened and the compounds that induce STF-1 can be easily identified.
    In another aspect of the present invention, a method of labeling insulin-producing pancreatic islet cells in vivo using the STF-1 promoter is provided. At this time, green fluorescent protein (GFP) plays an important role as a label. As described in the literature, expression of GFP can be confirmed without destroying cells (Ogawa et al., Proc. Natl. Acad. Sci., 1995, 92: 11899-11903). To facilitate the recovery of β cells from the pancreas of animals, the STF-1 promoter is fused to a gene encoding GFP. By introducing the STF-1-GFP trans gene into pigs, the pancreas can effectively and quickly recover insulin-producing cells. The pancreas is briefly recovered from pigs and then treated with collagenase to disperse the cells. Insulin-producing cells are recovered by fluorescence-active cell sorting (FACS) based on the expression of the STF-1-GFP trans gene. The purified β cell population is then used to treat the cells of the diabetic patient.
    In one aspect of the invention, there is provided a method comprising the following steps of measuring the ability of a test compound to stimulate pancreatic islet cells to induce transcription of STF-1: SEQ ID NO: 1 or SEQ ID NO: 2 STF-1 enhancers, promoters having sequences selected from the group of fragments, and reporter genes under the transcriptional control of the STF-1 enhancers and promoters, wherein the reporter genes may transmit detectable signals to the host cell. Providing a containing vector; Transferring this vector into said host cell; Culturing said host cell in the presence of a test compound to determine if a test agent can stimulate said host cell to generate said detectable signal; And analyzing said signal to determine if a test agent can stimulate said host cell to generate said detectable signal, wherein said test compound stimulates pancreatic islet cells if said signal is present. Means the induction of STF-1 transcription, and the absence of the signal indicates that the test compound does not stimulate pancreatic islet cells and thus does not induce transcription of STF-1).
    In a preferred embodiment for the purposes of the present invention, an enzyme is used as the reporter gene. In a more preferred embodiment, this enzyme is selected from the group consisting of luciferase and β-galactosidase.
    In another embodiment of the invention, said metastasis step is carried out by introduction of the transgene into the animal via transfection or micro injection.
    In another object of the present invention, there is provided a method comprising the steps of labeling insulin-producing pancreatic cells in vivo: a vector containing a reporter gene under the transcriptional control of the STF-1 promoter and STF-1 promoter Providing a gene wherein the reporter gene can deliver detectable signals to pancreatic islet cells; Introducing the vector as a transgene into an animal embryo; Growing this embryo into an animal having pancreatic islet cells; And analyzing said detection signal to determine whether said pancreatic islet cells of said animal express said reporter gene (wherein, if present, it means that insulin-producing pancreatic islet cells are present, and Is not present, insulin-producing pancreas also means no cells).
    In a preferred embodiment for the purposes of the present invention, the reporter gene produces a fluorescent protein and further comprises the step of sorting insulin-producing pancreatic islet and non-insulin-producing pancreatic islet cells by fluorescence-activated cell sorting (FACS). It provides a method to include.
    The human genome contains four families of genes called Hox or homeotic selector genes, which are the major determinants of axial body formation during embryonic development (Krumlauf, 1994 Cell 78 : 191-201). The four gene families contain up to 13 genes each, and genes in one gene group usually show significant homology with genes in other gene groups. Genes with this association are called paralogs; Thus, HoxA1, HoxB1, HoxC1 and HoxD1 are paralogs with close correlations with each other in different Hox groups on different chromosomes, respectively. HoxB complex is located on the long arm of chromosome 17 and HoxB1 to HoxB9 have been identified.
    Regulation of the glucose-dependent insulin gene is likely to be accompanied by an increase in insulin release mediated by glucose. This increase in insulin release is likely due in part to an increase in intra-calcium concentrations. In addition, by modulating the activity of the FLAT-binding protein, at least a portion of the glucose-reactive insulin promoter can operate. HoxB13 binds strongly to functionally important FLAT elements on the insulin promoter. In addition, the addition of HoxB13 and the insulin ICE / Nir element-binding factor Pan-1 together strongly activates the insulin promoter. This is consistent with the observation that FLAT and Nir elements are synergistic in insulin-producing cells. These data suggest entirely that calcium-dependent signaling pathways can modulate the function of HoxB13.
    In accordance with the present invention, recombinant DNA techniques can be applied that fall within the technical scope of conventional molecular biology, microbiology and related fields. Such techniques are explained fully in the literature (see, eg, Maniatis, Frisch & Sambrook, "Molecular Cloning: A Laboratory Manual (1982)"; "DNA Cloning: A Practical Approach," Volume I and II (D.N. Glover ed. 1985); "Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid Hybridization" (B.D. Hames & S. J. Higgins eds. 1985); "Transcription and Translation" (B.D. Hames & S. J. Higgins eds. 1984); "Animal Cell Culture" (R. I. Freshney, ed. 1986); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, "A Practical Guide To Molecular Cloning" (1984).
    Definitions of terms expressed herein are described below.
    A "vector" is a replicon, such as a plasmid, phage, or cosmid, that other DNA fragments bind to and allow the linked fragments to replicate.
    "DNA molecule" means a polymer form of single or double stranded deoxyribonucleotides (adenine, guanine, thymine or cytosine). This means only primary and secondary structures, but is not limited to any particular tertiary structure. Thus, the term includes, among other things, double-stranded DNA found in linear DNA molecules (eg, restriction fragments), viruses, plasmids and chromosomes. As used herein, the term structure refers to a conventional one having a sequence from 5 'to 3' along untransferred DNA.
    "Replica start point" refers to a DNA sequence involved in DNA synthesis.
