Journal of Biological Chemistry · 2001 · 104 citations · 40 references
Life SciencesGeneticsMolecular BiologyCell SignalingGene ExpressionTranscriptional RegulationChromatinCis-regulatory ElementsEnhancer FunctionPromoter RegulationTranscription FactorsTranscriptional Regulation In EukaryotesCell Type-specific Enhancer ActivityCell Type-specific Transcriptional RegulationDevelopmental EnhancersDistal Regulatory ElementsEnhancer-driven Transcriptional RegulationEnhancer-mediated ExpressionTranscription Factor Binding At EnhancersTranscriptional Control Elements
The PDX-1 transcription factor plays a key role in pancreas development. Although expressed in all cells at the early stages, in the adult it is mainly restricted to the β-cell. To characterize the regulatory elements and potential transcription factors necessary for human PDX-1 gene expression in β-cells, we constructed a series of 5′ and 3′ deletion fragments of the 5′-flanking region of the gene, fused to the luciferase reporter gene. In this report, we identify by transient transfections in β- and non-β-cells a novel β-cell-specific distal enhancer element located between −3.7 and −3.45 kilobases. DNase I footprinting analysis revealed two protected regions, one binding the transcription factors SP1 and SP3 and the other hepatocyte nuclear factor 3β (HNF-3β) and HNF-1α. Cotransfection experiments suggest that HNF-3β, HNF-1α, and SP1 are positive regulators of the herein-described human PDX-1 enhancer element. Furthermore, mutations within each motif abolished the binding of the corresponding factor(s) and dramatically impaired the enhancer activity, therefore suggesting cooperativity between these factors. The PDX-1 transcription factor plays a key role in pancreas development. Although expressed in all cells at the early stages, in the adult it is mainly restricted to the β-cell. To characterize the regulatory elements and potential transcription factors necessary for human PDX-1 gene expression in β-cells, we constructed a series of 5′ and 3′ deletion fragments of the 5′-flanking region of the gene, fused to the luciferase reporter gene. In this report, we identify by transient transfections in β- and non-β-cells a novel β-cell-specific distal enhancer element located between −3.7 and −3.45 kilobases. DNase I footprinting analysis revealed two protected regions, one binding the transcription factors SP1 and SP3 and the other hepatocyte nuclear factor 3β (HNF-3β) and HNF-1α. Cotransfection experiments suggest that HNF-3β, HNF-1α, and SP1 are positive regulators of the herein-described human PDX-1 enhancer element. Furthermore, mutations within each motif abolished the binding of the corresponding factor(s) and dramatically impaired the enhancer activity, therefore suggesting cooperativity between these factors. pancreatic duodenal homeobox kilobase(s) base pair(s) Luciferase hepatocyte nuclear factor Chinese hamster ovary thymidine kinase electrophoretic mobility shift assay The mammalian pancreas develops by fusion of dorsal and ventral buds which form as evaginations of the upper duodenal part of the gut. Identification of the homeodomain-containing transcription factor PDX-11 as the first molecular marker temporally correlates with the pancreatic commitment of the epithelial cells in this region (1Guz Y. Montminy M.R. Stein R. Leonard J. Gamer L.W. Wright C.V. Teitelman G. Development. 1995; 121: 11-18Crossref PubMed Google Scholar, 2Ahlgren U. Jonsson J. Edlund H. Development. 1996; 122: 1409-1416Crossref PubMed Google Scholar, 3Ahlgren U. Jonsson J. Jonsson L. Simu K. Edlund H. Genes Dev. 1998; 12: 1763-1768Crossref PubMed Scopus (792) Google Scholar). Targeted inactivation of this gene in the mouse (4Jonsson J. Carlsson L. Edlund T. Edlund H. Nature. 1994; 371: 606-609Crossref PubMed Scopus (1589) Google Scholar) as well as its mutation in man (5Stoffers D.A. Zinkin N.T. Stanojevic V. Clarke W.L. Habener J.F. Nat. Genet. 1997; 15: 106-110Crossref PubMed Scopus (956) Google Scholar) results in agenesis of the pancreas. The gene is expressed both in endocrine and exocrine cells of the developing pancreas; however in the adult islet, its expression is predominantly restricted to the β-cell (1Guz Y. Montminy M.R. Stein R. Leonard J. Gamer L.W. Wright C.V. Teitelman G. Development. 1995; 121: 11-18Crossref PubMed Google Scholar, 2Ahlgren U. Jonsson J. Edlund H. Development. 