A Composite Ets/Pit-1 Binding Site in the Prolactin Gene Can Mediate Transcriptional Responses to Multiple Signal Transduction Pathways
1995; Elsevier BV; Volume: 270; Issue: 36 Linguagem: Inglês
10.1074/jbc.270.36.20930
ISSN1083-351X
AutoresPaul W. Howard, Richard A. Maurer,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoBinding sites for the tissue-specific transcription factor, Pit-1, are required for basal and hormonally induced prolactin gene transcription. Although Pit-1 is phosphorylated in response to several signaling pathways, the mechanism by which Pit-1 contributes to hormonal induction of gene transcription has not been defined. Recent reports suggest that phosphorylation of Pit-1 may not be required for hormonal regulation of the prolactin promoter. Analysis of the contribution of individual Pit-1 binding sites has been complicated due to the fact that some of the elements appear to be redundant. To better understand the role of Pit-1 sites in mediating hormonal regulation of the prolactin gene, we have performed enhancer tests using the three most proximal Pit-1 binding sites of the rat prolactin gene which are designated the 1P, 2P, and 3P sites. The results demonstrate that multimers of the 3P Pit-1 binding site are much more responsive to several hormonal and intracellular signaling pathways than multimers of the 1P or 2P sites. The 3P DNA element was found to contain a consensus binding site for the Ets family of proteins. Mutation of the Ets binding site greatly decreased the ability of epidermal growth factor, phorbol esters, Ras, or the Raf kinase to induce reporter gene activity. Mutation of the Ets site had little effect on basal enhancer activity. In contrast, mutation of the consensus Pit-1 binding site in the 3P element essentially abolished all basal enhancer activity. Overexpression of Ets-1 in GH3 pituitary cells enhanced both basal and Ras induced activity from the 3P enhancer. These data describe a composite element in the prolactin gene containing binding sites for two different factors and the studies suggest a mechanism by which Ets proteins and Pit-1 functionally cooperate to permit transcriptional regulation by different signaling pathways. Binding sites for the tissue-specific transcription factor, Pit-1, are required for basal and hormonally induced prolactin gene transcription. Although Pit-1 is phosphorylated in response to several signaling pathways, the mechanism by which Pit-1 contributes to hormonal induction of gene transcription has not been defined. Recent reports suggest that phosphorylation of Pit-1 may not be required for hormonal regulation of the prolactin promoter. Analysis of the contribution of individual Pit-1 binding sites has been complicated due to the fact that some of the elements appear to be redundant. To better understand the role of Pit-1 sites in mediating hormonal regulation of the prolactin gene, we have performed enhancer tests using the three most proximal Pit-1 binding sites of the rat prolactin gene which are designated the 1P, 2P, and 3P sites. The results demonstrate that multimers of the 3P Pit-1 binding site are much more responsive to several hormonal and intracellular signaling pathways than multimers of the 1P or 2P sites. The 3P DNA element was found to contain a consensus binding site for the Ets family of proteins. Mutation of the Ets binding site greatly decreased the ability of epidermal growth factor, phorbol esters, Ras, or the Raf kinase to induce reporter gene activity. Mutation of the Ets site had little effect on basal enhancer activity. In contrast, mutation of the consensus Pit-1 binding site in the 3P element essentially abolished all basal enhancer activity. Overexpression of Ets-1 in GH3 pituitary