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W2015128553 abstract "Expression of the somatostatin gene in endocrine pancreatic cells is controlled by several regulatorycis-elements located in the promoter region. Among these, the adjacent UE-A and TSEI elements, located from −113 to −85 relative to the transcription initiation site, function in combination and act as a pancreas-specific mini-enhancer. The TSEI element is recognized by the pancreatic homeodomain factor PDX1. In the present study, we show that the UE-A element binds a heterodimeric complex composed of a Pbx factor and the Prep1 protein, both belonging to the atypical three-amino acid loop extension homeodomain family. Recombinant Pbx1 and Prep1 proteins bind cooperatively to the UE-A site, whereas neither protein can bind this site alone. Transient transfection experiments reveal that both Pbx1 and Prep1 are required to generate a strong transcriptional activation from the UE-A element when this element is inserted close to the TATA box. In contrast, in the context of the intact somatostatin promoter or mini-enhancer, Pbx1 and Prep1 alone have no effect, but they produce a drastic activation when the pancreatic homeodomain factor PDX1 is also coexpressed. Thus, the activity of the somatostatin mini-enhancer is mediated by a cooperative interaction between the Pbx-Prep1 heterodimeric complex and the pancreatic factor PDX1. Expression of the somatostatin gene in endocrine pancreatic cells is controlled by several regulatorycis-elements located in the promoter region. Among these, the adjacent UE-A and TSEI elements, located from −113 to −85 relative to the transcription initiation site, function in combination and act as a pancreas-specific mini-enhancer. The TSEI element is recognized by the pancreatic homeodomain factor PDX1. In the present study, we show that the UE-A element binds a heterodimeric complex composed of a Pbx factor and the Prep1 protein, both belonging to the atypical three-amino acid loop extension homeodomain family. Recombinant Pbx1 and Prep1 proteins bind cooperatively to the UE-A site, whereas neither protein can bind this site alone. Transient transfection experiments reveal that both Pbx1 and Prep1 are required to generate a strong transcriptional activation from the UE-A element when this element is inserted close to the TATA box. In contrast, in the context of the intact somatostatin promoter or mini-enhancer, Pbx1 and Prep1 alone have no effect, but they produce a drastic activation when the pancreatic homeodomain factor PDX1 is also coexpressed. Thus, the activity of the somatostatin mini-enhancer is mediated by a cooperative interaction between the Pbx-Prep1 heterodimeric complex and the pancreatic factor PDX1. Somatostatins are peptides of 14 and 28 amino acid residues that regulate the secretion of various hormones, including growth hormone, thyrotropin, glucagon, and insulin (1Reichlin S. N. Engl. J. Med. 1983; 309: 1495-1501Crossref PubMed Scopus (1218) Google Scholar). The gene encoding the preprosomatostatin is strongly expressed in the δ endocrine cells of the pancreas and in neurons of the hypothalamus, but its expression is also detected in other cell types, such as the D-cells of the digestive tract, the C-cells of the thyroid gland, and sensory neurons (1Reichlin S. N. Engl. J. Med. 1983; 309: 1495-1501Crossref PubMed Scopus (1218) Google Scholar).Previous studies have shown that transcription of the somatostatin gene is controlled by several cis-elements present in its 5′ flanking sequence (see Fig. 1). A crucial activating sequence is the CRE element located between −35 and −55 relative to the transcription start site, which is recognized by the factor CREB and other related nuclear proteins (2Montminy M.R. Gonzalez G.A. Yamamoto K.K. Recent Prog. Horm. Res. 1990; 46: 219-229PubMed Google Scholar, 3Vallejo M. Ron D. Miller C.P. Habener J.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4679-4683Crossref PubMed Scopus (208) Google Scholar). In addition, the activity of the somatostatin promoter in pancreatic cell lines is stimulated by two tissue-specific elements, TSEI and TSEII, located respectively at −85/−99 and at −280/−300 (4Vallejo M. Miller C.P. Habener J.F. J. Biol. Chem. 1992; 267: 12868-12875Abstract Full Text PDF PubMed Google Scholar, 5Leonard J. Serup P. Gonzalez G. Edlund T. Montminy M.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6247-6251Crossref PubMed Scopus (102) Google Scholar, 6Leonard J. Peers B. Johnson T. Ferreri K. Lee S. Montminy M.R. Mol. Endocrinol. 1993; 7: 1275-1283Crossref PubMed Google Scholar) (see Fig. 1). These TSEs are both recognized by the pancreatic-specific homeodomain factor PDX1 (also named STF1, IDX1, and IPF1) (6Leonard J. Peers B. Johnson T. Ferreri K. Lee S. Montminy M.R. Mol. Endocrinol. 1993; 7: 1275-1283Crossref PubMed Google Scholar, 7Miller C.P. McGehee Jr., R.E. Habener J.F. EMBO J. 1994; 13: 1145-1156Crossref PubMed Scopus (376) Google Scholar, 8Ohlsson H. Karlsson K. Edlund T. EMBO J. 1993; 12: 4251-4259Crossref PubMed Scopus (765) Google Scholar), which plays an important role in pancreas organogenesis (9Jonsson J. Carlsson L. Edlund T. Edlund H. Nature. 1994; 371: 606-609Crossref PubMed Scopus (1552) Google Scholar). Whereas PDX1 binds the TSEIelement as a monomer, it recognizes the TSEII element mainly as a heterodimer with the Pbx factors (6Leonard J. Peers B. Johnson T. Ferreri K. Lee S. Montminy M.R. Mol. Endocrinol. 