Transcription Factor BETA2 Acts Cooperatively with E2A and PDX1 to Activate the Insulin Gene Promoter

The insulin gene is efficiently expressed only in pancreatic beta cells. Using reverse transcriptase-polymerase chain reaction analysis, we show that insulin mRNA levels are at least 105-fold higher in beta cells than non-beta cells. To examine the underlying mechanisms, we expressed beta cell transcription factors by transfection of non-beta cells. Separate expression of BETA2, E2A, or PDX1 led to modest (<10-fold) activation of the insulin promoter, whereas co-expression of the three proteins produced synergistic, high level activation (160-fold). This level of activity is ∼25% that observed in transfected beta cell lines. Of the three factors studied, BETA2 appears to play a dominant role. Efficient transcription required a C-terminal activation domain of BETA2 and an N-terminal region, which does not function as an independent activation domain. The myogenic basic helix-loop-helix (bHLH) protein MyoD was unable to bind and activate the promoter, even when its DNA binding region was replaced with that of BETA2. Our results demonstrate the central importance of BETA2 in insulin gene transcription and the importance of sequences outside the canonical DNA binding domain in permitting efficient DNA binding and cell-specific activity of the insulin gene promoter. The insulin gene is efficiently expressed only in pancreatic beta cells. Using reverse transcriptase-polymerase chain reaction analysis, we show that insulin mRNA levels are at least 105-fold higher in beta cells than non-beta cells. To examine the underlying mechanisms, we expressed beta cell transcription factors by transfection of non-beta cells. Separate expression of BETA2, E2A, or PDX1 led to modest (<10-fold) activation of the insulin promoter, whereas co-expression of the three proteins produced synergistic, high level activation (160-fold). This level of activity is ∼25% that observed in transfected beta cell lines. Of the three factors studied, BETA2 appears to play a dominant role. Efficient transcription required a C-terminal activation domain of BETA2 and an N-terminal region, which does not function as an independent activation domain. The myogenic basic helix-loop-helix (bHLH) protein MyoD was unable to bind and activate the promoter, even when its DNA binding region was replaced with that of BETA2. Our results demonstrate the central importance of BETA2 in insulin gene transcription and the importance of sequences outside the canonical DNA binding domain in permitting efficient DNA binding and cell-specific activity of the insulin gene promoter. basic helix-loop-helix reverse transcriptase-polymerase chain reaction cytomegalovirus chloramphenicol acetyltransferase muscle creatine kinase tetracycline electrophoretic mobility shift assay Expression of the insulin gene in adult mammals is restricted with great specificity to the pancreatic beta cells (1.Steiner D.F. Chan S.J. Welsh J.M. Kwok S.C.M. Annu. Rev. Genet. 1985; 19: 463-484Crossref PubMed Scopus (197) Google Scholar). The mechanisms involved are primarily transcriptional and operate through a number of well studied cis elements located in the proximal promoter region of the gene (2.Walker M.D. Edlund T. Boulet A.M. Rutter W.J. Nature. 1983; 306: 557-561Crossref PubMed Scopus (291) Google Scholar, 3.German M. Ashcroft S. Docherty K. Edlund T. Edlund H. Goodison S. Imura H. Kennedy G. Madsen O. Melloul D. Moss L. Olson K. Permutt A. Philippe J. Robertson R.P. Rutter W.J. Serup P. Stein R. Steiner D. Tsai M.-J. Walker M.D. Diabetes. 1995; 44: 1002-1004Crossref PubMed Scopus (148) Google Scholar). Several transcription factors have been shown to bind to these cis elements and are implicated in regulation of insulin gene transcription. The best characterized of these are the basic helix-loop-helix (bHLH)1proteins E2A (4.Aronheim A. Ohlsson H. Park C.W. Edlund T. Walker M.D. Nucleic Acids Res. 1991; 19: 3893-3899Crossref PubMed Scopus (66) Google Scholar, 5.Cordle S.R. Henderson E. Masuoka H. Weil P.A. Stein R. Mol. Cell. Biol. 1991; 11: 1734-1738Crossref PubMed Google Scholar) and BETA2 (NeuroD1) (6.Naya F.J. Stellrecht C.M.M. Tsai M.J. Genes Dev. 1995; 9: 1009-1019Crossref PubMed Scopus (525) Google Scholar, 7.Lee J.E. Hollenberg S.M. Snider L. Turner D.L. Lipnick N. Weintraub H. Science. 