    A "coding sequence" of DNA refers to a double stranded DNA sequence that is translated into transcription and polypeptide in vivo when under the control of appropriate regulatory sequences. The boundaries of this coding sequence are defined by the translation initiation codon located at the 5 '(amino) end and the translation end codon located at the 3' (carboxyl) end. Code sequences may include, but are not limited to, prokaryotic sequences, cDNAs from eukaryotic mRNAs, genomic DNA sequences from eukaryotic (eg, mammalian cell) DNAs, and synthetic DNA sequences. The polyadenylation signal and transcription termination sequence are usually located 3 'of the coding sequence.
    Transcriptional and translational control sequences are DNA regulatory sequences such as promoters, enhancers, polyadenylation signals, terminators, and the like for expression of coding sequences in host cells.
    A "promoter sequence" is a DNA regulatory site that allows RNA synthetases in a cell to bind so that the coding sequence in the lower (3 'direction) can initiate transcription. To define the present invention, the promoter sequence includes the minimum base or element necessary to initiate transcription at a detectable level towards the upper (5 'direction), with the transcription initiation site at the 3' terminal border. Within the promoter sequence is a transcription initiation site (simply defined by a genetic map using a nucleic acid hydrolase S1), and a protein binding domain (common sequence) to which an RNA synthetase binds. Promoters of eukaryotic cells often contain "TATA" boxes and "CAT" boxes, which are not always the case. The prokaryotic promoter contains a shine-delgano sequence in addition to the -10 and -35 consensus sequences.
    An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of other DNA sequences. When a coding sequence is transcribed into mRNA by an RNA synthetase and then translated into a protein encoded by the coding sequence, the coding sequence is controlled by intracellular transcriptional and translation control sequences.
    A "signal sequence" can be included before a coding sequence. This signal sequence encodes a signal peptide at the N terminus of the polypeptide, which is signaled with the host cell to direct the polypeptide to the cell surface or release it into the medium, which is then removed by the host cell before the protein leaves the cell. do. The signal sequence may be associated with various proteins in prokaryotic and eukaryotic cells.
    The term “oligonucleotide” as used when referring to a probe of the present invention refers to a molecule comprising two or more (preferably eight or more) ribonucleotides. The exact size of an oligonucleotide depends on many factors depending on the final function and use of this nucleotide.
    As used herein, the term “primer” refers to the conditions under which the synthesis of a primer extension product complementary to the nucleic acid chain is induced (ie, when an inducer such as appropriate temperature and pH, nucleotides and DNA synthetase is present) Oligonucleotides that can act as starting points for synthesis, which are naturally occurring or synthesized as cleavage of purified restriction enzymes. The primer is single or double stranded and should be long enough to prepare for the synthesis of the desired kidney product in the presence of an inducing agent. The exact length of the primer is determined by several factors, including temperature, source and use of the primer. For example, when used for identification, depending on how complex the target sequence is, oligonucleotide primers usually contain 15-25 or more nucleotides, but may also contain fewer.
    The primers selected herein are "substantially" complementary to the different specific DNA sequence chains that they target. This means that the primer is complementary enough to hybridize with the corresponding chain. Thus, the primer sequence need not be exactly complementary to the sequence of the template. For example, a non-complementary nucleotide fragment may be bound to the 5 'end of the primer so that only the remainder is complementary to the model chain. Alternatively, non-complementary bases or longer sequences may be interspersed in the primers if the primer sequences are sufficiently complementary or hybridized to the corresponding sequences to form a template for the synthesis of the extension product.
    As used herein, the terms “limiting endonuclease” and “limiting enzyme” refer to enzymes present in bacteria that cleave double-stranded DNA at or near a specific nucleotide sequence, respectively.
    When exogenous or heterologous DNA is introduced into a cell, the cell is "transformed" by this DNA. The transforming DNA may or may not be integrated (via covalent bonds) into the genome of the cell. For example, in prokaryotic cells, yeast and mammalian cells, the transforming DNA may be conserved on episomes such as plasmids. In eukaryotic cells, in stably transformed cells, the transforming DNA can be integrated into the chromosome and inherited into daughter cells through chromosomal replication. Whether or not the eukaryotic cells can form a cell line or a clone including the daughter cell group containing the transformed DNA can determine whether the transformation is stable. A "clone" is a cell group derived from a single cell or a common ancestor through mitosis. A "cell line" is a primary cell clone that can stably grow for generations in vitro.
    If at least about 75% (preferably at least about 80%, most preferably at least about 90 to 95%) of nucleotides in a given DNA sequence match, the two DNA sequences are "substantially homologous". By comparing the sequences using standard software obtained from Southern hybridization experiments under stringent conditions defined for the sequence data bank or for this particular system, it is possible to confirm that the sequences are substantially homologous. Conditions suitable for hybridization are within the skill of the art. See, eg, Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual (1982)"; "DNA Cloning: A Practical Approach" Volume I and II (D.N. Glover ed. 1985); "Nucleic Acid Hybridization" (B.D. Hames & S. J. Higgins eds. 1985).
    The "heterologous" site of a DNA construct is an identifiable DNA fragment within a larger DNA molecule that is not found in nature in combination with this larger DNA molecule. Thus, when a heterologous region encodes a mammalian gene, the gene usually has a flanking DNA in the genome of the organism from which it does not have the genomic DNA of the flanking. In another example, the coding sequence is a construct that is not found in nature (eg, cDNA when the genomic coding sequence contains a synthetic sequence with a codon different from an intron or natural gene). Heterologous regions of DNA as defined herein are not generated by allelic variation or naturally occurring mutations.