cells enhanced both basal and Ras induced activity from the 3P enhancer. These data describe a composite element in the prolactin gene containing binding sites for two different factors and the studies suggest a mechanism by which Ets proteins and Pit-1 functionally cooperate to permit transcriptional regulation by different signaling pathways. INTRODUCTIONThe transcription of the prolactin gene is modulated by a number of hormones which bind to plasma membrane receptors. Hormones which regulate the transcription of the prolactin gene include dopamine(1Maurer R.A. Nature. 1981; 294: 94-97Crossref PubMed Scopus (193) Google Scholar), epidermal growth factor(2Supowit S.C. Potter E. Evans R.M. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2975-2979Crossref PubMed Scopus (47) Google Scholar, 3Stanley F. J. Biol. Chem. 1988; 263: 13444-13448Abstract Full Text PDF PubMed Google Scholar), and thyrotropin releasing hormone(4Murdoch G.H. Franco R. Evans R.M. Rosenfeld M.G. J. Biol. Chem. 1983; 258: 15329-15335Abstract Full Text PDF PubMed Google Scholar, 5Murdoch G.H. Waterman M. Evans R.M. Rosenfeld M.G. J. Biol. Chem. 1985; 260: 11852-11858Abstract Full Text PDF PubMed Google Scholar). The transcriptional effects of these hormones likely involves the activation of protein kinases leading to the phosphorylation of specific transcription factors. It has been demonstrated that activation of the cAMP-dependent protein kinase(6Maurer R.A. J. Biol. Chem. 1989; 264: 6870-6873Abstract Full Text PDF PubMed Google Scholar), the Ca2+/calmodulin-dependent protein kinase type II(7Kapiloff M.S. Mathis J.M. Nelson C.A. Lin C.R. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3710-3714Crossref PubMed Scopus (106) Google Scholar), or the MAPK 1The abbreviations used are: MAPKmitogen activated protein kinaseTRHthyrotropin releasing hormonePMAphorbol myristate acetateEGFepidermal growth factor. cascade (8Conrad K.E. Oberwetter J.M. Vaillancourt R. Johnson G.L. Gutierrez-Hartmann A. Mol. Cell. Biol. 1994; 14: 1553-1565Crossref PubMed Scopus (71) Google Scholar) is sufficient to stimulate transcription of the prolactin gene. Despite rather extensive studies of the prolactin promoter, the mechanisms which permit transcriptional responses to different hormones and signal transduction pathways have not been clearly defined. It is clear that the prolactin promoter contains multiple binding sites for the tissue-specific transcription factor Pit-1(9Nelson C. Albert V.R. Elsholtz H.P. Lu L.I.-W. Rosenfeld M.G. Science. 1988; 239: 1400-1405Crossref PubMed Scopus (416) Google Scholar, 10Mangalam H.J. Albert V.R. Ingraham H.A. Kapiloff M. Wilson L. Nelson C. Elsholtz H. Rosenfeld M.G. Genes & Dev. 1989; 3: 946-958Crossref PubMed Scopus (267) Google Scholar), and there is evidence that Pit-1 binding sites may contribute to both basal and hormonally regulated transcription(11Iverson R.A. Day K.H. d'Emden M. Day R.N. Maurer R.A. Mol. Endocrinol. 1990; 4: 1564-1571Crossref PubMed Scopus (103) Google Scholar, 12Shepard A.R. Zhang W. Eberhardt N.L. J. Biol. Chem. 1994; 269: 1804-1814Abstract Full Text PDF PubMed Google Scholar, 13Yan G.-z. Bancroft C. Mol. Endocrinol. 1991; 5: 1488-1497Crossref PubMed Scopus (45) Google Scholar, 14Yan G.-z. Pan W.T. Bancroft C. Mol. Endocrinol. 1991; 5: 535-541Crossref PubMed Scopus (68) Google Scholar, 15Kim M.K. McClaskey J.H. Bodenner D.L. Weintraub B.D. J. Biol. Chem. 1993; 268: 23366-23375Abstract Full Text PDF PubMed Google Scholar, 16Hoggard N. Davis J.R.E. Berwaer M. Monget P. Belayew B.P.A. Martial J.A. Mol. Endocrinol. 1991; 5: 1748-1754Crossref PubMed Scopus (38) Google Scholar). The observation that treatment of GH3 cells with cAMP or phorbol esters stimulates phosphorylation of Pit-1 (17Kapiloff M.S. Farkash Y. Wegner M. Rosenfeld M.G. Science. 