1995; 268: 836-844Crossref PubMed Scopus (939) Google Scholar) and the homeodomain protein PDX1 (IPF1/STF1/IDX1) (8.Ohlsson H. Karlsson K. Edlund T. EMBO J. 1993; 12: 4251-4259Crossref PubMed Scopus (775) Google Scholar, 9.Leonard J. Peers B. Johnson T. Ferreri K. Lee S. Montminy M.R. Mol. Endocrinol. 1993; 7: 1275-1283Crossref PubMed Google Scholar, 10.Miller C.P. McGehee R.E. Habener J.F. EMBO J. 1994; 13: 1145-1156Crossref PubMed Scopus (378) Google Scholar, 11.Peers B. Leonard J. Sharma S. Teitelman G. Montminy M.R. Mol. Endocrinol. 1994; 8: 1798-1806PubMed Google Scholar, 12.Serup P. Jensen J. Andersen F.G. Jorgensen M.C. Blume N. Holst J.J. Madsen O.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9015-9020Crossref PubMed Scopus (130) Google Scholar). The E2A proteins E12/E47 and the products of the related genes E2–2 and HEB are distributed in many or all cell types (13.Murre C. Bain G. Vandijk M.A. Engel I. Furnari B.A. Massari M.E. Matthews J.R. Quong M.W. Rivera R.R. Stuiver M.H. Biochim. Biophys. Acta. 1994; 1218: 129-135Crossref PubMed Scopus (412) Google Scholar). On the other hand BETA2 and PDX1 are found in beta cells and a very restricted subset of additional cells (14.Sander M. German M.S. J. Mol. Med. 1997; 75: 327-340Crossref PubMed Scopus (285) Google Scholar). E2A and BETA2 form a heterodimeric complex, which binds at two sites (E1 and E2) on the insulin promoter (6.Naya F.J. Stellrecht C.M.M. Tsai M.J. Genes Dev. 1995; 9: 1009-1019Crossref PubMed Scopus (525) Google Scholar, 15.Park C.W. Walker M.D. J. Biol. Chem. 1992; 267: 15642-15649Abstract Full Text PDF PubMed Google Scholar). Likewise PDX1 binds to the A1 and A3/4 regions of the insulin promoter. The phenotypes of mice bearing disrupted alleles of BETA2, PDX1, and other potential insulin gene transcription factors Pax4, Pax6, Nkx2.2, and Nkx6.1 indicate that these proteins may play an important role in beta cell differentiation also (reviewed in Refs. 16.Edlund H. Diabetes. 1998; 47: 1817-1823Crossref PubMed Scopus (276) Google Scholar and 17.St-Onge L. Wehr R. Gruss P. Curr. Opin. Genet. Dev. 1999; 9: 295-300Crossref PubMed Scopus (79) Google Scholar). Previous studies have focused on the activity of individual factors or a limited subset of the known factors, primarily using artificial promoter fragments (6.Naya F.J. Stellrecht C.M.M. Tsai M.J. Genes Dev. 1995; 9: 1009-1019Crossref PubMed Scopus (525) Google Scholar, 11.Peers B. Leonard J. Sharma S. Teitelman G. Montminy M.R. Mol. Endocrinol. 1994; 8: 1798-1806PubMed Google Scholar, 12.Serup P. Jensen J. Andersen F.G. Jorgensen M.C. Blume N. Holst J.J. Madsen O.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9015-9020Crossref PubMed Scopus (130) Google Scholar, 18.German M.S. Wang J.H. Chadwick R.B. Rutter W.J. Genes Dev. 1992; 6: 2165-2176Crossref PubMed Scopus (362) Google Scholar, 19.Sharma A. Moore M. Marcora E. Lee J.E. Qiu Y. Samaras S. Stein R. Mol. Cell. Biol. 1999; 19: 704-713Crossref PubMed Scopus (82) Google Scholar). The aim of the present study was to analyze a more physiologically relevant situation, namely the action of several of the known factors (E2A, BETA2, and PDX1) on the insulin promoter. We show that in transfected non-beta cells, combined expression of these three factors leads to dramatically elevated expression as compared with levels obtained in the presence of each factor alone. This level corresponds to ∼25% of the promoter activity observed in transfected beta cells. In turn, this represents at least a 100-fold lower specificity as compared with differential steady state insulin mRNA levels as measured by reverse transcriptase-polymerase chain reaction (RT-PCR) reaction. We have further examined the sequence requirements of the BETA2 protein for this activity and find that efficient activation requires distinct functional domains of the BETA2 molecule. Substitution with domains derived from the muscle bHLH protein MyoD cannot generate efficient activation of the insulin promoter, in part because binding to the promoter fragment is inefficient. Expression vectors encoding E2A (E47) (20.Aronheim A. Shiran R. Rosen A. Walker M.