    The most commonly used labels in these studies include radioactive elements, enzymes, and chemicals that fluoresce when exposed to ultraviolet light. Numerous fluorescent materials are known and can be used as labels. For example, such fluorescent materials include fluorescein, rhodamine, auramin, Texas red, AMCA blue and lucifera yellow. Special detection agents include anti-rabbit antibodies prepared from chlorine and fused with fluorescein via isothiocyanate.
    Enzyme labels are also useful and can be detected using any of the currently used colorimetric techniques, spectrophotometric techniques, fluorescence photometric techniques, amperometric techniques or gas measurement techniques. The particles are fused to selected particles through reaction with bridge molecules such as carbodiimide, diisocyanate, glutaraldehyde and the like. Many enzymes that can be used in this process are known and can be used. Peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase and peroxidase, alkaline phosphatase are preferred. In US Pat. Nos. 3,654,090, 3,850,752 and 4,016,043, mention is made as an example to disclose other labeling substances and methods.
    The following examples are intended to illustrate various aspects of the invention and are in no way intended to limit the invention.
    Example 1
    Chromosome Mapping
    Gene maps of STF-1 were prepared using the (B6 × SPRET) F1 × SPRET backcross DNA panel available from the Jackson Laboratory Backcross DNA Panel Map Service, which used a 32 P-labeled STF-1 cDNA fragment as a probe. do.
    Example 2
    Preparation and β-galactosidase staining of transformed mice
    Using standard cloning techniques, a fusion gene containing 6500bp of STF-1 sequence on top of the β-galactosidase reporter gene was constructed and then injected into the male pronuclei of fertilized oocytes. Founder rats are identified using Southern blot and PCR amplification techniques. Expression of the STF-1 / β-galactosidase gene in transformed rat tissues is measured on a 20 μM cross section of paraformaldehyde immobilized tissue using X-gal as a pigment source substrate.
    Example 3
    Antibodies
    Non-specific USF-1,2 antibodies used in supershift experiments are available from Santa Cruz Biotechnology. TFE3 antibodies can be obtained from K.Jones. In particular, antibodies that recognize USF-1 or USF-2 can be obtained from M. Sawadogo (see citations 17 and 18).
    Example 4
    Isolation of STF-1 Genomic Clones
    STF-1 cDNA fragments labeled with 32 P were used as hybridization probes to isolate the STF-1 gene from the genomic library of EMBL 3 mice. The STF-1 positive genomic fragment is inserted into the EcoRI site of the SK II plasmid (stratagene).
    Example 5
    RNase protection and primer extension
    Poly A RNA is prepared using an Oligotex (dT) 30 system (Quiagen). Oligonucleotide primers used for primer extension analysis use γ- 32 P ATP and T4 polynucleotide kinase to label the 5 ′ terminal sites. 5 μg of poly A RNA was incubated at 80 ° C. for 5 minutes with the terminally labeled antisense primer, followed by 16 hours at 42 ° C. Primer extension reactions are performed using AMV reverse transcriptase at 37 ° C. for 1 hour, and the product is analyzed on 5% denaturing polyacrylamide. RNase protection assays are performed using 25 μg of total RNA extracted from Tu6 cells (see citation 19). Antisense STF-1 RNA probes are prepared using STF-1 genomic fragments ranging from -185 to +93 based on the translation initiation point. Antisense STF-1 RNA labeled with 32 P is synthesized in vitro using T7 RNA synthase and 32 P-UTP. STF-1 antisense RNA probe and mRNA are annealed at 80 ° C. for 5 minutes and then incubated at 65 ° C. for 16 hours. The anneal reaction is then treated with RNase 40 μg / ml for 1 hour at room temperature and then analyzed on 5% modified polyacrylamide electrophoresis.
    Example 6
    Reporter clones and transient transfection
    All promoter fragments are inserted into the p (A) luciferase backbone provided by D. Helinski using standard cloning methods (see citation 20). The 5 'side of the STF-1 promoter is fused to +68 based on the luciferase gene and major transcription initiation (-6500 STF Luc, -3500 STF Luc, -540 STF Luc, -410 STF Luc, 225 STF Luc, -140 STF Luc) or +78 (-190 STF Luc, -130 STF Luc, -120 STF Luc, -95 STF Luc, -35 STF Luc). Through calcium phosphate precipitation, the plasmids are transfected into HIT-T15 cells (ATCC), βTC3 (provided by D. Hananahan (see citation 21)), PC12, COS and HeLa cells. Luciferase levels are measured on a single photoluminescence instrument and then normalized to CAT activity derived from the transfected RSV-CAT internal control plasmid.
    Example 7
    Gel Transfer Assay and DNase Protection Assay
    For electrophoretic mobility shift assay, oligonucleotide probes are labeled with α 32 P-dCTP via a fill-in reaction using a Klenow fragment. 4 μg of nuclear extract is incubated with 0.5 ng of double stranded oligonucleotides labeled with 32 P, followed by non-modified polyacrylamide electrophoresis as previously described (see citation 9). For hypermigration assays, proteins are preincubated with antibodies, then incubated with radioactive double stranded oligonucleotides, followed by electrophoresis. DNase protection assays are as previously described above (see citation 22).
    The drawings for DNA binding analysis were scanned from the first pictures taken with the HP ScanJet 3C and then assembled with Canvas software on the Macintosh. The scanned image is played back on a Tektronix Phaser II SDX.
    Example 8
    Location and Genome Composition of Chromosome of the STF-1 Gene
    Using a STF-1 cDNA as a hybridization probe on a DNA backbone panel obtained from the Jackson Laboratory, a single copy of a single copy of the STF-1 gene map was generated at the terminal region of mouse chromosome 5 (FIG. 1A). No recombinants were found in the terminal markers Pmv12 or Iapls3-9, whereas six recombinants were observed in the Actb region, which was further terminal (FIG. 1B). From these results, it is expected that the SFT-1 gene will be found on chromosome 14 of mouse and 7q chromosome of human unrelated to four HOX gene groups. These results show that STF-1 should be classified as an "orphan" homeo box gene.