1991; 253: 786-789Crossref PubMed Scopus (173) Google Scholar) is consistent with a role for Pit-1 in mediating hormonal regulation of transcription. However, recent studies have shown that phosphorylation of Pit-1 may not be necessary for hormonal induction of prolactin gene transcription (18Okimura Y. Howard P.W. Maurer R.A. Mol. Endocrinol. 1994; 8: 1559-1565PubMed Google Scholar, 19Fischberg D.J. Chen X. Bancroft C. Mol. Endocrinol. 1994; 8: 1566-1573PubMed Google Scholar, 20Howard P.W. Maurer R.A. J. Biol. Chem. 1994; 269: 28662-28669Abstract Full Text PDF PubMed Google Scholar). This led to the suggestion that other factors which interact with Pit-1 binding sites may mediate transcriptional regulation of the prolactin gene or that co-activators may interact with Pit-1 in a regulated fashion(21Gutierrez-Hartmann A. Mol. Endocrinol. 1994; 8: 1447-1454PubMed Google Scholar). There is evidence that cooperative interactions between Pit-1 and other transcription factors may be crucial for some transcriptional responses(12Shepard A.R. Zhang W. Eberhardt N.L. J. Biol. Chem. 1994; 269: 1804-1814Abstract Full Text PDF PubMed Google Scholar, 15Kim M.K. McClaskey J.H. Bodenner D.L. Weintraub B.D. J. Biol. Chem. 1993; 268: 23366-23375Abstract Full Text PDF PubMed Google Scholar, 22Day R.N. Koike S. Sakai M. Muramatsu M. Maurer R.A. Mol. Endocrinol. 1990; 4: 1964-1971Crossref PubMed Scopus (193) Google Scholar, 23Schaufele F. West B.L. Baxter J.D. Mol. Endocrinol. 1992; 6: 656-665PubMed Google Scholar). It has also been shown that factors other than Pit-1 can interact with Pit-1 binding sites. For instance, Oct-1(24Voss J.W.L.W. Rosenfeld M.G. Genes & Dev. 1991; 5: 1309-1320Crossref PubMed Scopus (171) Google Scholar), Zn-15(25Lipkin S.M. Näär A.M. Kalla K.A. Sack R.A. Rosenfeld M.G. Genes & Dev. 1993; 7: 1674-1687Crossref PubMed Scopus (116) Google Scholar), and TEF (26Drolet D.W. Scully K.M. Simmons D.M. Wegner M. Chu K. Swanson L.W. Rosenfeld M.G. Genes & Dev. 1991; 5: 1739-1753Crossref PubMed Scopus (206) Google Scholar) can all interact with Pit-1 binding sites and activate transcription through these sites. However, it is not clear if any of these other factors play a role in mediating hormonally regulated transcriptional activation.In the present study we have examined the ability of individual Pit-1 binding sites to permit responses to hormones and activators of specific signal transduction pathways. Previous studies have suggested that the 5′-flanking region of the rat prolactin gene contains multiple, redundant DNA elements which mediate responses to TRH(11Iverson R.A. Day K.H. d'Emden M. Day R.N. Maurer R.A. Mol. Endocrinol. 1990; 4: 1564-1571Crossref PubMed Scopus (103) Google Scholar). This functional redundancy has complicated analysis of the role of specific DNA elements in mediating hormonal responsiveness. Because of this problem, we have used an enhancer test to compare the ability of the three most proximal Pit-1 binding sites of the prolactin gene to respond to hormones, intracellular second messengers and activated components of signal transduction pathways. We find that one site, the 3P site, is particularly responsive to several signal transduction pathways including activation of the MAPK cascade. We have further examined the DNA sequences of the 3P DNA element which are important for transcriptional regulation. These studies suggest that the 3P element contains a binding site for a member of the Ets family of transcription factors in addition to a Pit-1 binding site. The Ets site in the 3P DNA element is crucial for transcriptional responses to hormones and second messengers.MATERIALS AND METHODSTransfection of GH3 and Rat-1 CellsGH3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 15% equine serum and 2.5% fetal bovine serum. Rat-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. Reporter genes containing 0.6 kilobase pairs of 5′-flanking region of the rat prolactin gene fused to the firefly luciferase coding sequence (11Iverson R.A. Day K.H. d'Emden M. Day R.N. Maurer R.A. Mol. Endocrinol. 