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• W2122520123 abstract "In Caenorhabditis elegans, the predicted transcription factor SKN-1 is required for embryonic endodermal and mesodermal specification and for maintaining differentiated intestinal cells post-embryonically. The SKN-1 DNA-binding region is related to the Cap'n'Collar (CNC) family of basic leucine zipper proteins, but uniquely, SKN-1 binds DNA as a monomer. CNC proteins are absent in C. elegans, however; and their involvement in the endoderm and mesoderm suggests some functional parallels to SKN-1. Using a cell culture assay, we show that SKN-1 induces transcription and contains three potent activation domains. The functional core of one domain is a short motif, the DIDLID element, which is highly conserved in a subgroup of vertebrate CNC proteins. The DIDLID element is important for SKN-1-driven transcription, suggesting a likely significance in other CNC proteins. SKN-1 binds to and activates transcription through the p300/cAMP-responsive element-binding protein-binding protein (CBP) coactivator, supporting the genetic prediction that SKN-1 recruits theC. elegans p300/CBP ortholog, CBP-1. The DIDLID element appears to act independently of p300/CBP, however, suggesting a distinct conserved target. The evolutionarily preservation of the DIDLID transcriptional element supports the model that SKN-1 and some CNC proteins interact with analogous cofactors and may have preserved some similar functions despite having divergent DNA-binding domains. In Caenorhabditis elegans, the predicted transcription factor SKN-1 is required for embryonic endodermal and mesodermal specification and for maintaining differentiated intestinal cells post-embryonically. The SKN-1 DNA-binding region is related to the Cap'n'Collar (CNC) family of basic leucine zipper proteins, but uniquely, SKN-1 binds DNA as a monomer. CNC proteins are absent in C. elegans, however; and their involvement in the endoderm and mesoderm suggests some functional parallels to SKN-1. Using a cell culture assay, we show that SKN-1 induces transcription and contains three potent activation domains. The functional core of one domain is a short motif, the DIDLID element, which is highly conserved in a subgroup of vertebrate CNC proteins. The DIDLID element is important for SKN-1-driven transcription, suggesting a likely significance in other CNC proteins. SKN-1 binds to and activates transcription through the p300/cAMP-responsive element-binding protein-binding protein (CBP) coactivator, supporting the genetic prediction that SKN-1 recruits theC. elegans p300/CBP ortholog, CBP-1. The DIDLID element appears to act independently of p300/CBP, however, suggesting a distinct conserved target. The evolutionarily preservation of the DIDLID transcriptional element supports the model that SKN-1 and some CNC proteins interact with analogous cofactors and may have preserved some similar functions despite having divergent DNA-binding domains. Cap'n'Collar cAMP-responsive element-binding protein-binding protein cytomegalovirus thymidine kinase chloramphenicol acetyltransferase glutathione S-transferase p300/CBP-associated factor During development, establishment of cell fates frequently involves conserved regulatory pathways. In the earlyCaenorhabditis elegans embryo, endodermal (intestinal) fates are specified by GATA family transcription factors (END-1, END-3, ELT-2) that are related to those that mediate endoderm development inDrosophila (Serpent) and vertebrates (GATA-4/5/6) (1.Zhu J. Fukushige T. McGhee J.D. Rothman J.H. Genes Dev. 1998; 12: 3809-3814Crossref PubMed Scopus (120) Google Scholar, 2.Zhu J. Hill R.J. Heid P.J. Fukuyama M. Sugimoto A. Priess J.R. Rothman J.H. Genes Dev. 1997; 11: 2883-2896Crossref PubMed Scopus (178) Google Scholar, 3.Fukushige T. Hawkins M.G. McGhee J.D. Dev. Biol. 1998; 198: 286-302PubMed Google Scholar). This program is triggered by maternally expressed SKN-1, a predicted transcription factor (4.Blackwell T.K. Bowerman B. Priess J. Weintraub H. Science. 