    To isolate the gene encoding STF-1, screening 106 bacteriophage clones from the EMBL 3 genomic library of rats using a 32 P-labeled STF-1 cDNA probe, followed by two containing 15 kb genomic inserts each. Obtain positive clones. The 15 kb STF-1 genomic fragment contains a 6.5 kb 5 'side, a 3.5 kb 3' side, and the entire STF-1-coding site, directly above the homeo box-coding sequence (amino acids 140-215). One intron (Ala 135) of 4 kb is inserted in FIG.
    The 5 'side of the STF-1 genomic clone (Figure 2B) lacks the consensus TATA box and starting sequence. A map of the transcription start site for this gene is prepared. Using RNase protection and primer extension analysis of mRNA extracted from the insulin-producing cell lines RIN and Tu6 (FIGS. 2C and 2D), three major initiation sites (called S1, S2, S3) differed from the translation initiation sites, respectively. Toward 91, 107 and 120/125 nucleotides. A secondary fourth transcription initiation site, located 137 nucleotides upwards from the onset of translation, is also observed. Like other promoters without a TATA box, the STF-1 promoter contains G / A and G / C-rich sequences at 30bp upstream from the S1 and S2 initiation sites (see citations 23-25).
    Example 9
    Activity of the STF-1 Promoter in Pancreatic Islet Cells
    To determine whether a sequence within the 5 'side of the STF-1 gene is sufficient for targeted expression of STF-1 in pancreatic islet cells, 6500 bp of the 5' side of the STF-1 gene was β-galactosidase. After fusion to the gene, expression of the STF-1-lacZ reporter in the transgenic mice is measured. Using X-gal as a pigment source substrate, β-galactosidase activity is detected in the pancreatic islets of the transformed littermates of three independent founder lines, but not in the litterates used as controls. (FIG. 3A).
    When maintaining the expression pattern of the endogenous STF-1 protein as described above, in exocrine acinar cells (FIG. 3B) of transgenic mice or in non-pancreatic tissues such as liver or spleen (not shown) There was no significant activity of β-galactosidase. If reportedly maintaining the expression of the endogenous STF-1 gene in the duodenum (see citations 8 and 13), in situ hybridization studies using anti-sense β-galactosidase RNA probes were performed. Transgene expression was also seen in the epidermal cells of the duodenum of transgenic animals (not here). These results indicate that 6500bp of STF-1 promoter is sufficient to target expression of STF-1 in pancreatic islet cells and duodenal cells.
    To define functional elements that induce STF-1 expression on pancreatic islet cells, the activity of the -6500 STF Luc reporter is tested in two distinct pancreatic islet cell lines (βTC3, HIT). As can be expected from the results in transgenic mice, the STF-1 reporter shows 20 to 100-fold activity in pancreatic islet cells compared to non-pancreatic islet cell lines such as HeLA, PC12 and COS ( 4A). On the other hand, when the intron 4 kb and the 3 'side 3 kb of the STF-1 gene were inserted into a small amount of the SV40 CAT promoter plasmid, this activity was not shown (not shown here), indicating that the STF-1 promoter 6.5 kb fragment was obtained from the pancreas. It also suggests specifically targeting and expressing STF-1 in cells.
    To describe the sequences in the STF-1 promoter that lead to expression in pancreatic islet cells, a series of 5 ′ deletion constructs are made and then these reporters are analyzed via transfection with HIT cells (FIG. 4B). Deleting sequences from -6500 bp to -3500 bp of the -6500 STF reporter construct reduced the activity of the STF-1 reporter by 4-fold, suggesting the presence of terminal active sequences within this site. When the STF-1 promoter was further deleted from -3500 bp to -190 bp, the activity of the reporter in HIT cells was not significantly affected (FIG. 4B). On the other hand, the deletion of the sequence from -190bp to -95bp of the STF-1 promoter significantly reduced the activity of the reporter in HIT cells, indicating that a proximal element is also required for STF-1 promoter function. Examination of the STF-1 promoter sequences -190bp to -95bp revealed that there were three consensus sequence E box motifs (FIG. 2A). Removing the two tandem E boxes located at -177 did not reduce promoter activity, but removing the E box at the base located at -104 (-95 SFT luc) resulted in no STF-1 in HIT cells. It is not expressed.
    Example 10
    Recognition of USF-containing complexes of the base E box in the STF-1 promoter
    To characterize upstream factors that bind to functional elements in the STF-1 promoter, a DNase I protection assay is performed using nuclear extracts from HIT cells and HeLa cells (FIG. 5A). Footprinting activity was prominent in both extracts, and their boundaries were consistent with the functionally major base E box motif (-118 / -95). The gel mobility change analysis describes in more detail the properties of the important -104 E box motif-binding proteins in HIT and HeLa extracts. Using double-stranded STF-1 oligonucleotides ranging from -118 to -95, complexes C1, C2 and C3 were observed with the two nuclear extracts above (FIG. 5B). Formation of complexes C1, C2 and C3 is inhibited in the binding reaction by an unlabeled 50-fold excess of STF-1 E box competitor oligonucleotides. On the other hand, mutant E box oligonucleotides or non-specific competitor DNA did not affect this binding activity, meaning that C1, C2 and C3 have specificity for the STF-1 E box sequence. No qualitative difference was observed in this complex pattern between HeLa and HIT extracts, suggesting that the E box motif located at -104 can recognize factors that are equally expressed in these two cell types.