1990; 4: 1564-1571Crossref PubMed Scopus (103) Google Scholar) and five copies of a GAL4 binding site upstream of the E1b TATA box linked to luciferase (27Sun P. Enslen H. Myung P.S. Maurer R.A. Genes & Dev. 1994; 8: 2527-2539Crossref PubMed Scopus (636) Google Scholar) have been described previously. To prepare a luciferase reporter gene (28d'Emden M.C. Okimura Y. Maurer R.A. Mol. Endocrinol. 1992; 6: 581-588Crossref PubMed Scopus (40) Google Scholar) containing multiple copies of the prolactin gene proximal Pit-1 binding sites, the rat prolactin 5′-flanking region was truncated at position −33 and a non-palindromic AvaI site was placed at the upstream termini. Multiple, tandem copies of Pit-1 binding sites with AvaI cohesive termini were ligated upstream of the minimal prolactin promoter and luciferase gene. GH3 and Rat-1 cells were transfected by electroporation with 10-15 μg of luciferase reporter genes using conditions described previously(29Kim K.E. Day R.N. Maurer R.A. Mol. Endocrinol. 1988; 3: 1374-1381Crossref Scopus (25) Google Scholar, 30Liang J. Kim K.E. Schoderbek W.E. Maurer R.A. Mol. Endocrinol. 1992; 6: 885-892PubMed Google Scholar). In some experiments the cells were transfected with expression vectors encoding globin, Pit-1(11Iverson R.A. Day K.H. d'Emden M. Day R.N. Maurer R.A. Mol. Endocrinol. 1990; 4: 1564-1571Crossref PubMed Scopus (103) Google Scholar), Raf-BXB, a constitutively active form of the Raf kinase(31Bruder J.T. Heidecker G. Rapp U.R. Genes & Dev. 1992; 6: 545-556Crossref PubMed Scopus (396) Google Scholar), human Ets-1(32Watson D.K. McWilliams M.J. Lapis P. Lautenberger J.A. Schweinfest C.W. Papas T.S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7862-7866Crossref PubMed Scopus (206) Google Scholar), or a H-ras valine 12 mutant(33Shih C. Weinberg R.A. Cell. 1982; 29: 161-169Abstract Full Text PDF PubMed Scopus (533) Google Scholar). After electroporation, the cells were divided equally into 60-mm tissue culture plates. At 18 h after transfection, some of the cultures were treated with 0.5 mM 8-(4-chlorophenylthio)cAMP, 100 nM TRH, 10 nM murine EGF, or 100 nM PMA for an additional 6 h. The cultured cells were collected and assayed for luciferase activity as described (34de Wet J.R. Wood K.V. DeLuca M. Helinski D.R. Subramani S. Mol. Cell. Biol. 1987; 7: 725-737Crossref PubMed Scopus (2473) Google Scholar).Mobility Shift Assays for Analysis of Protein-DNA InteractionsRecombinant Pit-1 containing a six histidine amino-terminal tag was expressed in Pichia pastoris and purified on nickel chelate agarose as recommended by the manufacturer (Invitrogen). Recombinant histidine-tagged ER81 was produced in Escherichia coli and purified by nickel chelate chromatography. Nuclear extracts were prepared from GH3 cells as described previously(30Liang J. Kim K.E. Schoderbek W.E. Maurer R.A. Mol. Endocrinol. 1992; 6: 885-892PubMed Google Scholar). Binding reactions contained 10,000 counts/min of 32P-labeled DNA probe, varying amounts of Pit-1, 2 μg of sheared salmon sperm DNA, 20 μg of bovine serum albumin, 10 mM Tris, pH 7.5, 5% glycerol, 50 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol in a total volume of 25 μl. In some reactions 1 μl of preimmune serum or antiserum to Pit-1 was included. The antiserum to Pit-1 has been described previously (18Okimura Y. Howard P.W. Maurer R.A. Mol. Endocrinol. 