1996; 122: 1409-1416Crossref PubMed Google Scholar, 3Ahlgren U. Jonsson J. Jonsson L. Simu K. Edlund H. Genes Dev. 1998; 12: 1763-1768Crossref PubMed Scopus (792) Google Scholar), where it acts as the mediator of glucose action on insulin gene expression (6Melloul D. Ben-Neriah Y. Cerasi E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3865-3869Crossref PubMed Scopus (160) Google Scholar, 7Petersen H.V. Serup P. Leonard J. Michelsen B.K. Madsen O.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10465-10469Crossref PubMed Scopus (191) Google Scholar, 8Marshak S. Totary H. Cerasi E. Melloul D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15057-15062Crossref PubMed Scopus (153) Google Scholar, 9MacFarlane W.M. Read M.L. Gilligan M. Bujalska I. Docherty K. Biochem. J. 1994; 303: 625-631Crossref PubMed Scopus (125) Google Scholar). In mice, β-cell-selective disruption ofpdx-1 leads to diabetes associated with reduced insulin and glucose transporter 2 expression (3Ahlgren U. Jonsson J. Jonsson L. Simu K. Edlund H. Genes Dev. 1998; 12: 1763-1768Crossref PubMed Scopus (792) Google Scholar). Development, cell fate, and cell differentiation are complex events that depend on switching on and off the expression of specific sets of genes. Such regulation operates mainly at the transcriptional level by the assembly of multiprotein complexes at the enhancer(s) and the promoter regions of the gene. These complexes are formed and stabilized through multiple protein-DNA and protein-protein interactions. Since PDX-1 plays such a central role in β-cell differentiation and function, the molecular basis of its regulation and, hence, the DNA elements and the interacting proteins involved in this process must be clarified. To this end, a 6.5-kb fragment upstream of the transcription start site of rat pdx-1/stf-1 (10Sharma S. Leonard J. Lee S. Chapman H.D. Leiter E.H. Montminy M.R. J. Biol. Chem. 1996; 271: 2294-2299Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) and a fragment extending from the −4.5 to +8.2-kb region of mouse pdx-1(11Wu K.L. Gannon M. Peshavaria M. Offield M.F. Henderson E. Ray M. Marks A. Gamer L.W. Wright C.V. Stein R. Mol. Cell. Biol. 1997; 17: 6002-6013Crossref PubMed Google Scholar) were shown to direct the expression of the β-galactosidase reporter gene to pancreatic islet cells in transgenic mice. In transiently transfected β-cells, appropriate expression of the ratpdx-1 gene depended in part on a proximal E box that predominantly binds the ubiquitous transcription factor USF1 (10Sharma S. Leonard J. Lee S. Chapman H.D. Leiter E.H. Montminy M.R. J. Biol. Chem. 1996; 271: 2294-2299Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Tissue-specific regulation also appeared to require a distal enhancer sequence located between the −6.2- and −5.67-kb region of the ratpdx-1 gene. Analysis of the factors bound to this element indicated that the endodermal factors HNF-3β and Neurod/Beta2 act cooperatively to induce pdx-1 expression in islet cells. Furthermore, it was shown that glucocorticoids reduce pdx-1gene expression by interfering with HNF-3β activity (12Sharma S. Jhala U.S. Johnson T. Ferreri K. Leonard J. Montminy M. Mol. Cell. Biol. 1997; 17: 2598-2604Crossref PubMed Scopus (99) Google Scholar). Studies on the mouse pdx-1 promoter revealed that the region from −2560 to −1880 bp regulates β-cell-specific transcription and directs the appropriate developmental and adult-specific expressions in transgenic animals. It was found that an HNF-3-like element contained within this region is important for the β-cell specificity (11Wu K.L. Gannon M. Peshavaria M. Offield M.F. Henderson E. Ray M. Marks A. Gamer L.W. Wright C.V. Stein R. Mol. Cell. Biol. 1997; 17: 6002-6013Crossref PubMed Google Scholar). Additional studies indicate that two highly conserved sequences in the 5′-flanking region of the PDX-1 gene (PH1/area1 and PH2/area2) confer β-cell-specific transcriptional activity (13Gerrish K. Gannon M. Shih D. Henderson E. Stoffel M. Wright C.V. Stein R. J. Biol. Chem. 2000; 275: 3485-3492Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 14Marshak S. Benshushan E. Shoshkes M. Havin L. Cerasi E. Melloul D. Mol. Cell. Biol. 2000; 20: 7583-7590Crossref PubMed Scopus (109) Google Scholar) on a heterologous promoter. DNase I footprinting and binding analyses revealed that both sequences bind and are transactivated by HNF-3β; this