    According to previous reports, E-boxes such as the -118 / -95 motif (CACGTG) preferentially bind to BHLH-ZIP proteins such as myc, max, TFE-3, TFE-B and USF. The presence of any of these proteins in the C1, C2 and C3 complexes (see citations 26 and 27) is determined. Since USF, a -118 / -95 E box-binding protein, can withstand thermal denaturation, it has been tested whether USF, a higher-factor of stability against heat, is a component of C1, C2 and C3 (Citation Document 28). Reference). In particular, the addition of anti-USF antiserum to the gel mobility change-reactant interfered with the formation of all three complexes (FIG. 5C). In contrast, anti-TFE-3 antiserum had no effect on complexes C1, C2 or C3, suggesting that these complexes are more likely to be formed by USF proteins. In gel transfer assays, recombinant USF-1 forms a protein-DNA complex, which moves to the same position relative to complex C2 (not here). In studies through protection from DNase I, recombinant USF-1 footprinting activity is consistent with that observed in HIT cells (FIG. 5A).
    Two forms of USF (called USF-1, USF-2) seem to be expressed in most cell types (see citation 18). To distinguish which of the USF proteins are contained in C1, C2 and C3, the HIT or HeLa extract is incubated with either anti-USF-1 specific antiserum or anti-USF-2 specific antiserum (FIG. 5D). . Although USF-1 antiserum can "supershift" all three complexes, only USF-2 specific antiserum inhibits formation of complexes C1 and C3. Through this result, complex C2 contains a USF-1 homodimer, while complexes C1 and C3 correspond to USF-1 / USF-2 heterodimers.
    To confirm whether the CACGTG E box sequence is essential for STF-1 promoter activity, a mutant STF-1 oligonucleotide is constructed in which two base pairs are substituted in the E box (-118 / -95). In the analysis of the gel mobility change of HIT nuclear extract, this mutant E box motif (AACGCG) failed to form complexes C1, C2 and C3 and could compete with USF-1 binding to wild type E box oligonucleotides. Could not (Figure 6A). Similarly, unshortened STF-1 (6.5 kb) and shortened (-190 STF) reporter plasmids containing the mutant STF E motifs are nearly 1 / 10-fold more active compared to the wild type in pancreatic islet cells. Dropped (Figure 6B). These results show that the E box of the base that binds to USF is of substantial importance for STF-1 promoter activity.
    Thus, two elements within the 6500 bp STF-1 5 'sequence appear to be important for pancreatic islet-specific expression: the terminal element located between -6500 and -3500 and the base element located at -104. The -104 element in the base consists of the E box motif, a consensus sequence that recognizes the USF, the ancestor of the parent. There is a wealth of evidence showing that USF is important for STF-1 promoter activity. First, USF-1 and USF-2 specific antibodies as well as USF-1,2 non- discriminatory antibodies recognize STF-1 E box-specific complexes. Second, the binding activity of the STF-1 E box in the HIT nuclear extract has properties that remind us of the USF: the complex is heat stable and shows a similar half-life to recombinant USF-1. Finally, point mutations that interfere with USF complex formation on the STF E box reduce STF-1 reporter activity. These results suggest that the USF complex is indeed important for STF-1 promoter activity and thus also for pancreatic organogenesis.
    In addition to USF, other nuclear factors (myc and max are most noted) can also bind strongly to the STF-1 E box (CACGTG) motif. myc is known to bind to the E box motif as a heterodimer together with max to transcribe the transcription of the target gene (see citations 32 and 33). Since the expression of the myc gene is usually undetectable in cells after mitosis (eg pancreatic islet cells), the myc-max complex may not be involved in STF-1 promoter regulation. On the other hand, during development, STF-1 expression focuses on proliferating ductal cells (see citation 6), so myc can promote STF-1 expression under these conditions. In this regard, the large changes in STF-1 expression observed during pancreatic development partially reflect that the changes in E box binding activity ultimately limit STF-1 production to pancreatic islet cells.
    Example 11
    Gel Transfer Assay and DNase I Protection Assay
    For electrophoretic mobility change analysis, double stranded oligonucleotides are labeled using a 32 PdCTP fill-in reaction with cleno fragments. 5 μg of the nuclear extract was incubated on 0.5 ng of labeled oligonucleotides and 1 μg of non-specific competition DNA on ice for 30 minutes, followed by non-modified polyacrylamide electrophoresis. For the hypermigration assay, the nuclear extract is pre-incubated with antisera for 30 minutes to 1 hour on ice before adding the labeled probe. Anti-HNF-3α, HNF-3β and HNF-3γ antibodies were provided by S. Duncan and J. Darnell. Anti-beta-2 antibodies were provided by MJTsai. Anti-E2A antibodies were purchased from Santa Cruz Biochemicals. DNase I protection assays are performed as previously described (see citation 37). Recombinant HNF-3α was provided by K.Zaret.
    Example 12
    Northern Blotting
    The soma monohydrate Tatiana Norma / Insulated Reno town Tu6 cells incubated with different times in ethanol or 10-7 deksa meth respect. The entire RNA is extracted using a guanidinium-phenol process. 15 μg of total RNA is transferred on agarose gels containing formaldehyde and then transferred to Zeta-Probe. Randomly primed STF-1 and tubulin cDNAs are prepared using an Amersham random priming kit.