1993; 7: 1275-1283Crossref PubMed Google Scholar, 10Peers B. Sharma S. Johnson T. Kamps M. Montminy M.R Mol. Cell. Biol. 1995; 15: 7091-7097Crossref PubMed Scopus (145) Google Scholar). The Pbx proteins, including the proto-oncogene Pbx1 and the closely related factors Pbx2 and Pbx3, contain an atypical three-amino acid loop extension-class homeodomain and share extensive sequence homology with the Drosophila protein extradenticle (EXD) 1The abbreviations used are: EXD, extradenticle; EMSA, electrophoretic mobility shift assay.1The abbreviations used are: EXD, extradenticle; EMSA, electrophoretic mobility shift assay. (11Monica K. Galili N. Nourse J. Saltman D. Cleary M.L. Mol. Cell. Biol. 1991; 11: 6149-6157Crossref PubMed Scopus (259) Google Scholar, 12Rauskolb C. Peifer M. Wieschaus E. Cell. 1993; 74: 1101-1112Abstract Full Text PDF PubMed Scopus (213) Google Scholar, 13Burglin T.R. Nucleic Acids Res. 1997; 25: 4173-4180Crossref PubMed Scopus (486) Google Scholar). Genetic and biochemical studies in Drosophila have shown that EXD acts as a co-factor for the homeotic selector proteins (HOM-C) to regulate their target genes (14Chan S.K. Jaffe L. Capovilla M. Botas J. Mann R.S. Cell. 1994; 78: 603-615Abstract Full Text PDF PubMed Scopus (331) Google Scholar, 15Rauskolb C. Smith K.M. Peifer M. Wieschaus E. Development. 1995; 121: 3663-3673PubMed Google Scholar, 16Sun B. Hursh D.A. Jackson D. Beachy P.A. EMBO J. 1995; 14: 520-535Crossref PubMed Scopus (77) Google Scholar, 17Gonzalez-Crespo S. Morata G. Development. 1995; 121: 2117-2125PubMed Google Scholar). EXD binds cooperatively with HOM-C proteins to DNA target sites, thereby increasing their DNA binding specificity (18van Dijk M.A. Murre C. Cell. 1994; 78: 617-624Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 19Mann R.S. Chan S.K. Trends Genet. 1996; 12: 258-262Abstract Full Text PDF PubMed Scopus (389) Google Scholar). Similarly, in mammals, the Pbx proteins interact with the Hox factors (mammalian homologs of HOM-C factors) and modulate their DNA binding activity (20Popperl H. Bienz M. Studer M. Chan S.K. Aparicio S. Brenner S. Mann R.S. Krumlauf R. Cell. 1995; 81: 1031-1042Abstract Full Text PDF PubMed Scopus (450) Google Scholar, 21Neuteboom S.T. Peltenburg L.T. van Dijk M.A. Murre C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9166-9170Crossref PubMed Scopus (101) Google Scholar, 22Phelan M.L. Rambaldi I. Featherstone M.S. Mol. Cell. Biol. 1995; 15: 3989-3997Crossref PubMed Scopus (202) Google Scholar, 23Chang C.P. Shen W.F. Rozenfeld S. Lawrence H.J. Largman C. Cleary M.L. Genes Dev. 1995; 9: 663-674Crossref PubMed Scopus (351) Google Scholar, 24Lu Q. Kamps M.P. Mol. Cell. Biol. 1996; 16: 1632-1640Crossref PubMed Scopus (100) Google Scholar). The conserved pentapeptide motif YPWMK present in many metazoan Hox/HOM-C proteins is necessary for the interaction with the Pbx/EXD factors (21Neuteboom S.T. Peltenburg L.T. van Dijk M.A. Murre C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9166-9170Crossref PubMed Scopus (101) Google Scholar, 22Phelan M.L. Rambaldi I. Featherstone M.S. Mol. Cell. Biol. 1995; 15: 3989-3997Crossref PubMed Scopus (202) Google Scholar, 23Chang C.P. Shen W.F. Rozenfeld S. Lawrence H.J. Largman C. Cleary M.L. Genes Dev. 1995; 9: 663-674Crossref PubMed Scopus (351) Google Scholar). The pancreatic factor PDX1, which is very similar to the Antennapedia class of homeodomain proteins, also contains this pentapeptide motif, and this motif is absolutely required for the cooperative binding of PDX1 and Pbx to the somatostatin TSEII element (10Peers B. Sharma S. Johnson T. Kamps M. Montminy M.R Mol. Cell. Biol. 1995; 15: 7091-7097Crossref PubMed Scopus (145) Google Scholar). All of these results show that the function of the Pbx/EXD proteins is to act as co-factors for the Hox/HOM-C proteins but also for orphan homeodomain proteins, such as PDX1.Full expression of the somatostatin gene in pancreatic cells results from the synergistic actions of the different cis-elements present in the promoter. Indeed, previous reports have shown that the activity of the TSEI element is dependent of the immediately adjacent sequence UE-A (located from −100 to −113; see Fig. 1) (4Vallejo M. Miller C.P. Habener J.F. J. Biol. Chem. 1992; 267: 12868-12875Abstract Full Text PDF PubMed Google Scholar). The bipartite element UE-A/TSEI acts as a pancreatic δ-cell-specific mini-enhancer. The UE-A site, although devoid of intrinsic activation capacity, is required for optimal mini-enhancer activity (4Vallejo M. Miller C.P. Habener J.F. J. Biol. Chem. 1992; 267: 12868-12875Abstract Full Text PDF PubMed Google Scholar). The bipartite UE-A/TSEI element acts also in synergy with the nearby CRE sequence to generate high somatostatin expression levels in pancreatic cells (4Vallejo M. Miller C.P. Habener J.F. J. Biol. Chem. 1992; 267: 12868-12875Abstract Full Text PDF PubMed Google Scholar, 5Leonard J. Serup P. Gonzalez G. Edlund T. Montminy M.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6247-6251Crossref PubMed Scopus (102) Google Scholar). The protein binding the TSEI element was identified as a monomer of the pancreatic factor PDX1 (6Leonard J. Peers B. Johnson T. Ferreri K. Lee S. Montminy M.R. Mol. Endocrinol. 1993; 7: 1275-1283Crossref PubMed Google Scholar, 7Miller C.P. McGehee Jr., R.E. Habener J.F. EMBO J. 1994; 13: 1145-1156Crossref PubMed Scopus (376) Google Scholar). However, characterization of the cellular factor(s) recognizing the UE-A element has not been reported to date (25Vallejo M. Penchuk L. Habener J.F. J. Biol. Chem. 1992; 267: 12876-12884Abstract Full Text PDF PubMed Google Scholar).In the present study, we have identified the factors binding the UE-A element. This sequence recognizes a heterodimeric complex containing a Pbx factor and the Prep1/pKnox protein (36Berthelsen J. Zappavigna V. Mavilio F. Blasi F. EMBO J. 1998; 17: 1423-1433Crossref PubMed Scopus (149) Google Scholar, 40Chen H. Rossier C. Nakamura Y. Lynn A. Chakravarti A. Antonarakis S.E. Genomics. 