1994; 8: 1559-1565PubMed Google Scholar) (20). Reactions were incubated for 30 min at room temperature and then analyzed on non-denaturing, 4% polyacrylamide gels. Quantitation of mobility shift gels was performed using a Molecular Dynamics PhosphorImager Si and Imagequant Software. Relative dissociation constants and Hill cooperativity coefficients were determined by non-linear curvefitting of the Hill equation to the binding data.RESULTSEnhancer Activity of Multimers of Pit-1 Binding SitesIn an effort to provide a relatively simple test system for analysis of possible mechanisms mediating hormonal responsiveness of the prolactin promoter, we tested the ability of individual Pit-1 binding sites to function as hormone-responsive enhancers. This approach is based on the observation that there appears to be functional redundancy in the DNA elements which permit hormonal regulation of the prolactin promoter (11Iverson R.A. Day K.H. d'Emden M. Day R.N. Maurer R.A. Mol. Endocrinol. 1990; 4: 1564-1571Crossref PubMed Scopus (103) Google Scholar, 35Day R.N. Maurer R.A. Mol. Endocrinol. 1989; 3: 3-9Crossref PubMed Scopus (90) Google Scholar, 36Keech C.A. Jackson S.M. Siddiqui S.K. Ocran K.W. Gutierrez-Hartmann A. Mol. Endocrinol. 1992; 6: 2059-2070PubMed Google Scholar) and the finding that multimers of individual Pit-1 binding sites can support transcriptional responses to some hormones or second messengers(14Yan G.-z. Pan W.T. Bancroft C. Mol. Endocrinol. 1991; 5: 535-541Crossref PubMed Scopus (68) Google Scholar, 18Okimura Y. Howard P.W. Maurer R.A. Mol. Endocrinol. 1994; 8: 1559-1565PubMed Google Scholar). The proximal region of the prolactin gene is sufficient to mediate transcriptional responses to several different hormones(2Supowit S.C. Potter E. Evans R.M. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2975-2979Crossref PubMed Scopus (47) Google Scholar, 35Day R.N. Maurer R.A. Mol. Endocrinol. 1989; 3: 3-9Crossref PubMed Scopus (90) Google Scholar, 37Camper S.A. Yao Y.A.S. Rottman F.M. J. Biol. Chem. 1985; 260: 12246-12251Abstract Full Text PDF PubMed Google Scholar, 38Elsholtz H.P. Mangalam H.J. Potter E. Albert V.R. Supowit S. Evans R.M. Rosenfeld M.G. Science. 1986; 234: 1552-1557Crossref PubMed Scopus (107) Google Scholar), and this region contains four Pit-1 binding sites which are designated P1 through P4(9Nelson C. Albert V.R. Elsholtz H.P. Lu L.I.-W. Rosenfeld M.G. Science. 1988; 239: 1400-1405Crossref PubMed Scopus (416) Google Scholar, 10Mangalam H.J. Albert V.R. Ingraham H.A. Kapiloff M. Wilson L. Nelson C. Elsholtz H. Rosenfeld M.G. Genes & Dev. 1989; 3: 946-958Crossref PubMed Scopus (267) Google Scholar). We have focused our attention on the three most proximal Pit-1 binding sites (P1, P2, and P3) as we previously found that mutation of the P4 Pit-1 binding site had little effect on hormonal responsiveness(11Iverson R.A. Day K.H. d'Emden M. Day R.N. Maurer R.A. Mol. Endocrinol. 1990; 4: 1564-1571Crossref PubMed Scopus (103) Google Scholar). We prepared multimerized, direct repeats of the P1, P2, and P3 Pit-1 binding sites which were placed upstream of the minimal TATA box of the prolactin gene and the luciferase coding sequence. The reporter genes containing the multimers were then tested for hormonal regulation by transfection of the GH3, rat pituitary cell line (Fig. 1). The response of the multimers was compared to a reporter gene containing 600 base pairs of the proximal region and promoter of the rat prolactin gene (0.6PRL). A reporter gene containing the thymidine kinase (TK) promoter was included as a control for nonspecific effects of agonist treatments. The minimal prolactin promoter (TATA box alone) was essentially inactive. All three Pit-1 binding site multimers enhanced basal expression of the reporter gene. The 1P site is the highest affinity Pit-1 binding site in the proximal prolactin promoter(9Nelson C. Albert V.R. Elsholtz H.P. Lu L.I.