    Example 13
    Regulation of STF-1 Expression of Endoderm Factors HNF-3β and Beta-2 Via Pancreatic Islet-Specific Enhancers
    Promoter constructs containing 530 bp fragments from -6200 to -5670 of the STF-1 gene are completely inhibited by dexamethasone treatment, while small amounts of STF-1 promoter constructs containing ubiquitous USF-1 recognition sites are not. Not (FIG. 7A). The activity of this 530 bp STF-1 site in HIT-T15 cells is 5 to 10 times greater than that of COS-7 cells, indicating that this fragment has pancreatic islet-specific activity (FIG. 7B). ). Examination of the nucleotide sequence within the pancreatic cell-specific STF-1 enhancer revealed that there is a consensus sequence motif for E box-binding proteins (-5.98 to -5.963) and HNF-3 (-5.927 to -5.907). (FIG. 7C). The E box motif, called the B element, is identical in sequence to the NIR and FAR elements in the insulin I and II promoters of rats (see citation 34). The HNF-3 site, called the H element, is identical to 9 out of 12 with the HNF-3 consensus sequence binding site.
    To determine whether the B and H elements are actually recognized by pancreatic islet-specific nuclear proteins, a DNase I protection assay is performed using STF-1 enhancer fragments labeled with 32 P ranging from -5870 to -6100. Nuclear extracts prepared from HIT T15 cells were found to have DNA binding activity at the B site (E box motif) and H site (HNF-3 motif) (compare lanes 1 and 2 of FIG. 8A). B elements were equally protected in DNase I protection assays using nuclear extracts of HIT, HeLa and COS 7 cells, while H elements were only observed in extracts of HIT cells (compare FIG. 8A, lanes 2 and 4). High-sensitivity sites that interfere with the footprint of the H element were also seen in reactions containing purified recombinant HNF-3α protein, a member of the HNF-3 family of regulators in HIT-T15 cells. It can be assumed that it binds to the STF-1 H element (compare FIG. 8A, lanes 2 and 5).
    To further characterize the nuclear factor that recognizes the H element of the STF-1 enhancer, gel mobility change analysis is performed using a 32 P labeled STF-1 H element probe. Major complexes with poor mobility were observed in reactions containing HIT or RIN nuclear extracts, and the formation of such complexes was specifically inhibited by adding 100-fold molar excess of non-labeled wild type H element competitor DNA, but mutant H element Not by competitor DNA (compare FIG. 8B, lanes 4-5, 9-10, 14-15). No specific protein-DNA complexes were observed in the reaction containing HeLa nuclear extract, indicating that the H element would recognize nuclear factors with limited expression patterns (compare FIG. 8B, lanes 1 and 6). On the other hand, the presence of the H element-specific complex in HepG2 hepatoma extract with the same mobility as the RIN / HIT-T15 complex suggests that the H element-binding protein can generally be expressed in cells of endoderm origin. (FIG. 8B, comparing lanes 3 to 5).
    The nuclear activator HNF-3 group consists of three genes (α, β and γ) that bind DNA through a highly conserved winged helix domain. To determine whether the binding activity of H elements in HIT extracts corresponds to members of the HNF-3 group, gel mobility change assays are performed using antisera specific to each of the HNF-3 members. Although three HNF-3 proteins were detected in nuclear extracts of HIT-T15 insulinoma cells by Western blot analysis (not here), only HNF-3β antiserum using the same extract with H element probe It was found to prevent the formation of protein-DNA complexes (FIG. 8C, lanes 1-4). The same results were obtained in gel transfer assays of nuclear extracts prepared from primary cultures of mature mouse pancreatic cells (FIG. 8C, lanes 5 to 8).
    The B element in the STF-1 enhancer contains the E box motif of the same consensus sequence as the E box in the insulin promoter. Beta-2, a pancreatic islet-specific factor, together with the ubiquitous factor E47, enhances the activity of the insulin promoter by binding to the insulin promoter as a heterodimer. The formation of an E47 heterodimer was tested. In gel mobility change analysis of HIT nuclear extracts, four specific DNA-protein complexes were competed by the addition of a non-labeled wild type B element oligonucleotide, but not by the addition of a mutant B element oligonucleotide. Formation of the B element probe labeled 32 P was observed (compare lanes FIG. 8D, 1, 5, 6). The slowest moving complex is in position with the recombinant E2A / beta 2 heterodimer complex (compare FIG. 8D, lanes 1 and 7). Although unrelated antiserum had no effect on B element binding activity (lane 4 in FIG. 8D), beta-2 or E2A antiserum specifically inhibited the formation of the slowest moving complex (Fig. 8). 8D, lanes 2 and 3), which shows that the beta-2 / E2A heterodimer actually recognizes the B element in the HIT nuclear extract.
    To determine the importance of HNF-3β and E2A / beta-2 recognition sites in mediating the activity of the STF-1 enhancer, point mutations in the B and H elements that disrupt the binding of these syngeneic factors in vitro. Caused. When tested at an enhancer of 530 bp, STF-1 reporter plasmids containing mutations in the B or H elements were significantly less active in HIT T15 cells compared to wild type constructs (FIG. 9A). In contrast, mutant B and H constructs have the same activity as the wild type -6500 STF-1 reporter in COS-7 cells; However, these mutations show that the pancreas also destroys transcriptional activity that is specifically related to cells. Thus, the B and H element mutant STF-1 reporter plasmids also do not respond to dexamethasone induction in HIT T15 cells, suggesting that this hormone may specifically interfere with E2A / beta 2 or HNF-3β activity. It can be seen (Fig. 9B).