1997; 41: 193-200Crossref PubMed Scopus (40) Google Scholar) (referred to here as Prep1). Although these two factors cannot bind separately the UE-A element, they bind strongly when added together. Furthermore, transfection experiments show that full activation of the intact somatostatin promoter is only obtained when the three transcription factors PDX1, Pbx1, and Prep1 are coexpressed. Thus, we have been able to reconstitute the strong pancreatic-specific enhancer complex of the somatostatin gene by using the two ubiquitous factors Pbx1 and Prep1 and the pancreatic homeodomain factor PDX1.DISCUSSIONThe UE-A element is a well conserved regulatory element of the somatostatin gene and is required for the optimal function of the promoter in pancreatic cells. Whereas the UE-A site is part of a pancreas-specific mini-enhancer, it has no intrinsic transactivating capacity (data not shown and Ref. 4Vallejo M. Miller C.P. Habener J.F. J. Biol. Chem. 1992; 267: 12868-12875Abstract Full Text PDF PubMed Google Scholar). This element acts by potentiating the transcriptional activation of the nearby TSEIelement.In the present study, we demonstrated that the UE-A site is recognized by heterodimers composed of a Pbx family member and the Prep1 protein. The two protein-DNA complexes L and S formed on UE-A with the cell extracts correspond to Pbx-Prep1 heterodimers containing, respectively, the long and short isoforms of Pbx proteins. This is demonstrated by the supershift obtained using the specific Pbx and Prep1 antibodies and the comigration of the L complex with the in vitrotranslated Pbx1a and Prep1 proteins. In transient transfection experiments, we observed that the somatostatin enhancer was strongly stimulated by the co-expression of the three factors, PDX1, Prep1, and Pbx1a. Similar stimulations were obtained by replacing the Pbx1a expression vector by a Pbx1b or Pbx2 expression plasmid (data not shown). Taken together, these results indicate that somatostatin gene expression is under the control of Pbx-Prep1 heterodimers.In addition to the somatostatin gene, the only known target of the Prep1 factor is the urokinase plasminogen activator gene (36Berthelsen J. Zappavigna V. Mavilio F. Blasi F. EMBO J. 1998; 17: 1423-1433Crossref PubMed Scopus (149) Google Scholar). Similar to the somatostatin UE-A element, Prep1 binds the COM element of the urokinase plasminogen activator enhancer as a heterodimer with Pbx factors, and the COM element is not able to activate transcription on its own. In fact, COM seems to act by increasing the transcriptional activation produced by the transcription factors bound to neighboring sites (i.e. Jun, ATF, and Ets factors) (38De Cesare D. Palazzolo M. Blasi F. Oncogene. 1996; 13: 2551-2562PubMed Google Scholar). In the present study, we also observed that the Pbx-Prep1 heterodimer, bound on the UE-A element, strongly synergizes with the PDX1 factor bound to the nearby TSEI site, producing a full activation of the somatostatin mini-enhancer. EMSA failed to show that this synergism is due to a cooperative binding of Pbx-Prep1 heterodimer and PDX1 to the bipartite UE-A/TSEI element (data not shown). However, we cannot completely rule out the possibility that DNA binding cooperativity might occur within the cell, perhaps requiring the presence of an unknown cellular factor or eventually requiring the natural context of chromatin.In this study, we show for the first time that the UE-A element can generate a transcriptional activation, but only when Pbx and Prep1 are overexpressed and if the UE-A sequence is inserted immediately upstream the TATA box. Increasing the distance between the UE-A site and the TATA box strongly reduces (see Fig. 9, UE-A/TSEI reporter plasmid) or completely abolishes the activation (see Fig. 9, pSRIF-Luc reporter). These observations suggest that the Pbx-Prep1 heterodimers possesses an activation capacity that is highly dependent on spatial organization. We can also postulate that in the context of the somatostatin promoter, one function of PDX1 is to promote functional interactions between the basal machinery and the Pbx-Prep1 heterodimer. However, further experiments are required to determine the mechanism of the transactivation mediated by the Pbx-Prep1 heterodimers.The protein Prep1 shares sequence similarity with the Meis proteins (Meis1, Meis2, Meis3, mrg1, and mrg2) not only in the homeodomain, but also in the two N-terminal domains involved in dimerization with Pbx factors (36Berthelsen J. Zappavigna V. Mavilio F. Blasi F. EMBO J. 1998; 17: 1423-1433Crossref PubMed Scopus (149) Google Scholar, 39Nakamura T. Jenkins N.A. Copeland N.G. Oncogene. 1996; 13: 2235-2242PubMed Google Scholar, 40Chen H. Rossier C. Nakamura Y. Lynn A. Chakravarti A. Antonarakis S.E. Genomics. 1997; 41: 193-200Crossref PubMed Scopus (40) Google Scholar, 41Steelman S. Moskow J. Muzinski K. North C.D.T. Montgomery J. Huebner K. Daar I. Buchberg A.M. Genome Res. 1997; 7: 142-156Crossref PubMed Scopus (49) Google Scholar). Despite this sequence similarity, the Pbx1-Meis1 heterodimer binds much less efficiently to UE-A than the Pbx1-Prep1 heterodimer. Furthermore, we were unable to detect binding of Pbx-Meis heterodimers on UE-A using cell extracts, and all protein complexes observed were Pbx-Prep1 heterodimers. This indicates that Pbx-Prep1 and Pbx-Meis1 heterodimers could have different DNA binding specificity and could regulate distinct target genes. Actually, it has been shown that the Pbx1-Meis complex preferentially recognizes the TGATTGACAG motif (34Chang C.P. Jacobs Y. Nakamura T. Jenkins N.A. Copeland N.G. Cleary M.L. Mol. Cell. Biol. 1997; 17: 5679-5687Crossref PubMed Scopus (207) Google Scholar). We are currently investigating in more detail this DNA binding specificity.The mechanism of action of the Pbx factors has been remarkably maintained through animal evolution. Indeed, both in mammals and inDrosophila, Pbx/EXD factors interact and cooperate with the Hox/HOM-C-like factors (reviewed in Ref. 19Mann R.S. Chan S.K. Trends Genet. 1996; 12: 258-262Abstract Full Text PDF PubMed Scopus (389) Google Scholar), and the interaction between mammalian Pbx and the Meis/Prep1 related factors also occurs inDrosophila as EXD interacts with the homothorax protein, which seems to be the ortholog of the murine Meis factors (42Rieckhof G.E. Casares F. Ryoo H.D. Abu-Shaar M. Mann R.S. Cell. 1997; 91: 171-183Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 43Casares F. Mann R.S. Nature. 