1994; 266: 621-628Crossref PubMed Scopus (137) Google Scholar) that also specifies mesodermal lineages (pharynx and some body wall muscle) (5.Bowerman B. Eaton B.A. Priess J.R. Cell. 1992; 68: 1061-1075Abstract Full Text PDF PubMed Scopus (298) Google Scholar). The presence of consensus SKN-1-binding sites adjacent to the end-1 gene along with the timing of its expression suggests that SKN-1 may activateend-1 directly (2.Zhu J. Hill R.J. Heid P.J. Fukuyama M. Sugimoto A. Priess J.R. Rothman J.H. Genes Dev. 1997; 11: 2883-2896Crossref PubMed Scopus (178) Google Scholar). SKN-1 is also required in the endoderm post-embryonically, to prevent differentiated intestinal cells from undergoing severe atrophy (5.Bowerman B. Eaton B.A. Priess J.R. Cell. 1992; 68: 1061-1075Abstract Full Text PDF PubMed Scopus (298) Google Scholar).Apparent SKN-1 orthologs have not been identified outside of nematodes, but in its DNA-binding region, SKN-1 is related to a subgroup of basic leucine zipper transcription factors (4.Blackwell T.K. Bowerman B. Priess J. Weintraub H. Science. 1994; 266: 621-628Crossref PubMed Scopus (137) Google Scholar, 5.Bowerman B. Eaton B.A. Priess J.R. Cell. 1992; 68: 1061-1075Abstract Full Text PDF PubMed Scopus (298) Google Scholar), the CNC1 proteins. A basic DNA-binding region at the SKN-1 COOH terminus is particularly similar to those of CNC proteins, but SKN-1 lacks a zipper dimerization domain (Fig. 1, A and C) and, uniquely, binds DNA as a monomer (4.Blackwell T.K. Bowerman B. Priess J. Weintraub H. Science. 1994; 266: 621-628Crossref PubMed Scopus (137) Google Scholar). Its DNA binding requires the adjacent α-helical “CNC region” (Fig. 1, A andC) (4.Blackwell T.K. Bowerman B. Priess J. Weintraub H. Science. 1994; 266: 621-628Crossref PubMed Scopus (137) Google Scholar, 6.Carroll A.S. Gilbert D.E. Liu X. Cheung J.W. Michnowicz J.E. Wagner G. Ellenberger T.E. Blackwell T.K. Genes Dev. 1997; 11: 2227-2238Crossref PubMed Scopus (37) Google Scholar, 7.Lo M.-C. Ha S. Pelczer I. Pal S. Walker S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8455-8460Crossref PubMed Scopus (15) Google Scholar, 8.Rupert P.B. Daughdrill G.W. Bowerman B. Matthews B.W. Nat. Struct. Biol. 1998; 5: 484-491Crossref PubMed Scopus (49) Google Scholar), which is otherwise found only in CNC proteins. An adjacent SKN-1 element that is lacking in CNC proteins, the NH2-terminal arm (Fig. 1, A and C), contributes additional binding affinity and specificity (6.Carroll A.S. Gilbert D.E. Liu X. Cheung J.W. Michnowicz J.E. Wagner G. Ellenberger T.E. Blackwell T.K. Genes Dev. 1997; 11: 2227-2238Crossref PubMed Scopus (37) Google Scholar, 9.Kophengnavong T. Carroll A.S. Blackwell T.K. Mol. Cell. Biol. 1999; 19: 3039-3050Crossref PubMed Scopus (19) Google Scholar). These similarities and differences pose the question of whether SKN-1- and CNC-related proteins simply bind DNA through related but divergent mechanisms or might share a closer functional relationship.Like SKN-1, many CNC proteins are involved in the development or function of endodermal or mesodermal cells. DrosophilaCNC is required for specification of pharyngeal segments (10.Mohler J. Mahaffey J.W. Deutsch E. Vani K. Development. 