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8063-8067Crossref PubMed Scopus (106) Google Scholar) and PDX1 (8.Ohlsson H. Karlsson K. Edlund T. EMBO J. 1993; 12: 4251-4259Crossref PubMed Scopus (775) Google Scholar) were generated by insertion of the full-length protein-coding sequences to the vector pcDNA3 (Stratagene). A BETA2 (6.Naya F.J. Stellrecht C.M.M. Tsai M.J. Genes Dev. 1995; 9: 1009-1019Crossref PubMed Scopus (525) Google Scholar) expression vector was generated by insertion of the full-length protein-coding sequences to the vector pCGN-HA (21.Tanaka M. Herr W. Cell. 1990; 60: 375-386Abstract Full Text PDF PubMed Scopus (517) Google Scholar). In both vectors, expression is under the control of the cytomegalovirus (CMV) promoter. Gal4-BETA2 hybrid constructs were generated by PCR reaction using Pwo DNA polymerase (Roche Molecular Biochemicals). The resulting BETA2 fragments were introduced into an expression vector encoding the Gal4 DNA binding domain under the control of the CMV promoter. The constructs were named according to the N- and C-terminal amino acid. DNA fragments encoding deleted and substituted BETA2 were generated by PCR and introduced into pCGN-HA. The mouse MyoD cDNA (22.Davis R.L. Weintraub H. Lassar A.B. Cell. 1987; 51: 987-1000Abstract Full Text PDF PubMed Scopus (2499) Google Scholar) was used for generating hybrid proteins with BETA2. pOK1 (23.Karlsson O. Edlund T. Moss J.B. Rutter W.J. Walker M.D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8819-8823Crossref PubMed Scopus (184) Google Scholar) contains 410 base pairs of the rat insulin 1 gene promoter upstream of the chloramphenicol acetyltransferase (CAT) reporter. To generate prIns-LUC, the SV40 promoter of the plasmid pGL3.promoter (Promega) was replaced by the 410 base pairs of rat insulin I gene promoter. The plasmids 5Gal4.E1b.CAT (24.Lillie J.W. Green M.R. Nature. 1989; 338: 39-44Crossref PubMed Scopus (472) Google Scholar) and pRSV-LUC (25.De Wet J.R. Wood K.V. DeLuca M. Helinski D.R. Subramani S. Mol. Cell. Biol. 1987; 7: 725-737Crossref PubMed Scopus (2482) Google Scholar) have been described previously. The insulinM-CAT reporter plasmid was constructed by substituting the insulin E box motifs in pOK1 by the muscle creatine kinase (MCK) E box motifs using PCR with primers containing the MCK E box sequence 5′-AACACCTGCT-3′ (26.Davis R.L. Cheng P.F. Lassar A.B. Weintraub H. Cell. 1990; 60: 733-746Abstract Full Text PDF PubMed Scopus (528) Google Scholar). The relevant regions of plasmids constructed by PCR were verified by DNA sequencing. Cells were grown in Dulbecco's modified Eagle's medium in the presence of 10% fetal calf serum, penicillin (100 unit/ml), and streptomycin (100 μg/ml). The hybrid cell lines Rin-m × L were established and cultured as described (27.Leshkowitz D. Walker M.D. Mol. Cell. Biol. 1991; 11: 1547-1552Crossref PubMed Google Scholar). The cell line HeLa (Tet-off) PDX1 was generated by stable transfection of the line HeLa Tet-off (CLONTECH) with pTet-splice-PDX1, a plasmid containing the PDX1-coding sequence under the control of TRE (tetracycline response element) in the vector pTet-splice (28.Yin D.X. Zhu L. Schimke R.T. Anal. Biochem. 