1998; 392: 723-726Crossref PubMed Scopus (243) Google Scholar, 44Kurant E. Pai C.Y. Sharf R. Halachmi N. Sun Y.H. Salzberg A. Development. 1998; 125: 1037-1048Crossref PubMed Google Scholar, 45Pai C.Y. Kuo T.S. Jaw T.J. Kurant E. Chen C.T. Bessarab D.A. Salzberg A. Sun Y.H. Genes Dev. 1998; 12: 435-446Crossref PubMed Scopus (269) Google Scholar). Homothorax was shown to be necessary for the nuclear localization of EXD and for its function. Remarkably, the murine Meis1 was able to rescue the homothorax mutant phenotype (42Rieckhof G.E. Casares F. Ryoo H.D. Abu-Shaar M. Mann R.S. Cell. 1997; 91: 171-183Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar) and to induce nuclear translocation of EXD. Therefore, it is probable that the nuclear translocation of Pbx factors in mammals is controlled by the Meis factors. Thus, it will be interesting to test whether Prep1 could have such a role. As Rieckhof et al. (42Rieckhof G.E. Casares F. Ryoo H.D. Abu-Shaar M. Mann R.S. Cell. 1997; 91: 171-183Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar) observed nuclear EXD without homothorax expression in some cells of Drosophilaembryo (42Rieckhof G.E. Casares F. Ryoo H.D. Abu-Shaar M. Mann R.S. Cell. 1997; 91: 171-183Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar), it is possible that another factor, similar to homothorax, exists in these cells. This factor could eventually correspond to the ortholog of Prep1. A sequence comparison of homothorax, Meis, and Prep1 reveals that Meis1 is much more related to homothorax than to Prep1, suggesting that before the divergence of vertebrates and invertebrates, an ancestral gene duplicated and generated the Prep1 and the Meis/homothorax genes. Thus, this is consistent with the hypothesis that a Prep1-like gene could exist in Drosophila.The somatostatin promoter is a good model to study the regulation of gene expression by the Pbx factors. This promoter contains two distinct Pbx binding sites, the TSEII and the UE-A element. Pbx binds TSEII cooperatively with the pancreatic factor PDX1, and the formation of this heterodimer requires the FPMWK motif of PDX1 (10Peers B. Sharma S. Johnson T. Kamps M. Montminy M.R Mol. Cell. Biol. 1995; 15: 7091-7097Crossref PubMed Scopus (145) Google Scholar). On the UE-A element, Pbx binds as a heterodimer with the Prep1 factor. This Pbx-Prep1 heterodimer functionally cooperates with the PDX1 factor bound to the adjacent TSEI site. The somatostatin promoter is the only regulatory sequence shown to bind the three factors Pbx, Prep1, and PDX1 (Hox-like) with a high affinity; this gene is thus a good model in order to investigate, at the molecular level, the mechanisms of action of the Pbx/EXD, Meis/Prep/homothorax and Hox-like homeodomain factors. Somatostatins are peptides of 14 and 28 amino acid residues that regulate the secretion of various hormones, including growth hormone, thyrotropin, glucagon, and insulin (1Reichlin S. N. Engl. J. Med. 1983; 309: 1495-1501Crossref PubMed Scopus (1218) Google Scholar). The gene encoding the preprosomatostatin is strongly expressed in the δ endocrine cells of the pancreas and in neurons of the hypothalamus, but its expression is also detected in other cell types, such as the D-cells of the digestive tract, the C-cells of the thyroid gland, and sensory neurons (1Reichlin S. N. Engl. J. Med. 1983; 309: 1495-1501Crossref PubMed Scopus (1218) Google Scholar). Previous studies have shown that transcription of the somatostatin gene is controlled by several cis-elements present in its 5′ flanking sequence (see Fig. 1). A crucial activating sequence is the CRE element located between −35 and −55 relative to the transcription start site, which is recognized by the factor CREB and other related nuclear proteins (2Montminy M.R. Gonzalez G.A. Yamamoto K.K. Recent Prog. Horm. Res. 1990; 46: 219-229PubMed Google Scholar, 3Vallejo M. Ron D. Miller C.P. Habener J.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4679-4683Crossref PubMed Scopus (208) Google Scholar). In addition, the activity of the somatostatin promoter in pancreatic cell lines is stimulated by two tissue-specific elements, TSEI and TSEII, located respectively at −85/−99 and at −280/−300 (4Vallejo M. Miller C.P. Habener J.F. J. Biol. Chem. 1992; 267: 12868-12875Abstract Full Text PDF PubMed Google Scholar, 5Leonard J. Serup P. Gonzalez G. Edlund T. Montminy M.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6247-6251Crossref PubMed Scopus (102) Google Scholar, 6Leonard J. Peers B. Johnson T. Ferreri K. Lee S. Montminy M.R. Mol. Endocrinol. 1993; 7: 1275-1283Crossref PubMed Google Scholar) (see Fig. 1). These TSEs are both recognized by the pancreatic-specific homeodomain factor PDX1 (also named STF1, IDX1, and IPF1) (6Leonard J. Peers B. Johnson T. Ferreri K. Lee S. Montminy M.R. Mol. Endocrinol. 1993; 7: 1275-1283Crossref PubMed Google Scholar, 7Miller C.P. McGehee Jr., R.E. Habener J.F. EMBO J. 1994; 13: 1145-1156Crossref PubMed Scopus (376) Google Scholar, 8Ohlsson H. Karlsson K. Edlund T. EMBO J. 1993; 12: 4251-4259Crossref PubMed Scopus (765) Google Scholar), which plays an important role in pancreas organogenesis (9Jonsson J. Carlsson L. Edlund T. Edlund H. Nature. 1994; 371: 606-609Crossref PubMed Scopus (1552) Google Scholar). Whereas PDX1 binds the TSEIelement as a monomer, it recognizes the TSEII element mainly as a heterodimer with the Pbx factors (6Leonard J. Peers B. Johnson T. Ferreri K. Lee S. Montminy M.R. Mol. Endocrinol. 