-W. Rosenfeld M.G. Science. 1988; 239: 1400-1405Crossref PubMed Scopus (416) Google Scholar), and the 1P multimers were the strongest basal enhancer. Multimers of the 1P site also supported a modest response to cAMP as reported previously(18Okimura Y. Howard P.W. Maurer R.A. Mol. Endocrinol. 1994; 8: 1559-1565PubMed Google Scholar). Although it has been reported that multimers of the 1P site are able to permit a response to TRH(14Yan G.-z. Pan W.T. Bancroft C. Mol. Endocrinol. 1991; 5: 535-541Crossref PubMed Scopus (68) Google Scholar), we could not confirm this observation. This may reflect differences in culture conditions as the previous studies used serum-free medium while we used serum-containing medium. Multimers of the 2P site were found to be a rather weak basal enhancer and did not appear to permit responses to hormones or second messenger treatments. Although Pit-1 binding to the 2P site has been demonstrated(10Mangalam H.J. Albert V.R. Ingraham H.A. Kapiloff M. Wilson L. Nelson C. Elsholtz H. Rosenfeld M.G. Genes & Dev. 1989; 3: 946-958Crossref PubMed Scopus (267) Google Scholar), there is also evidence that this DNA element can interact with a ubiquitous factor (39Gutierrez-Hartmann A. Siddiquid S. Loukin S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5211-5215Crossref PubMed Scopus (62) Google Scholar) and that it plays a role in repressing the activity of the prolactin promoter in non-pituitary cell types(40Jackson S.M. Keech C.A. Williamson D.J. Gutierrez-Hartmann A. Mol. Cell. Biol. 1992; 12: 2708-2719Crossref PubMed Scopus (53) Google Scholar). Multimers of the 3P site demonstrated substantial basal enhancer activity and also permitted responses to cAMP, TRH, and PMA. Indeed, a reporter gene containing seven copies of the 3P site was substantially more responsive to phorbol esters than the native prolactin promoter. These findings demonstrate that multimers of the 3P element are sufficient to confer responses to at least two different signal transduction pathways and suggest that this element may contribute to the ability of the prolactin promoter to respond to several different hormones. We were particularly interested in the finding that the 3P multimer was more responsive to TRH than the 1P multimer. TRH has been reported to activate the MAPK pathway in GH3 cells(41Ohmichi M. Sawada T. Kanda Y. Koike K. Hirota K. Miyake A. Saltiel A.R. J. Biol. Chem. 1994; 269: 3783-3788Abstract Full Text PDF PubMed Google Scholar), and the prolactin promoter has been shown to be responsive to activation of MAPK cascade(8Conrad K.E. Oberwetter J.M. Vaillancourt R. Johnson G.L. Gutierrez-Hartmann A. Mol. Cell. Biol. 1994; 14: 1553-1565Crossref PubMed Scopus (71) Google Scholar). Therefore, we tested the ability of the multimerized DNA elements to mediate responses to activation of the MAPK pathway (Fig. 1B). For these studies we used expression vectors encoding activated forms of ras (33Shih C. Weinberg R.A. Cell. 1982; 29: 161-169Abstract Full Text PDF PubMed Scopus (533) Google Scholar) or the raf kinase (31Bruder J.T. Heidecker G. Rapp U.R. Genes & Dev. 1992; 6: 545-556Crossref PubMed Scopus (396) Google Scholar) which would be expected to stimulate the activity of the MAPK cascade. As previously reported(8Conrad K.E. Oberwetter J.M. Vaillancourt R. Johnson G.L. Gutierrez-Hartmann A. Mol. Cell. Biol. 1994; 14: 1553-1565Crossref PubMed Scopus (71) Google Scholar), the prolactin promoter is quite responsive to expression vectors for activated Ras or Raf. While neither the 1P nor 2P multimers permitted a substantial