is in accordance with the fact that its absence in mouse embryonic stem cells had a dramatic effect on pdx-1 gene expression (13Gerrish K. Gannon M. Shih D. Henderson E. Stoffel M. Wright C.V. Stein R. J. Biol. Chem. 2000; 275: 3485-3492Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Thus, data from studies with the rat (12Sharma S. Jhala U.S. Johnson T. Ferreri K. Leonard J. Montminy M. Mol. Cell. Biol. 1997; 17: 2598-2604Crossref PubMed Scopus (99) Google Scholar), the mouse (11Wu K.L. Gannon M. Peshavaria M. Offield M.F. Henderson E. Ray M. Marks A. Gamer L.W. Wright C.V. Stein R. Mol. Cell. Biol. 1997; 17: 6002-6013Crossref PubMed Google Scholar, 13Gerrish K. Gannon M. Shih D. Henderson E. Stoffel M. Wright C.V. Stein R. J. Biol. Chem. 2000; 275: 3485-3492Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), and the human (13Gerrish K. Gannon M. Shih D. Henderson E. Stoffel M. Wright C.V. Stein R. J. Biol. Chem. 2000; 275: 3485-3492Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 14Marshak S. Benshushan E. Shoshkes M. Havin L. Cerasi E. Melloul D. Mol. Cell. Biol. 2000; 20: 7583-7590Crossref PubMed Scopus (109) Google Scholar) promoters suggest that HNF-3β is an important regulator of pdx-1 gene transcription. Interestingly, we found that PDX-1 itself binds to the PH1/area 1 element and cooperates with HNF-3β to activate transcription (14Marshak S. Benshushan E. Shoshkes M. Havin L. Cerasi E. Melloul D. Mol. Cell. Biol. 2000; 20: 7583-7590Crossref PubMed Scopus (109) Google Scholar). To further identify key components of importance for the expression of the human PDX-1 gene in β-cells, we sequenced about 4.5 kb of the 5′-flanking region of the gene, constructed a series of 5′ deletion fragments fused to the luciferase reporter gene, and tested them in β- and non-β-cells. In this report we provide evidence for a novel β-cell-specific distal enhancer element that appears to be specific to the human PDX-1 gene. DNase I footprinting analysis revealed two protected regions: one binding the proteins identified as SP1 and SP3 and the second, the transcription factors HNF-3β and HNF-1α. These are thus candidate transcription factors that are involved in regulating selective expression of the human PDX-1 gene in the adult β-cell. Hamster insulinoma HIT-T15, mouse insulinoma βTC6, and mouse glucagonoma αTC1 cells were cultured in Dulbecco's modified Eagle's medium with 15% horse serum and 2.5% fetal calf serum (FCS), AR42J, HepG2, CHO, HeLa, and NIH 3T3 cells with 10% FCS. 100 units/ml penicillin and 100 mg/ml streptomycin were added to the media. HIT-T15, HepG2, NIH3T3, COS, and CHO cells were transfected using the calcium phosphate coprecipitation method (15Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989)Molecular Cloning: A Laboratory Manual, pp. 16.33–16.36 and 16.66–16.67, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar), and αTC1, βTC6, and AR42J cells were transfected using the Fugene transfection reagent (Roche Molecular Biochemicals) according to the manufacturer's recommendations with 1.5 μg of humanPDX-1 luciferase derivatives and 0.5 μg of the internal control cytomegalovirus-β-galactosidase DNA plasmid (CMV-βGal). In co-transfection experiments 1.5 μg of the reporter plasmid and 0.1–1 μg (as indicated) of the expression plasmids HNF-3α, HNF-3β, HNF-1α, HNF-1β, SP1, and/or SP3 were used. The cells were harvested 48 h after transfection, and about 100 μg of protein extracts were used to measure luciferase activity with the luciferase assay system (Promega, Madison, WI) and about 10 μg for the β-galactosidase assay as described (15Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989)Molecular Cloning: A Laboratory Manual, pp. 16.33–16.36 and 16.66–16.67, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar). Luciferase activity was measured with a luminometer (EG&G Berthold, Bad Wildbad, Germany) and normalized to β-galactosidase values. Plasmids containing fragments of the human PDX-1 promoter were kindly by of The fragment the sequences from to kb was in the luciferase A series of 5′ and 3′ were The PDX-1 enhancer element was to the thymidine kinase promoter of the were by and each and was by extracts were as described E. P. 17: PubMed Scopus Google Scholar). cell extracts were by of the cells in 2 1 and 10 was by and and the was by at for at were by the method Biochem. PubMed Scopus Google Scholar). DNA