1993; 7: 1275-1283Crossref PubMed Google Scholar, 10Peers B. Sharma S. Johnson T. Kamps M. Montminy M.R Mol. Cell. Biol. 1995; 15: 7091-7097Crossref PubMed Scopus (145) Google Scholar). The Pbx proteins, including the proto-oncogene Pbx1 and the closely related factors Pbx2 and Pbx3, contain an atypical three-amino acid loop extension-class homeodomain and share extensive sequence homology with the Drosophila protein extradenticle (EXD) 1The abbreviations used are: EXD, extradenticle; EMSA, electrophoretic mobility shift assay.1The abbreviations used are: EXD, extradenticle; EMSA, electrophoretic mobility shift assay. (11Monica K. Galili N. Nourse J. Saltman D. Cleary M.L. Mol. Cell. Biol. 1991; 11: 6149-6157Crossref PubMed Scopus (259) Google Scholar, 12Rauskolb C. Peifer M. Wieschaus E. Cell. 1993; 74: 1101-1112Abstract Full Text PDF PubMed Scopus (213) Google Scholar, 13Burglin T.R. Nucleic Acids Res. 1997; 25: 4173-4180Crossref PubMed Scopus (486) Google Scholar). Genetic and biochemical studies in Drosophila have shown that EXD acts as a co-factor for the homeotic selector proteins (HOM-C) to regulate their target genes (14Chan S.K. Jaffe L. Capovilla M. Botas J. Mann R.S. Cell. 1994; 78: 603-615Abstract Full Text PDF PubMed Scopus (331) Google Scholar, 15Rauskolb C. Smith K.M. Peifer M. Wieschaus E. Development. 1995; 121: 3663-3673PubMed Google Scholar, 16Sun B. Hursh D.A. Jackson D. Beachy P.A. EMBO J. 1995; 14: 520-535Crossref PubMed Scopus (77) Google Scholar, 17Gonzalez-Crespo S. Morata G. Development. 1995; 121: 2117-2125PubMed Google Scholar). EXD binds cooperatively with HOM-C proteins to DNA target sites, thereby increasing their DNA binding specificity (18van Dijk M.A. Murre C. Cell. 1994; 78: 617-624Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 19Mann R.S. Chan S.K. Trends Genet. 1996; 12: 258-262Abstract Full Text PDF PubMed Scopus (389) Google Scholar). Similarly, in mammals, the Pbx proteins interact with the Hox factors (mammalian homologs of HOM-C factors) and modulate their DNA binding activity (20Popperl H. Bienz M. Studer M. Chan S.K. Aparicio S. Brenner S. Mann R.S. Krumlauf R. Cell. 1995; 81: 1031-1042Abstract Full Text PDF PubMed Scopus (450) Google Scholar, 21Neuteboom S.T. Peltenburg L.T. van Dijk M.A. Murre C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9166-9170Crossref PubMed Scopus (101) Google Scholar, 22Phelan M.L. Rambaldi I. Featherstone M.S. Mol. Cell. Biol. 1995; 15: 3989-3997Crossref PubMed Scopus (202) Google Scholar, 23Chang C.P. Shen W.F. Rozenfeld S. Lawrence H.J. Largman C. Cleary M.L. Genes Dev. 1995; 9: 663-674Crossref PubMed Scopus (351) Google Scholar, 24Lu Q. Kamps M.P. Mol. Cell. Biol. 1996; 16: 1632-1640Crossref PubMed Scopus (100) Google Scholar). The conserved pentapeptide motif YPWMK present in many metazoan Hox/HOM-C proteins is necessary for the interaction with the Pbx/EXD factors (21Neuteboom S.T. Peltenburg L.T. van Dijk M.A. Murre C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9166-9170Crossref PubMed Scopus (101) Google Scholar, 22Phelan M.L. Rambaldi I. Featherstone M.S. Mol. Cell. Biol. 1995; 15: 3989-3997Crossref PubMed Scopus (202) Google Scholar, 23Chang C.P. Shen W.F. Rozenfeld S. Lawrence H.J. Largman C. Cleary M.L. Genes Dev. 1995; 9: 663-674Crossref PubMed Scopus (351) Google Scholar). The pancreatic factor PDX1, which is very similar to the Antennapedia class of homeodomain proteins, also contains this pentapeptide motif, and this motif is absolutely required for the cooperative binding of PDX1 and Pbx to the somatostatin TSEII element (10Peers B. Sharma S. Johnson T. Kamps M. Montminy M.R Mol. Cell. Biol. 1995; 15: 7091-7097Crossref PubMed Scopus (145) Google Scholar). All of these results show that the function of the Pbx/EXD proteins is to act as co-factors for the Hox/HOM-C proteins but also for orphan homeodomain proteins, such as PDX1. Full expression of the somatostatin gene in pancreatic cells results from the synergistic actions of the different cis-elements present in the promoter. Indeed, previous reports have shown that the activity of the TSEI element is dependent of the immediately adjacent sequence UE-A (located from −100 to −113; see Fig. 1) (4Vallejo M. Miller C.P. Habener J.F. J. Biol. Chem. 1992; 267: 12868-12875Abstract Full Text PDF PubMed Google Scholar). The bipartite element UE-A/TSEI acts as a pancreatic δ-cell-specific mini-enhancer. The UE-A site, although devoid of intrinsic activation capacity, is required for optimal mini-enhancer activity (4Vallejo M. Miller C.P. Habener J.F. J. Biol. Chem. 1992; 267: 12868-12875Abstract Full Text PDF PubMed Google Scholar). The bipartite UE-A/TSEI element acts also in synergy with the nearby CRE sequence to generate high somatostatin expression levels in pancreatic cells (4Vallejo M. Miller C.P. Habener J.F. J. Biol. Chem. 1992; 267: 12868-12875Abstract Full Text PDF PubMed Google Scholar, 5Leonard J. Serup P. Gonzalez G. Edlund T. Montminy M.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6247-6251Crossref PubMed Scopus (102) Google Scholar). The protein binding the TSEI element was identified as a monomer of the pancreatic factor PDX1 (6Leonard J. Peers B. Johnson T. Ferreri K. Lee S. Montminy M.R. Mol. Endocrinol. 1993; 7: 1275-1283Crossref PubMed Google Scholar, 7Miller C.P. McGehee Jr., R.E. Habener J.F. EMBO J. 1994; 13: 1145-1156Crossref PubMed Scopus (376) Google Scholar). However, characterization of the cellular factor(s) recognizing the UE-A element has not been reported to date (25Vallejo M. Penchuk L. Habener J.F. J. Biol. Chem. 1992; 267: 12876-12884Abstract Full Text PDF PubMed Google Scholar). In the present study, we have identified the factors binding the UE-A element. This sequence recognizes a heterodimeric complex containing a Pbx factor and the Prep1/pKnox protein (36Berthelsen J. Zappavigna V. Mavilio F. Blasi F. EMBO J. 1998; 17: 1423-1433Crossref PubMed Scopus (149) Google Scholar, 40Chen H. Rossier C. Nakamura Y. Lynn A. Chakravarti A. Antonarakis S.E. Genomics. 