response to the Ras or Raf expression vectors, the 3P multimers conferred a vigorous response. The results shown in Fig. 1were obtained using multimers containing seven copies of the 1P, 2P, and 3P elements. Similar results have been obtained using multimers containing four copies of the DNA elements (data not shown). These enhancer tests suggest that the 3P element is particularly capable of responding to multiple signal transduction pathways including the MAPK cascade.Presumably, the ability of the 3P multimer to facilitate transcriptional responses is dependent on binding of Pit-1 to these elements. On the other hand, although Pit-1 is known to bind to both the 1P and 3P elements, the 3P element multimer was much more responsive to activation of the MAPK pathway. Pit-1 expression is restricted to somatotrophs, lactotophs, and a subset of thyrotroph cells of the anterior pituitary(42Ingraham H.A. Chen R. Mangalam H.J. Elsholtz H.P. Flynn S.E. Lin C.R. Simmons D.M. Swanson L. Rosenfeld M.G. Cell. 1988; 55: 519-529Abstract Full Text PDF PubMed Scopus (787) Google Scholar, 43Simmons D.M. Voss J.W. Ingraham H.A. Holloway J.M. Broide R.S. Rosenfeld M.G. Swanson L.W. Genes & Dev. 1990; 4: 695-711Crossref PubMed Scopus (561) Google Scholar, 44Bodner M. Karin M. Cell. 1987; 50: 267-275Abstract Full Text PDF PubMed Scopus (209) Google Scholar). To directly assess the role in Pit-1 in mediating transcriptional responses, we transfected the same reporter genes in the presence or absence of a Pit-1 expression vector into Rat-1 cells which do not contain endogenous Pit-1 (Fig. 2). In the absence of the Pit-1 expression vector, the proximal prolactin promoter, the 1P multimer construct and the 2P multimer construct were all essentially inactive in Rat-1 cells. The 3P multimer did support a very low level of basal expression. Transfection of the Pit-1 expression vector substantially activated the proximal prolactin reporter gene (23-fold) as well as the constructs containing the 1P (49-fold) or the 3P multimers (43-fold). The 2P multimer did not respond to the Pit-1 expression vector in Rat-1 cells. Transfection of the Pit-1 expression vector enabled the 1P multimer to support a modest response to cAMP as has been reported previously(18Okimura Y. Howard P.W. Maurer R.A. Mol. Endocrinol. 1994; 8: 1559-1565PubMed Google Scholar). Although both the proximal prolactin promoter and the 3P multimer supported responses to phorbol esters in GH3 cells, these reporter genes did not respond to phorbol esters in Rat-1 cells even in the presence of the Pit-1 expression vector. This difference may indicate that tissue-specific factors other than Pit-1 are required for these regulatory responses. Alternatively, there may be differences in the signal transduction machinery of GH3 and Rat-1 cells.Figure 2:Enhancer activity of Pit-1 binding site multimers in Rat-1 fibroblast cells. The reporter genes described in Fig. 1were transfected by electroporation into Rat-1 cells along with an expression vector for globin (panel A) or for Pit-1 (panel B). Each transfection was divided evenly into three plates. The next day the cells were untreated (Control) or treated with 0.5 mM CPT-cAMP or 100 nM PMA for 6 hours prior to collection and assaying for luciferase activity. Values are the average ± standard error of three independent transfections.