binding were by in for 10 μg of cell extracts nuclear extracts with the sequence in the of 10 10% 2 μg of and The binding contained 10% 1 and 1 μg were in a and in the for 10 the of the were by a using the fragment of DNA I. 1 of were added the The used SP1 and binding DNase I footprinting a fragment to was at by a using the fragment of DNA I and to a specific activity of were with μg of cell extracts in a containing 10 and μg of of at of DNase I in and 10 was added for 1 The was by of containing and μg of DNA was with and on a of each were using the and PubMed Scopus Google Scholar). To the DNA sequences gene we a fragment extending from about to kb of the 5′-flanking region of humanPDX-1 to luciferase reporter gene and constructed a series of 5′ as in The were transiently transfected and CHO cells. was β-cell as shown in of sequences between and −3.7 kb to an in luciferase activity in in CHO the of a regulatory an deletion of the distal region located between −3.7 and kb was the activity by about suggesting the of a positive regulatory element. of sequences to bp had In deletion of the proximal region between and bp abolished the transcriptional activity in both cell S. Leonard J. Lee S. Chapman H.D. Leiter E.H. Montminy M.R. J. Biol. Chem. 1996; 271: 2294-2299Abstract Full Text Full Text PDF PubMed Scopus (79) Google and data In this data indicate that the fragment a positive regulatory region that β-cell-specific expression on the reporter gene. To the sequences for the activity in the fragment extending from −3.7 to kb was upstream of the PDX-1 promoter fused to the luciferase gene. In transiently transfected an in transcriptional activity was 5′ deletion of sequences between −3.7 and kb reduced the transcriptional activity to promoter To further the regulatory sequences contained within this 3′ were transient transfections revealed the of a positive regulatory element the region from −3.7 to −3.45 element had the of an enhancer as it transactivated the PDX-1 promoter in upstream of the thymidine kinase promoter its role as a this fragment β-cell-specific expression of the luciferase reporter gene in transfected β-cells, and β-cells, the exocrine AR42J the glucagonoma αTC1 CHO and the cells 2 this analysis it that is a distal β-cell-specific enhancer located between −3.7 and −3.45 element appears to be specific to the human PDX-1 gene as sequence was found in the of this region in the mouse gene The transcriptional activity by the distal enhancer element of human PDX-1 the of regulatory elements in this To such elements with specific we DNase I footprinting analysis using the fragment extending from −3.7 to kb as a and extracts from and CHO cells. shown in two protected regions were The of the first protected sequence a site in the of cell The between and within a region and a in and CHO cell The sequence of the human PDX-1 enhancer element is in with the regions To further characterize the factors binding to the regions, these sequences were and used as to and CHO proteins by electrophoretic mobility shift the sequence as a two binding complexes and in were in cell analysis for potential binding sequences E. P. H. R. 1996; PubMed Scopus Google Scholar) revealed a element. To this motif with the of the containing the SP1 motif was added to the binding The DNA complexes were by the SP1 by a one To that SP1 are involved in specific SP1 and SP3 were added and to the binding The results that the complex a was by complex with SP3 In using the sequences with cell a complex and a complex were complex was in all pancreatic cells the glucagonoma αTC1 and the exocrine AR42J as well as in the cells. In the was mainly in a complex in AR42J and cells is contained within an region and analysis for transcription factors potential binding for homeodomain-containing proteins E. P. H. R. 1996; PubMed Scopus Google Scholar) for and To is involved in the electrophoretic mobility shift were using the sequence the form containing a modified motif The DNA complexes a and in cells were by of In the containing the a the sequence was used as the a and complexes were To the of HNF-3β and in the to with a series of was that the complex is by HNF-3β with the proteins Furthermore, the a complex with with extracts from cells transfected with an expression plasmid for HNF-3β were for with the sequence to that the protein contained in complex to the HNF-3β and