1997; 41: 193-200Crossref PubMed Scopus (40) Google Scholar) (referred to here as Prep1). Although these two factors cannot bind separately the UE-A element, they bind strongly when added together. Furthermore, transfection experiments show that full activation of the intact somatostatin promoter is only obtained when the three transcription factors PDX1, Pbx1, and Prep1 are coexpressed. Thus, we have been able to reconstitute the strong pancreatic-specific enhancer complex of the somatostatin gene by using the two ubiquitous factors Pbx1 and Prep1 and the pancreatic homeodomain factor PDX1. DISCUSSIONThe UE-A element is a well conserved regulatory element of the somatostatin gene and is required for the optimal function of the promoter in pancreatic cells. Whereas the UE-A site is part of a pancreas-specific mini-enhancer, it has no intrinsic transactivating capacity (data not shown and Ref. 4Vallejo M. Miller C.P. Habener J.F. J. Biol. Chem. 1992; 267: 12868-12875Abstract Full Text PDF PubMed Google Scholar). This element acts by potentiating the transcriptional activation of the nearby TSEIelement.In the present study, we demonstrated that the UE-A site is recognized by heterodimers composed of a Pbx family member and the Prep1 protein. The two protein-DNA complexes L and S formed on UE-A with the cell extracts correspond to Pbx-Prep1 heterodimers containing, respectively, the long and short isoforms of Pbx proteins. This is demonstrated by the supershift obtained using the specific Pbx and Prep1 antibodies and the comigration of the L complex with the in vitrotranslated Pbx1a and Prep1 proteins. In transient transfection experiments, we observed that the somatostatin enhancer was strongly stimulated by the co-expression of the three factors, PDX1, Prep1, and Pbx1a. Similar stimulations were obtained by replacing the Pbx1a expression vector by a Pbx1b or Pbx2 expression plasmid (data not shown). Taken together, these results indicate that somatostatin gene expression is under the control of Pbx-Prep1 heterodimers.In addition to the somatostatin gene, the only known target of the Prep1 factor is the urokinase plasminogen activator gene (36Berthelsen J. Zappavigna V. Mavilio F. Blasi F. EMBO J. 1998; 17: 1423-1433Crossref PubMed Scopus (149) Google Scholar). Similar to the somatostatin UE-A element, Prep1 binds the COM element of the urokinase plasminogen activator enhancer as a heterodimer with Pbx factors, and the COM element is not able to activate transcription on its own. In fact, COM seems to act by increasing the transcriptional activation produced by the transcription factors bound to neighboring sites (i.e. Jun, ATF, and Ets factors) (38De Cesare D. Palazzolo M. Blasi F. Oncogene. 1996; 13: 2551-2562PubMed Google Scholar). In the present study, we also observed that the Pbx-Prep1 heterodimer, bound on the UE-A element, strongly synergizes with the PDX1 factor bound to the nearby TSEI site, producing a full activation of the somatostatin mini-enhancer. EMSA failed to show that this synergism is due to a cooperative binding of Pbx-Prep1 heterodimer and PDX1 to the bipartite UE-A/TSEI element (data not shown). However, we cannot completely rule out the possibility that DNA binding cooperativity might occur within the cell, perhaps requiring the presence of an unknown cellular factor or eventually requiring the natural context of chromatin.In this study, we show for the first time that the UE-A element can generate a transcriptional activation, but only when Pbx and Prep1 are overexpressed and if the UE-A sequence is inserted immediately upstream the TATA box. Increasing the distance between the UE-A site and the TATA box strongly reduces (see Fig. 9, UE-A/TSEI reporter plasmid) or completely abolishes the activation (see Fig. 9, pSRIF-Luc reporter). These observations suggest that the Pbx-Prep1 heterodimers possesses an activation capacity that is highly dependent on spatial organization. We can also postulate that in the context of the somatostatin promoter, one function of PDX1 is to promote functional interactions between the basal machinery and the Pbx-Prep1 heterodimer. However, further experiments are required to determine the mechanism of the transactivation mediated by the Pbx-Prep1 heterodimers.The protein Prep1 shares sequence similarity with the Meis proteins (Meis1, Meis2, Meis3, mrg1, and mrg2) not only in the homeodomain, but also in the two N-terminal domains involved in dimerization with Pbx factors (36Berthelsen J. Zappavigna V. Mavilio F. Blasi F. EMBO J. 1998; 17: 1423-1433Crossref PubMed Scopus (149) Google Scholar, 39Nakamura T. Jenkins N.A. Copeland N.G. Oncogene. 1996; 13: 2235-2242PubMed Google Scholar, 40Chen H. Rossier C. Nakamura Y. Lynn A. Chakravarti A. Antonarakis S.E. Genomics. 1997; 41: 193-200Crossref PubMed Scopus (40) Google Scholar, 41Steelman S. Moskow J. Muzinski K. North C.D.T. Montgomery J. Huebner K. Daar I. Buchberg A.M. Genome Res. 1997; 7: 142-156Crossref PubMed Scopus (49) Google Scholar). Despite this sequence similarity, the Pbx1-Meis1 heterodimer binds much less efficiently to UE-A than the Pbx1-Prep1 heterodimer. Furthermore, we were unable to detect binding of Pbx-Meis heterodimers on UE-A using cell extracts, and all protein complexes observed were Pbx-Prep1 heterodimers. This indicates that Pbx-Prep1 and Pbx-Meis1 heterodimers could have different DNA binding specificity and could regulate distinct target genes. Actually, it has been shown that the Pbx1-Meis complex preferentially recognizes the TGATTGACAG motif (34Chang C.P. Jacobs Y. Nakamura T. Jenkins N.A. Copeland N.G. Cleary M.L. Mol. Cell. Biol. 1997; 17: 5679-5687Crossref PubMed Scopus (207) Google Scholar). We are currently investigating in more detail this DNA binding specificity.The mechanism of action of the Pbx factors has been remarkably maintained through animal evolution. Indeed, both in mammals and inDrosophila, Pbx/EXD factors interact and cooperate with the Hox/HOM-C-like factors (reviewed in Ref. 19Mann R.S. Chan S.K. Trends Genet. 1996; 12: 258-262Abstract Full Text PDF PubMed Scopus (389) Google Scholar), and the interaction between mammalian Pbx and the Meis/Prep1 related factors also occurs inDrosophila as EXD interacts with the homothorax protein, which seems to be the ortholog of the murine Meis factors (42Rieckhof G.E. Casares F. Ryoo H.D. Abu-Shaar M. Mann R.S. Cell. 1997; 91: 171-183Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 43Casares F. Mann R.S. Nature. 