1996; 235: 195-201Crossref PubMed Scopus (76) Google Scholar); HeLa (Tet-off) cells were transfected with the plasmids pTet-splice-PDX1 (30 μg) and pSV2-HYG (1.5 μg). Clones were selected in the presence of 200 μg/ml hygromycin, 100 μg/ml G418, and 2 μg/ml tetracycline (tet). Surviving clones were tested for their ability to express PDX1 in a tet-repressible manner. Cells were grown in the absence or presence (2 μg/ml) of tet for 24–48 h, and PDX1 expression was tested by immunoblotting using a monoclonal antibody directed against PDX1. One of the clones obtained, designated HeLa (Tet-off) PDX1, showed no detectable PDX1 protein in the presence of 2 μg/ml tet but strongly inducible PDX1 in the absence of tet (data not shown); the clone also showed closely similar ability to activate the transfected insulin CAT reporter plasmid pOK1 as compared with that obtained following transient transfection of HeLa cells with a PDX1 expression vector, pcDNA3-PDX1 (data not shown). HeLa (Tet-off), HIT cells (hamster insulinoma (29.Santerre R.F. Cook R.A. Crisel R.M.D. Sharp J.D. Schmidt R.J. Williams D.C. Wilson C.P. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4339-4343Crossref PubMed Scopus (307) Google Scholar)) and 293T cell lines were transfected using the calcium phosphate co-precipitation procedure (30.Wigler M. Pellicer A. Silverstein S. Axel R. Urlaub G. Chasin L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: PubMed Scopus Google Scholar). HeLa (Tet-off) PDX1 cells were transfection and cultured in the absence of tet to PDX1 Cells were with of CAT reporter plasmid and the of expression plasmids and control CAT activity was measured by the procedure R. R.E. Moore K. in Molecular Scholar) and according to the activity of the control was at least three shown are were from transfected cells as described (4.Aronheim A. Ohlsson H. Park C.W. Edlund T. Walker M.D. Nucleic Acids Res. 1991; 19: 3893-3899Crossref PubMed Scopus (66) Google Scholar). of protein were on 10% and to The was with the by antibody to and using the reaction. were with using DNA polymerase The activity obtained was × The protein μg) was for on in binding 10% containing of and of in a assay of was and was to for an additional were on at for 1 at binding and were at to The following sequences were from Rin-m cells insulinoma L cells and hybrid Rin-m × L cells were used to cDNA using reverse and was to to that at least 100 of was present in each reaction. The PCR reaction was with the following The of rat and mouse contains two insulin insulin I and insulin in both the insulin I gene contains a whereas the insulin gene contains 2 (1.Steiner D.F. Chan S.J. Welsh J.M. Kwok S.C.M. Annu. Rev. Genet. 1985; 19: 463-484Crossref PubMed Scopus (197) Google Scholar). The primers to sequences found in 1 and 2 I or 1 and and are to sequences of rat and mouse insulin The cDNA a PCR of to base pairs on the and gene gene I or whereas DNA PCR reaction products were by on and to to a region of the insulin mRNA the primers was with and kinase and used as a for containing PCR high levels of insulin mRNA in beta cells but levels in non-beta cell lines and insulinoma × hybrid cell lines (27.Leshkowitz D. Walker M.D. Mol. Cell. Biol. 1991; 11: 1547-1552Crossref PubMed Google Scholar, C. E. P. J. D. J. Cell. 1991; PubMed Scopus Google Scholar). We have used a more of lower of insulin The was with from the beta cell line the cell line and hybrid cells Rin-m × L the of the analysis, was on the of in the of The procedure is to of insulin mRNA in derived from of Rin-m by lower in with of from Rin-m × L cells or L cells, or no was observed from DNA was by the in insulin mRNA levels in hybrid cells and are at least 105-fold as compared with Rin-m cells. A similar differential was obtained of the beta cell HIT with (data not shown). To the role of the proteins PDX1, E2A, and BETA2 as potential insulin gene transcription factors, we expressed these proteins in a of HeLa cells, HeLa PDX1, and the activation of a reporter plasmid containing the rat insulin I gene promoter to expression of each transcription factor led to modest activity co-expression of all three led to a in expression Expression of BETA2 in with E2A or PDX1 led to expression levels The activity observed with BETA2 and PDX1 was BETA2 is to bind DNA as a (6.Naya F.J. Stellrecht C.M.M. Tsai M.J. Genes Dev. 1995; 9: 1009-1019Crossref PubMed Scopus (525) Google and E2A levels are under the of the transfection expression of BETA2 lower expression than BETA2 BETA2 to play a important role in activation of the insulin gene promoter, expression of these factors showed