    To determine whether dexamethasone inhibits STF-1 expression by disrupting HNF-3β or beta 2 / E47 activity on terminal enhancers, a transsient was performed using HNF-3β or beta 2 and E47 effector plasmids. ) Transfection analysis was performed. Overexpression of HNF-3β (FIG. 9B) or beta 2 / E47 (not here) has a slight effect on STF-1 reporter expression in unstimulated HIT T15 cells (FIG. 9B, No. 1 and Bar 5). HNF-3β has been shown to reduce the inhibitory effect of dexamethasone on wild-type STF-1 reporter activity (compare FIG. 9B, bar 1, 3, 7), beta-2 / E47, STF- Activators such as 1 and HNF-4 did not restore STF-1 promoter activity in cells treated with dexamethasone. Titration experiments performed with increasing amount of effector plasmid revealed that HNF-3β restored the activity of the -6500 STF-1 LUC reporter depending on the dose (FIG. 9C). On the other hand, HNF-3β does not enhance the activity of mutant STF-1 reporter plasmids containing mutations in the H element, suggesting that the inhibitory effect of this activator occurs through recognition sites in the STF-1 enhancer. (Compare bars 9, 2, 4, 6 and 8).
    The following are the references cited herein.
    Pictet, R. L., et al., Devlop. Biol .; 29,436-467 (1972).
    2. Gittes, G. K., et al., Proc. Natl. Acad. Sci .; 89, 1128-1132 (1992).
    3. Alpert, S., et al., Cell; 53,295-308, (1988).
    Jonsson, J., et al., Nature; 371,606-609, (1994).
    5. Ohlsson, H., et al., EMBO J .; 12,4251-4259, (1993).
    6. Guz, Y., et al., Development, 121, 11-18, (1995).
    7. Leonard, J., et al., Proc. Natl. Acad. Sci .; 89,6247-6251, (1992).
    8. Leonard, J., et al., Mol Endocrinol .; 7,1275-1283, (1993).
    9. Peers, B., et al., Mol Endocrinol .; 8,1798-1806, (1994).
    10. Walker, M. D., et al., Nature; 306,557-561, (1983).
    11. Vallejo, M., et al., J. Biol. Chem .; 267,12868-12875 (1992).
    12. Karlsson, O., et al., Proc. Natl. Acad. Sci .; 84,8819-8823, (1987).
    13. Miller, C., et al., EMBO J .; 13,1145-1156, (1994).
    14. Naya, F. J. et al., Genes and Dev .; 9,1009-1019 (1995).
    15. German, M., et al., Genes Dev .; 6,2165-2176, (1992).
    16. Peshavaria, M., et al., Mol Endocrinol .; 8,806-816, (1994).
    17. Van Dyke, M. W., et al., Gene III; 99-104, (1992).
    18. Sirito, M., et al., Nucleic Acids Res .; 22,427-433, (1994).
    19. Madsen, O. D., et al., J. Cell Biol .; 103,2025-2034, (1986).
    20. Maxwell, I. H., et al., Biotechniques; 7,276-280, (1989).
    21.Hanahan, D., Nature; 315,115-122, (1985).
    22. Montminy, M. R., et al., Nature; 328,175-178, (1987).
    23. Weis, L., et al., FASEB J .; 6,3300-3309, (1992).
    24. Ishi, S., et al., Science; 230, 1378-1381, (1985).
    25. Geng, Y., et al., Mol. Cell. Biol .; 13,4894-4903, (1993).
    26. Beckman, H., et al., Genes and Dev .; 4,167-179, (1990).
    27. Gregor, P. D., et al., Genes and Dev .; 4, 1730-1740, (1990).
    28. Sawadogo, M., et al., J. Biol. Chem .; 263, 11985-11993, (1988).
    29. Krumlauf, R., Cell; 78,191-201 (1994).
    30. Boncinelli, E., et al., Hum. Reproduc .; 3,880-886, (1988).
    31 Lewis, E. B., Nature; 276,565-570, (1978).
    32. Kretzner, L., et al., Nature; 359,426-429, (1992).
    33. Blackwood, E., et al., Science; 251, 1211-1217, (1991).
    34. Edlund, T., et al., Science; 230,912-916, (1985).
    35. Clark, K., et al., Nature; 364,412-420, (1993).
    36. Lai, E., et al., PNAS; 90,10421-10423, (1993).
    37. Sharma, et al., JBC; 271,2294-2299 (1996).
    Sequence list
    (1) General Information:
    (V) Applicants: Montmini and Sharma
    (Ii) Name of the Invention: A novel method describing the properties of a compound that promotes expression of STF-1 in pancreatic islet cells.
    (Iii) sequence number: 4
    (Ⅳ) contact address:
    (A) Address: Wahler James F., Attorney
    (B) Distance: Suit 1560 One Riverway
    (C) city: Houston
    (D) State: Texas
    (E) Country: United States
    (F) Zip code: 77056
    (Iii) computer-readable:
    (A) Media Type: DS, HD 1.44 Mb / 1.44 Mo
    (B) Computer: IBM PC compatible model
    (C) operating system: PC-DOS / MS-DOS
    (D) Software: WordPerfect 6.0
    (Iii) Application Information:
    (A) Application number: unspecified
    (B) filing date:
    (C) Classification: Usability
    (Iii) Agent Information:
    (A) Statement: Weiler James F.