1998; 392: 723-726Crossref PubMed Scopus (243) Google Scholar, 44Kurant E. Pai C.Y. Sharf R. Halachmi N. Sun Y.H. Salzberg A. Development. 1998; 125: 1037-1048Crossref PubMed Google Scholar, 45Pai C.Y. Kuo T.S. Jaw T.J. Kurant E. Chen C.T. Bessarab D.A. Salzberg A. Sun Y.H. Genes Dev. 1998; 12: 435-446Crossref PubMed Scopus (269) Google Scholar). Homothorax was shown to be necessary for the nuclear localization of EXD and for its function. Remarkably, the murine Meis1 was able to rescue the homothorax mutant phenotype (42Rieckhof G.E. Casares F. Ryoo H.D. Abu-Shaar M. Mann R.S. Cell. 1997; 91: 171-183Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar) and to induce nuclear translocation of EXD. Therefore, it is probable that the nuclear translocation of Pbx factors in mammals is controlled by the Meis factors. Thus, it will be interesting to test whether Prep1 could have such a role. As Rieckhof et al. (42Rieckhof G.E. Casares F. Ryoo H.D. Abu-Shaar M. Mann R.S. Cell. 1997; 91: 171-183Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar) observed nuclear EXD without homothorax expression in some cells of Drosophilaembryo (42Rieckhof G.E. Casares F. Ryoo H.D. Abu-Shaar M. Mann R.S. Cell. 1997; 91: 171-183Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar), it is possible that another factor, similar to homothorax, exists in these cells. This factor could eventually correspond to the ortholog of Prep1. A sequence comparison of homothorax, Meis, and Prep1 reveals that Meis1 is much more related to homothorax than to Prep1, suggesting that before the divergence of vertebrates and invertebrates, an ancestral gene duplicated and generated the Prep1 and the Meis/homothorax genes. Thus, this is consistent with the hypothesis that a Prep1-like gene could exist in Drosophila.The somatostatin promoter is a good model to study the regulation of gene expression by the Pbx factors. This promoter contains two distinct Pbx binding sites, the TSEII and the UE-A element. Pbx binds TSEII cooperatively with the pancreatic factor PDX1, and the formation of this heterodimer requires the FPMWK motif of PDX1 (10Peers B. Sharma S. Johnson T. Kamps M. Montminy M.R Mol. Cell. Biol. 1995; 15: 7091-7097Crossref PubMed Scopus (145) Google Scholar). On the UE-A element, Pbx binds as a heterodimer with the Prep1 factor. This Pbx-Prep1 heterodimer functionally cooperates with the PDX1 factor bound to the adjacent TSEI site. The somatostatin promoter is the only regulatory sequence shown to bind the three factors Pbx, Prep1, and PDX1 (Hox-like) with a high affinity; this gene is thus a good model in order to investigate, at the molecular level, the mechanisms of action of the Pbx/EXD, Meis/Prep/homothorax and Hox-like homeodomain factors. The UE-A element is a well conserved regulatory element of the somatostatin gene and is required for the optimal function of the promoter in pancreatic cells. Whereas the UE-A site is part of a pancreas-specific mini-enhancer, it has no intrinsic transactivating capacity (data not shown and Ref. 4Vallejo M. Miller C.P. Habener J.F. J. Biol. Chem. 1992; 267: 12868-12875Abstract Full Text PDF PubMed Google Scholar). This element acts by potentiating the transcriptional activation of the nearby TSEIelement. In the present study, we demonstrated that the UE-A site is recognized by heterodimers composed of a Pbx family member and the Prep1 protein. The two protein-DNA complexes L and S formed on UE-A with the cell extracts correspond to Pbx-Prep1 heterodimers containing, respectively, the long and short isoforms of Pbx proteins. This is demonstrated by the supershift obtained using the specific Pbx and Prep1 antibodies and the comigration of the L complex with the in vitrotranslated Pbx1a and Prep1 proteins. In transient transfection experiments, we observed that the somatostatin enhancer was strongly stimulated by the co-expression of the three factors, PDX1, Prep1, and Pbx1a. Similar stimulations were obtained by replacing the Pbx1a expression vector by a Pbx1b or Pbx2 expression plasmid (data not shown). Taken together, these results indicate that somatostatin gene expression is under the control of Pbx-Prep1 heterodimers. In addition to the somatostatin gene, the only known target of the Prep1 factor is the urokinase plasminogen activator gene (36Berthelsen J. Zappavigna V. Mavilio F. Blasi F. EMBO J. 1998; 17: 1423-1433Crossref PubMed Scopus (149) Google Scholar). Similar to the somatostatin UE-A element, Prep1 binds the COM element of the urokinase plasminogen activator enhancer as a heterodimer with Pbx factors, and the COM element is not able to activate transcription on its own. In fact, COM seems to act by increasing the transcriptional activation produced by the transcription factors bound to neighboring sites (i.e. Jun, ATF, and Ets factors) (38De Cesare D. Palazzolo M. Blasi F. Oncogene. 1996; 13: 2551-2562PubMed Google Scholar). In the present study, we also observed that the Pbx-Prep1 heterodimer, bound on the UE-A element, strongly synergizes with the PDX1 factor bound to the nearby TSEI site, producing a full activation of the somatostatin mini-enhancer. EMSA failed to show that this synergism is due to a cooperative binding of Pbx-Prep1 heterodimer and PDX1 to the bipartite UE-A/TSEI element (data not shown). However, we cannot completely rule out the possibility that DNA binding cooperativity might occur within the cell, perhaps requiring the presence of an unknown cellular factor or eventually requiring the natural context of chromatin. In this study, we show for the first time that the UE-A element can generate a transcriptional activation, but only when Pbx and Prep1 are overexpressed and if the UE-A sequence is inserted immediately upstream the TATA box. Increasing the distance between the UE-A site and the TATA box strongly reduces (see Fig. 9, UE-A/TSEI reporter plasmid) or completely abolishes the activation (see Fig. 9, pSRIF-Luc reporter). These observations suggest that the Pbx-Prep1 heterodimers possesses an activation capacity that is highly dependent on spatial organization. We can also postulate that in the context of the somatostatin promoter, one function of PDX1 is to promote functional interactions between the basal machinery and the Pbx-Prep1 heterodimer. However, further experiments are required to determine the mechanism of the transactivation mediated by the Pbx-Prep1 heterodimers. The protein Prep1 shares sequence similarity with the Meis proteins (Meis1, Meis2, Meis3, mrg1, and mrg2) not only in the homeodomain, but also in the two N-terminal domains involved in dimerization with Pbx factors (36Berthelsen J. Zappavigna V. Mavilio F. Blasi F. EMBO J. 1998; 17: 1423-1433Crossref PubMed Scopus (149) Google Scholar, 39Nakamura T. Jenkins N.A. Copeland N.G. Oncogene. 