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Analysis of Pit-1 Binding to the 1P and 3P ElementsMultimers of either the 1P or 3P Pit-1 binding site can provide basal enhancer activity. However, only the 3P multimer can support substantial transcriptional responses to TRH, phorbol esters, or activation of the MAPK cascade. It is certainly likely that this difference in transcriptional regulation mediated by the 1P and 3P multimers would involve differences in protein binding to these specific elements. This could reflect quantitative or qualitative differences in the binding of Pit-1 to these elements. Alternatively, the differential regulation could involve the binding of factors other than Pit-1 to the DNA elements. The possibility of qualitative differences in Pit-1 binding to these two sites is suggested by the organization of the two DNA elements (Fig. 3A). Comparison of the 1P and 3P DNA elements to the consensus Pit-1 binding site, TATTCAT (9Nelson C. Albert V.R. Elsholtz H.P. Lu L.I.-W. Rosenfeld M.G. Science. 1988; 239: 1400-1405Crossref PubMed Scopus (416) Google Scholar, 45Elsholtz H.P. Albert V.R. Treacy M.N. Rosenfeld M.G. Genes & Dev. 1990; 4: 43-51Crossref PubMed Scopus (62) Google Scholar, 46Voss J.W. Wilson L. Rhodes S.J. Rosenfeld M.G. Mol. Endocrinol. 1993; 7: 1551-1560PubMed Google Scholar) demonstrates that the 1P site contains two Pit-1 monomer sites arranged in an imperfect palindrome. The 3P site can be considered to contain a direct repeat of the monomer binding site. Previous studies have shown that Pit-1 interacts with the 1P element as both a monomer and a dimer with the dimer forming cooperatively as the concentration of Pit-1 is increased(47Ingraham H.A. Flynn S.E. Voss J.W. Albert V.R. Kapiloff M.S. Wilson L. Rosenfeld M.G. Cell. 1990; 61: 1021-1033Abstract Full Text PDF PubMed Scopus (319) Google Scholar). A detailed analysis of Pit-1 binding to the 3P element has not been performed. Therefore, we compared the ability of purified Pit-1 to bind to the 1P and 3P elements using a mobility shift assay (Fig. 3, B and C). With the 1P element, addition of increasing concentrations of Pit-1 resulted in the formation of two complexes (C1 and C2). Previous studies have shown that the C1 complex appears to represent the binding of Pit-1 monomers while the C2 complex represents binding of Pit-1 dimers(47Ingraham H.A. Flynn S.E. Voss J.W. Albert V.R. Kapiloff M.S. Wilson L. Rosenfeld M.G. Cell. 1990; 61: 1021-1033Abstract Full Text PDF PubMed Scopus (319) Google Scholar). As observed by Ingraham et al.(47Ingraham H.A. Flynn S.E. Voss J.W. Albert V.R. Kapiloff M.S. Wilson L. Rosenfeld M.G. Cell. 1990; 61: 1021-1033Abstract Full Text PDF PubMed Scopus (319) Google Scholar), the formation of the C2 complex on the 1P element is greatly facilitated over a narrow range of Pit-1 concentrations consistent with cooperative formation of Pit-1 dimers. Binding of Pit-1 to the 3P element also demonstrated the formation of two complexes which were similar in mobility to those observed with the 1P probe. However, the C1 complex persisted at much higher concentrations of Pit-1, and there was reduced formation of the C2 complex at all concentrations of Pit-1. These findings suggest reduced cooperativity in the binding of Pit-1 to the 3P element. Quantitative analysis of the mobility shifts shown in Fig. 2, A and B, by PhosphorImager confirmed these conclusions. The overall affinity of Pit-1 for the 3P element was about 60% of that for the 1P element. Binding to the 1P element occurred with a Hill coefficient of 1.5 consistent with substantial cooperativity while a Hill coefficient of 1.1 was obtained for the 3P DNA element. Similar results have been obtained in several independent binding experiments. The reduced cooperativity of binding and decreased apparent formation of Pit-1 dimers at the 3P site raises the possibility that Pit-1 may interact with this site as a monomer.Figure 3:Binding of purified Pit-1 to the 1P and 3P elements. The nucleotide sequences of the 1P and 3P DNA elements are indicated (A). Boxes indicate regions pr
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