lower activation when BETA2 was as compared with that observed when E2A or PDX1 were We obtained results using HeLa cells as compared with HeLa PDX1 (data not shown). gene promoter activity in HeLa cells was compared with that observed in transfected beta cells this to for in transfection cell we expression to that observed with the CMV promoter, which efficiently in both cell similar results were obtained with the promoter (data not shown). The activity of the insulin gene promoter in HeLa cells, observed expression of E2A, BETA2, and PDX1 was ∼25% that of the insulin gene promoter activity observed in the HIT cell line of its central importance in activation of the insulin promoter in this we examined the regions of the BETA2 protein required to high level We generated plasmids encoding of BETA2 the or N-terminal showed ability to activate the insulin gene promoter the BETA2 showed very activity when expressed (data not shown). C-terminal showed more than N-terminal that and BETA2 proteins were expressed at levels both N- and C-terminal domains of BETA2 are for efficient transcription by the insulin promoter. To the observed of the were to the presence of transcription activation expression plasmids were generated encoding of the BETA2 protein to the DNA binding domain of the transcriptional were to 293T cells with a CAT reporter plasmid bearing the Gal4 binding Gal4 proteins containing of the showed on the other N-terminal no activation but activity in this assay was to that the plasmids were efficiently expressed at the protein level This was also with other cell types cells and HIT (hamster beta The of the plasmids were similar to observed with 293T cells, in this protein expression levels were level of the assay (data not shown). these indicate that efficient transcription requires a domain located at the of the BETA2 protein and a domain at the which does not an independent activation To further examine the sequence requirements for BETA2 we generated additional expression plasmids on of the BETA2 protein with regions of the well studied MyoD The for this is that BETA2, is a bHLH protein that transcription as a heterodimeric with E2A and related A bHLH proteins (13.Murre C. Bain G. Vandijk M.A. Engel I. Furnari B.A. Massari M.E. Matthews J.R. Quong M.W. Rivera R.R. Stuiver M.H. Biochim. Biophys. Acta. 1994; 1218: 129-135Crossref PubMed Scopus (412) Google Scholar, H. Cell. 1993; 75: Full Text PDF PubMed Scopus Google Scholar) and in this with other proteins J.D. Olson Mol. Cell. Biol. 1998; PubMed Scopus (130) Google Scholar). The MyoD protein was unable to efficiently activate the insulin promoter A and to binding of to the E box sequences of the insulin promoter. To we generated a modified insulin promoter in which the two of the E box were replaced with sequences from the MCK promoter MyoD showed strongly ability to activate the modified promoter as compared with the promoter a further of we replaced the DNA binding domain of MyoD with that of BETA2 this showed transcriptional activity as compared with BETA2 The was not to expression levels but with DNA binding activity Previous studies with MyoD have that three amino in the basic and in the basic domain and the 1 of the play an role in myogenic activation R.L. Weintraub H. Science. 1992; PubMed Scopus Google Scholar). We tested the of the in BETA2 by to found in on the activity was observed the results indicate that the ability of BETA2 to bind DNA efficiently by sequences outside the canonical DNA binding the sequence of BETA2 is with efficient DNA whereas that of MyoD is To examine the of the in BETA2 to and in we generated two of the BETA2 substituting the amino and of BETA2 to in and as in A and two BETA2 proteins bind DNA with similar as the protein in to in was a in promoter activity observed appears that the in BETA2 to myogenic amino of MyoD are not for insulin gene promoter activity in The aim of this study was to the cell-specific expression of the insulin gene and to the role of known transcription factors in the We used to steady state mRNA levels in cell lines. The