1996; 13: 2235-2242PubMed Google Scholar, 40Chen H. Rossier C. Nakamura Y. Lynn A. Chakravarti A. Antonarakis S.E. Genomics. 1997; 41: 193-200Crossref PubMed Scopus (40) Google Scholar, 41Steelman S. Moskow J. Muzinski K. North C.D.T. Montgomery J. Huebner K. Daar I. Buchberg A.M. Genome Res. 1997; 7: 142-156Crossref PubMed Scopus (49) Google Scholar). Despite this sequence similarity, the Pbx1-Meis1 heterodimer binds much less efficiently to UE-A than the Pbx1-Prep1 heterodimer. Furthermore, we were unable to detect binding of Pbx-Meis heterodimers on UE-A using cell extracts, and all protein complexes observed were Pbx-Prep1 heterodimers. This indicates that Pbx-Prep1 and Pbx-Meis1 heterodimers could have different DNA binding specificity and could regulate distinct target genes. Actually, it has been shown that the Pbx1-Meis complex preferentially recognizes the TGATTGACAG motif (34Chang C.P. Jacobs Y. Nakamura T. Jenkins N.A. Copeland N.G. Cleary M.L. Mol. Cell. Biol. 1997; 17: 5679-5687Crossref PubMed Scopus (207) Google Scholar). We are currently investigating in more detail this DNA binding specificity. The mechanism of action of the Pbx factors has been remarkably maintained through animal evolution. Indeed, both in mammals and inDrosophila, Pbx/EXD factors interact and cooperate with the Hox/HOM-C-like factors (reviewed in Ref. 19Mann R.S. Chan S.K. Trends Genet. 1996; 12: 258-262Abstract Full Text PDF PubMed Scopus (389) Google Scholar), and the interaction between mammalian Pbx and the Meis/Prep1 related factors also occurs inDrosophila as EXD interacts with the homothorax protein, which seems to be the ortholog of the murine Meis factors (42Rieckhof G.E. Casares F. Ryoo H.D. Abu-Shaar M. Mann R.S. Cell. 1997; 91: 171-183Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 43Casares F. Mann R.S. Nature. 1998; 392: 723-726Crossref PubMed Scopus (243) Google Scholar, 44Kurant E. Pai C.Y. Sharf R. Halachmi N. Sun Y.H. Salzberg A. Development. 1998; 125: 1037-1048Crossref PubMed Google Scholar, 45Pai C.Y. Kuo T.S. Jaw T.J. Kurant E. Chen C.T. Bessarab D.A. Salzberg A. Sun Y.H. Genes Dev. 1998; 12: 435-446Crossref PubMed Scopus (269) Google Scholar). Homothorax was shown to be necessary for the nuclear localization of EXD and for its function. Remarkably, the murine Meis1 was able to rescue the homothorax mutant phenotype (42Rieckhof G.E. Casares F. Ryoo H.D. Abu-Shaar M. Mann R.S. Cell. 1997; 91: 171-183Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar) and to induce nuclear translocation of EXD. Therefore, it is probable that the nuclear translocation of Pbx factors in mammals is controlled by the Meis factors. Thus, it will be interesting to test whether Prep1 could have such a role. As Rieckhof et al. (42Rieckhof G.E. Casares F. Ryoo H.D. Abu-Shaar M. Mann R.S. Cell. 1997; 91: 171-183Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar) observed nuclear EXD without homothorax expression in some cells of Drosophilaembryo (42Rieckhof G.E. Casares F. Ryoo H.D. Abu-Shaar M. Mann R.S. Cell. 1997; 91: 171-183Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar), it is possible that another factor, similar to homothorax, exists in these cells. This factor could eventually correspond to the ortholog of Prep1. A sequence comparison of homothorax, Meis, and Prep1 reveals that Meis1 is much more related to homothorax than to Prep1, suggesting that before the divergence of vertebrates and invertebrates, an ancestral gene duplicated and generated the Prep1 and the Meis/homothorax genes. Thus, this is consistent with the hypothesis that a Prep1-like gene could exist in Drosophila. The somatostatin promoter is a good model to study the regulation of gene expression by the Pbx factors. This promoter contains two distinct Pbx binding sites, the TSEII and the UE-A element. Pbx binds TSEII cooperatively with the pancreatic factor PDX1, and the formation of this heterodimer requires the FPMWK motif of PDX1 (10Peers B. Sharma S. Johnson T. Kamps M. Montminy M.R Mol. Cell. Biol. 1995; 15: 7091-7097Crossref PubMed Scopus (145) Google Scholar). On the UE-A element, Pbx binds as a heterodimer with the Prep1 factor. This Pbx-Prep1 heterodimer functionally cooperates with the PDX1 factor bound to the adjacent TSEI site. The somatostatin promoter is the only regulatory sequence shown to bind the three factors Pbx, Prep1, and PDX1 (Hox-like) with a high affinity; this gene is thus a good model in order to investigate, at the molecular level, the mechanisms of action of the Pbx/EXD, Meis/Prep/homothorax and Hox-like homeodomain factors. We thank Dr. C. Murre and S. Neuteboom for the Flag-Pbx1a cDNA and Dr. N. Copeland for the Meis1 cDNA. We are grateful to Dr. M. Kamps for the Pbx antiserum, to Dr. M. Cleary for the Meis1 antiserum, and to Dr. F Blasi and J. Berthelsen for the Prep1 antibodies. We thank Dr. M. Montminy, M. Muller, and M. Alvarez for discussions and comments." @default.
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W2015128553 title "Functional and Cooperative Interactions between the Homeodomain PDX1, Pbx, and Prep1 Factors on the Somatostatin Promoter" @default.
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