    (B) registration number: 16,040
    (C) Reference / Event Record Number: D-5848
    (Iii) Communication Information:
    (A) Telephone: 713-626-8646
    (B) Fax: 713-963-5853
    (2) Information about SEQ ID NO: 1
    (Iii) sequence characteristics:
    (A) Length: 403 bp
    (B) Type: nucleic acid
    (C) chain: double strand
    (D) mode: linear
    (Ii) Molecular Type:
    (A) Technique: Other Nucleic Acids
    (Ⅲ) Assumption: No
    (Ⅳ) Antisense: No
    (Iii) Source:
    (B) strains:
    (C) Separated Objects:
    (D) Generation step:
    (E) Organization type:
    (F) cell type:
    (G) cell line:
    (xi) sequence description:
    (3) information about SEQ ID NO: 2
    (Iii) sequence characteristics:
    (A) Length: 490bp
    (B) Type: nucleic acid
    (C) chain: double strand
    (D) mode: linear
    (Ii) Molecular Type:
    (A) Technique: Other Nucleic Acids
    (Ⅲ) Assumption: No
    (Ⅳ) Antisense: No
    (Iii) Source:
    (B) strains:
    (C) Separated Objects:
    (D) Generation step:
    (E) Organization type:
    (F) cell type:
    (G) cell line:
    (xi) sequence description:
    (4) information about SEQ ID NO: 3
    (Iii) sequence characteristics:
    (A) Length: 20bp
    (B) Type: nucleic acid
    (C) chain: Japanese chain
    (D) mode: linear
    (Ii) Molecular Type:
    (A) Technique: Other Nucleic Acids
    (Ⅲ) Assumption: No
    (Ⅳ) Antisense: No
    (Iii) Source:
    (B) strains:
    (C) Separated Objects:
    (D) Generation step:
    (E) Organization type:
    (F) cell type:
    (G) cell line:
    (xi) sequence description:
    TCAGTGACAG ATGGAGTCCT 20
    (5) information about SEQ ID NO: 4
    (Iii) sequence characteristics:
    (A) Length: 20bp
    (B) Type: nucleic acid
    (C) chain: Japanese chain
    (D) mode: linear
    (Ii) Molecular Type:
    (A) Technique: Other Nucleic Acids
    (Ⅲ) Assumption: No
    (Ⅳ) Antisense: No
    (Iii) Source:
    (B) strains:
    (C) Separated Objects:
    (D) Generation step:
    (E) Organization type:
    (F) cell type:
    (G) cell line:
    (xi) sequence description:
    TCAGTGAAAG ACGGAGTCCT 20
    权利要求:
    Claims (9)
    [1" claim-type="Currently amended] (1) an STF-1 enhancer, a promoter having a sequence selected from the group consisting of SEQ ID NO: 1 or SEQ ID NO: 2 or fragments thereof, and a reporter gene under the transcriptional control of the STF-1 enhancer and the promoter, wherein the reporter gene is Providing a detectable signal to the cells of the pancreas, which is also a host cell);
    (2) transferring the vector into said host cell;
    (3) culturing said host cell in the presence of a test compound to determine if the test agent can stimulate said host cell to generate said detectable signal; And
    (4) analyzing said signal to determine if the test agent can stimulate this host cell to generate said detectable signal, wherein if said signal is present, the test compound is capable of Stimulating to induce transcription of STF-1, and the absence of said signal means that the test compound does not stimulate pancreatic islet cells to induce the transcription of STF-1). To measure the ability of a test compound to induce transcription of STF-1.
    [2" claim-type="Currently amended] The method of claim 1, wherein the reporter gene is an enzyme.
    [3" claim-type="Currently amended] The method of claim 2, wherein the enzyme is selected from the group of luciferase and β-galactosidase.
    [4" claim-type="Currently amended] The method of claim 1, wherein the metastasis step is performed by transfection.
    [5" claim-type="Currently amended] The method of claim 4, wherein the pancreatic islet cells are HIT cells.
    [6" claim-type="Currently amended] The method of claim 1, wherein the metastasis step is performed by trans gene introduction into the animal.
    [7" claim-type="Currently amended] (1) providing a vector containing an STF-1 promoter and a reporter gene under the transcriptional control of the STF-1 promoter, wherein the reporter gene can provide a detectable signal to the pancreatic islet cell as a host cell ;
    (2) transducing the vector into an animal embryo using this vector as a transgene;
    (3) growing the embryo into an animal with pancreatic islet cells; And
    (4) analyzing said detectable signal to determine whether said pancreatic islet cells of said animal express said reporter gene, wherein insulin-producing pancreatic islet cells are present if said signal is present. Means that no insulin-producing pancreatic cells are present if no such signal is present).
    [8" claim-type="Currently amended] 8. The method of claim 7, wherein the reporter gene produces a fluorescent protein.
    [9" claim-type="Currently amended] The method of claim 8, further comprising distinguishing insulin-producing pancreatic islet cells from non-insulin-producing pancreatic islet cells via fluorescence-active cell sorting (FACS).
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    同族专利:
    公开号 | 公开日
    NZ330866A|2001-04-27|
    ZA9700476B|1998-07-21|
    RU2183465C2|2002-06-20|
    WO1997026360A1|1997-07-24|
    US5969210A|1999-10-19|
    JP2000503211A|2000-03-21|
    EP0876492B1|2005-09-07|
    AT304055T|2005-09-15|
    AU1835797A|1997-08-11|
    CA2244176C|2008-07-15|
    IL125153D0|1999-01-26|
    CN1154738C|2004-06-23|
    CA2244176A1|1997-07-24|
    AU726356B2|2000-11-02|
    DE69734143D1|2005-10-13|
    IL125153A|2009-07-20|
    JP4472025B2|2010-06-02|
    CN1209843A|1999-03-03|
    DE69734143T2|2006-07-13|
    EP0876492A1|1998-11-11|
    EP0876492A4|2001-04-11|
    KR100454621B1|2005-01-13|
    AU726356C|2002-11-07|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1996-01-22|Priority to US1041496P
    1996-01-22|Priority to US60/010,414
    1997-01-22|Application filed by 와일러 제임스 에프., 리서치 디벨럽먼트 파운데이션
    1999-11-15|Publication of KR19990081853A
    2005-01-13|Application granted
    2005-01-13|Publication of KR100454621B1
    优先权:
    申请号 | 申请日 | 专利标题
    US1041496P| true| 1996-01-22|1996-01-22|
    US60/010,414|1996-01-22|
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