indicate a differential of at least In this a of mRNA differential insulin mRNA in beta non-beta cells not been been that transcriptional regulation the of the studies on the role of transcription factors on insulin gene transcription have used promoter We have established a on transfection of the insulin promoter and of efficient activation in a non-beta cell We have been to demonstrate that expression of the three insulin gene transcription factors BETA2, PDX1, and E2A in HeLa cells leads to efficient transcription of the insulin promoter. is to a level that observed in transfected pancreatic beta cells. The activity in beta cells may to the presence of additional beta cell transcription factors, and H. Diabetes. 1998; 47: 1817-1823Crossref PubMed Scopus (276) Google Scholar). we have shown that the differential expression of insulin mRNA levels in pancreatic beta cells as compared with non-beta cells is at least The higher specificity observed for the insulin gene as compared with a transfected gene is to additional levels of control on the for by To to the mechanisms involved in generating this high level of we have focused on the role of BETA2, which appears to play a dominant role in the In to the bHLH region of the protein known to for and DNA we that domains located both at the and are required for activation of the insulin promoter. The C-terminal domain function as an independent activation whereas the N-terminal domain the of BETA2 contains a transcription activation domain that only in the of the BETA2 or may to of the E2A, which two independent activation domains (20.Aronheim A. Shiran R. Rosen A. Walker M.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8063-8067Crossref PubMed Scopus (106) Google Scholar). and the results of Sharma A. Moore M. Marcora E. Lee J.E. Qiu Y. Samaras S. Stein R. Mol. Cell. Biol. 1999; 19: 704-713Crossref PubMed Scopus (82) Google Scholar) show activation domain activity in the of BETA2 using Gal4 To further the of BETA2, we compared its with of the related muscle bHLH protein The MyoD protein showed activation of the insulin promoter, at least in part because of its ability to bind the insulin promoter E MyoD efficiently a modified insulin promoter bearing MyoD binding On the other a hybrid bearing the DNA binding domain of BETA2 is unable to efficiently activate the insulin promoter because of DNA domains outside the canonical DNA binding domain have a dominant on BETA2 DNA This is of for the muscle bHLH and other transcription factors, important functional on DNA binding by sequences T. Olson Mol. Cell. Biol. 1991; 11: PubMed Google Scholar, M. R. J. Genet. 1995; Google Scholar). have also been for MyoD and other that sequences the DNA binding domain and a on transcription activation in a independent of DNA binding (26.Davis R.L. Cheng P.F. Lassar A.B. Weintraub H. Cell. 1990; 60: 733-746Abstract Full Text PDF PubMed Scopus (528) Google Scholar). The been to of protein by DNA binding and to of the activation domain J. Weintraub H. L. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). generating in BETA2, we have shown that the amino of BETA2 are not for insulin gene promoter activity in the are with a of transcription factors as with Nature. 1998; PubMed Scopus Google Scholar, M. A. Curr. Biol. 1998; 8: Full Text Full Text PDF PubMed Google Scholar). The mechanisms for efficient activation of the insulin promoter to BETA2 and PDX1 through or BETA2 been shown to with the transcriptional A. Moore M. Marcora E. Lee J.E. Qiu Y. Samaras S. Stein R. Mol. Cell. Biol. 1999; 19: 704-713Crossref PubMed Scopus (82) Google Scholar, H. F.J. Tsai M.J. A.B. Genes Dev. 1998; 12: PubMed Scopus Google Scholar). PDX1 on the other hand been shown to of binding to DNA in the presence of the homeodomain protein B. Sharma S. Johnson T. M. Montminy M. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). A of the mechanisms underlying cell-specific insulin gene expression more of the these and other transcription factors with of well studied as We D. and K. for E. and H. Edlund for the of and for and