complexes in cells to the complex in and were also by the corresponding these results that the and HNF-3β in cells bind the To the effect of the transcription factors on gene expression by the enhancer we transient transfection experiments in cells. To this end, the was with of the and plasmids in with HNF-3β expression plasmid In we experiments with SP1 and SP3 expression plasmids in HNF-3β and the activity in a Furthermore, with HNF-3β and of cooperatively the expression of the gene. HNF-3β and SP1 the gene, and with a of HNF-3β and of SP1 the expression of the gene in a In SP3 effect on the enhancer activity it dramatically the were to the SP1 in the of the human element to the luciferase reporter gene and by the promoter. The mutation that binding in enhancer activity in cells in the also an in enhancer The effect of HNF-3β on enhancer activity was further by experiments in CHO cells using the reporter the form a modified site with an HNF-3β expression was using the was with the reporter experiments that transactivated the enhancer by about had effect on the element The data thus suggest that the transcription factors HNF-3β, HNF-1α, SP1, and SP3 are regulators of the herein-described humanPDX-1 enhancer element. It is that PDX-1 as a regulator of the exocrine and endocrine pancreatic The pdx-1 gene is expressed early in cells of both it restricted mainly to containing and −4.5 sequences of the rat (10Sharma S. Leonard J. Lee S. Chapman H.D. Leiter E.H. Montminy M.R. J. Biol. Chem. 1996; 271: 2294-2299Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) and the mouse (11Wu K.L. Gannon M. Peshavaria M. Offield M.F. Henderson E. Ray M. Marks A. Gamer L.W. Wright C.V. Stein R. Mol. Cell. Biol. 1997; 17: 6002-6013Crossref PubMed Google gene, were in its expression to islet cells. to further characterize the potential regulatory elements of the human PDX-1 gene, we about 4.5 kb in the 5′-flanking region of the gene. transient transfections of β-cell and with 5′ and 3′ a β-cell-specific enhancer element located between −3.7 and −3.45 kb was The 5′-flanking sequences of the human and mouse are from the conserved proximal promoter highly located 3′ of the herein-described enhancer element are was between this enhancer and the one described further upstream in the rat gene (12Sharma S. Jhala U.S. Johnson T. Ferreri K. Leonard J. Montminy M. Mol. Cell. Biol. 1997; 17: 2598-2604Crossref PubMed Scopus (99) Google Scholar). These suggest that regulatory elements in pdx-1 gene to its β-cell-specific To identify the potential transcription factors that bind the distal enhancer DNase I analysis was and two protected regions and were The was found to bind the transcription factors SP1 and These factors bind DNA with specificity and SP1 is expressed in and inactivation of the gene in the mouse results in of the which early Mol. Cell. Biol. PubMed Scopus Google Scholar). are by SP1, which in acts cooperatively with other transcription factors R. Cell. Full Text PDF PubMed Scopus Google Scholar). SP3 shown to as an by with SP1 for binding to DNA M.R. J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google P. L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) as an on promoter and cell P. L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, H. M. 1997; PubMed Scopus (99) Google Scholar). It also that the of SP1 and SP3 thus the of S. G. PubMed Scopus Google Scholar). of SP1 were found in stem fetal and Mol. Cell. Biol. PubMed Scopus Google Scholar). also about as SP1 protein in pancreatic cells in of SP3 in all tested cells Furthermore, in this report we that in transfected SP1 a effect on the enhancer transcriptional activity, SP3 a effect on mainly by The protected is contained within an and factors binding this region were identified as HNF-3β and HNF-1α. The important role of HNF-3β in the conserved regulatory elements in the mouse and human region was shown (13Gerrish K. Gannon M. Shih D. Henderson E. Stoffel M. Wright C.V. Stein R. J. Biol. Chem. 2000; 275: 3485-3492Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 14Marshak S. Benshushan E. Shoshkes M. Havin L. Cerasi E. Melloul D. Mol. Cell. Biol. 2000; 20: 7583-7590Crossref PubMed Scopus (109) Google Scholar), and its absence in mouse embryonic stem cells dramatically impaired pdx-1 gene expression (13Gerrish K. Gannon M. Shih D. 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