1995; 121: 237-247Crossref PubMed Google Scholar), and vertebrate p45NF-E2 is involved in hematopoiesis (11.Shivdasani R.A. Rosenblatt M.F. Zucker-Franklin D. Jackson C.W. Hunt P. Saris C.J. Orkin S.H. Cell. 1995; 81: 695-704Abstract Full Text PDF PubMed Scopus (614) Google Scholar). In mice, different knockouts of the Nrf1(NF-E2 relatedfactor-1; also LCRF-1/TC11) gene either cause a fetal liver microenvironment defect (12.Chan J.Y. Kwong M. Lu R. Chang J. Wang B. Yen T.S. Kan Y.W. EMBO J. 1998; 17: 1779-1787Crossref PubMed Scopus (215) Google Scholar) or appear to block cell-to-cell induction of the mesoderm, a function usually ascribed to endodermal cells (13.Farmer S.C. Sun C.W. Winnier G.E. Hogan B.L. Townes T.M. Genes Dev. 1997; 11: 786-798Crossref PubMed Scopus (98) Google Scholar). Both NRF1 and NRF2 directly induce expression of detoxification enzymes (14.Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3127) Google Scholar, 15.Venugopal R. Jaiswal A.K. Oncogene. 1998; 17: 3145-3156Crossref PubMed Scopus (483) Google Scholar, 16.Kwong M. Kan Y.W. Chan J.Y. J. Biol. Chem. 1999; 274: 37491-37498Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 17.Alam J. Stewart D. Touchard C. Boinapally S. Choi A.M. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1051) Google Scholar), a pathway that is markedly stimulated in the liver and intestine (14.Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3127) Google Scholar). Supporting the idea that some CNC protein functions might parallel those of SKN-1, neither they nor their dimerization partners, the Maf basic leucine zipper proteins (18.Blank V. Andrews N.C. Trends Biochem. Sci. 1997; 22: 437-441Abstract Full Text PDF PubMed Scopus (219) Google Scholar, 19.Motohashi H. Shavit J.A. Igarashi K. Yamamoto M. Engel J.D. Nucleic Acids Res. 1997; 25: 2953-2959Crossref PubMed Scopus (237) Google Scholar), appear to be encoded in the complete C. elegans genome (data not shown).Genetic evidence suggests that SKN-1 may interact functionally with CBP-1, the C. elegans ortholog of the p300/CBP transcription coactivators. (20.Shi Y. Mello C. Genes Dev. 1998; 12: 943-955Crossref PubMed Scopus (126) Google Scholar). p300/CBP proteins are metazoan histone acetyltransferases that are involved in developmental and inducible gene expression and that are recruited by numerous activators (21.Shikama N.X. Lyon J. La Thangue N.B. Trends Cell Biol. 1997; 7: 230-236Abstract Full Text PDF PubMed Scopus (424) Google Scholar), including the CNC protein p45NF-E2 (22.Cheng X. Reginato M.J. Andrews N.C. Lazar M.A. Mol. Cell. Biol. 1997; 17: 1407-1416Crossref PubMed Scopus (97) Google Scholar). C. elegans CBP-1 is required for specification of all non-neuronal embryonic developmental lineages (20.Shi Y. Mello C. Genes Dev. 1998; 12: 943-955Crossref PubMed Scopus (126) Google Scholar). Endodermal differentiation can be restored in embryos that lack either CBP-1 or SKN-1, however, by inhibition of histone deacetylases (20.Shi Y. Mello C. Genes Dev. 1998; 12: 943-955Crossref PubMed Scopus (126) Google Scholar). This suggests that in the endoderm, SKN-1 might recruit the CBP-1 histone acetyltransferase activity directly. It remains possible, nevertheless, that lack of histone deacetylases simply restores expression of downstream genes independently of possible histone acetyltransferase recruitment by SKN-1.We have addressed these issues by investigating how SKN-1 regulates transcription. C. elegans cell lines have not been developed, but given the conservation of the eukaryotic mRNA transcription machinery (23.Hampsey M. Microbiol. Mol. Biol. Rev. 1998; 62: 465-503Crossref PubMed Google Scholar), SKN-1 would be predicted to be active in mammalian cells. Using transfection assays, we show here that SKN-1 is a powerful activator of transcription that contains three transactivation domains. The functional core of one domain consists of a short sequence (the DIDLID element) (Fig. 1 B) that is specific to SKN-1-related proteins and to the NRF group of CNC proteins. The p300/CBP proteins appear to be direct cofactors of SKN-1, but not to be critical for the activity of the DIDLID element. The conservation and transcriptional function of the DIDLID element suggest that SKN-1 and the NRF CNC proteins share a common transcriptional protein target in addition to p300/CBP and, during evolution, may have maintained some parallel functions in endodermal cells.DISCUSSIONThe presence of a DNA-binding domain in SKN-1 (4.Blackwell T.K. Bowerman B. Priess J. Weintraub H. Science. 1994; 266: 621-628Crossref PubMed Scopus (137) Google Scholar) and its localization to nuclei (36.Bowerman B. Draper B.W. Mello C. Priess J. Cell. 1993; 74: 443-452Abstract Full Text PDF PubMed Scopus (196) Google Scholar) suggested previously that SKN-1 is likely to regulate transcription. We have shown here that SKN-1 is a potent activator of transcription when it binds its cognate site (Fig. 2). SKN-1 interacts with two regions of human p300 in vitro(Fig. 5 A), and p300/CBP proteins contribute to its activity (Fig. 5, B and C). Given the extensive similarity between p300/CBP proteins and their C. elegans ortholog CBP-1, these data support the model that SKN-1 may recruit CBP-1 directly to promoters as a cofactor in vivo (20.Shi Y. Mello C. Genes Dev. 1998; 12: 943-955Crossref PubMed Scopus (126) Google Scholar). Overexpression of p300 potentiated SKN-1 activation of domains B and C (Fig. 5 D), suggesting that it can be recruited directly or indirectly by them. Domain B was not inhibited by E1A mutants in which binding to either p300/CBP (p300mut) or PCAF (E55) in particular was impaired (Fig. 5 E), implicating each of these histone acetyltransferases in its activity. Domain C was partially inhibited by these E1A mutants (Fig. 5 E), however, suggesting either that it may require both histone acetyltransferases simultaneously or might act on an independent E1A target. The activity of domain A, which contains the DIDLID element (Fig. 1 A), was not enhanced significantly by p300 expression (Fig. 5 D), and was inhibited by E1A p300mut (Fig. 5E), suggests that it has a target that is distinct from p300/CBP.The conservation of the DIDLID element across evolution (Fig. 1,B and C) supports the model that SKN-1 and the CNC proteins evolved from a common precursor (4.Blackwell T.K. Bowerman B. Priess J. Weintraub H. Science. 1994; 266: 621-628Crossref PubMed Scopus (137) Google Scholar). This conservation is particularly striking because the DIDLID motif is separate from the DNA-binding domain and was maintained despite divergences in how these proteins bind DNA (Fig. 1 C). Point mutants in the DIDLID element dramatically decreased SKN-1-driven transcription in mammalian cells (Fig. 4 A), supporting the idea that it mediates a highly specific interaction that is common to these proteins. In apparent contrast to our findings, an NRF2 fragment that contained the DIDLID element appeared to lack transcriptional activity in Gal4 fusion assays (29.Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2711) Google Scholar). This particular NRF2 fragment also included the inhibitory domain that can retain NRF2 in the cytoplasm (29.Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2711) Google Scholar), however, suggesting that in this context, the DIDLID element might have been masked or not present in the nucleus.Our findings suggest that SKN-1 and CNC proteins may have preserved some parallel functions in the endoderm and mesoderm. Also supporting this idea, in all of these proteins, hydrophobic residues on the CNC region surface are conserved that are not predicted to influence folding or DNA binding, but instead form a pocket, suggesting a common protein-protein interaction (8.Rupert P.B. Daughdrill G.W. Bowerman B. Matthews B.W. Nat. Struct. Biol. 1998; 5: 484-491Crossref PubMed Scopus (49) Google Scholar). The CNC protein most analogous to SKN-1 may be NRF1, which contains DIDLID (Fig. 1, B andC) and appears to be involved in endodermal and mesodermal differentiation and regulation of detoxification genes (13.Farmer S.C. Sun C.W. Winnier G.E. Hogan B.L. Townes T.M. Genes Dev. 1997; 11: 786-798Crossref PubMed Scopus (98) Google Scholar, 15.Venugopal R. Jaiswal A.K. Oncogene. 1998; 17: 3145-3156Crossref PubMed Scopus (483) Google Scholar, 16.Kwong M. Kan Y.W. Chan J.Y. J. Biol. Chem. 1999; 274: 37491-37498Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). These similarities suggest a particularly intriguing possibility, that the little understood requirement for SKN-1 to maintain the viability of differentiated intestinal cells (5.Bowerman B. Eaton B.A. Priess J.R. Cell. 1992; 68: 1061-1075Abstract Full Text PDF PubMed Scopus (298) Google Scholar) might involve functions that parallel the role of the NRF1 and NRF2 proteins in antioxidant responses. During development, establishment of cell fates frequently involves conserved regulatory pathways. In the earlyCaenorhabditis elegans embryo, endodermal (intestinal) fates are specified by GATA family transcription factors (END-1, END-3, ELT-2) that are related to those that mediate endoderm development inDrosophila (Serpent) and vertebrates (GATA-4/5/6) (1.Zhu J. Fukushige T. McGhee J.D. Rothman J.H. Genes Dev. 1998; 12: 3809-3814Crossref PubMed Scopus (120) Google Scholar, 2.Zhu J. Hill R.J. Heid P.J. Fukuyama M. Sugimoto A. Priess J.R. Rothman J.H. Genes Dev. 1997; 11: 2883-2896Crossref PubMed Scopus (178) Google Scholar, 3.Fukushige T. Hawkins M.G. McGhee J.D. Dev. Biol. 1998; 198: 286-302PubMed Google Scholar). This program is triggered by maternally expressed SKN-1, a predicted transcription factor (4.Blackwell T.K. Bowerman B. Priess J. Weintraub H. Science. 1994; 266: 621-628Crossref PubMed Scopus (137) Google Scholar) that also specifies mesodermal lineages (pharynx and some body wall muscle) (5.Bowerman B. Eaton B.A. Priess J.R. Cell. 1992; 68: 1061-1075Abstract Full Text PDF PubMed Scopus (298) Google Scholar). The presence of consensus SKN-1-binding sites adjacent to the end-1 gene along with the timing of its expression suggests that SKN-1 may activateend-1 directly (2.Zhu J. Hill R.J. Heid P.J. Fukuyama M. Sugimoto A. Priess J.R. Rothman J.H. Genes Dev. 1997; 11: 2883-2896Crossref PubMed Scopus (178) Google Scholar). SKN-1 is also required in the endoderm post-embryonically, to prevent differentiated intestinal cells from undergoing severe atrophy (5.Bowerman B. Eaton B.A. Priess J.R. Cell. 1992; 68: 1061-1075Abstract Full Text PDF PubMed Scopus (298) Google Scholar). Apparent SKN-1 orthologs have not been identified outside of nematodes, but in its DNA-binding region, SKN-1 is related to a subgroup of basic leucine zipper transcription factors (4.Blackwell T.K. Bowerman B. Priess J. Weintraub H. Science. 1994; 266: 621-628Crossref PubMed Scopus (137) Google Scholar, 5.Bowerman B. Eaton B.A. Priess J.R. Cell. 1992; 68: 1061-1075Abstract Full Text PDF PubMed Scopus (298) Google Scholar), the CNC1 proteins. A basic DNA-binding region at the SKN-1 COOH terminus is particularly similar to those of CNC proteins, but SKN-1 lacks a zipper dimerization domain (Fig. 1, A and C) and, uniquely, binds DNA as a monomer (4.Blackwell T.K. Bowerman B. Priess J. Weintraub H. Science. 1994; 266: 621-628Crossref PubMed Scopus (137) Google Scholar). Its DNA binding requires the adjacent α-helical “CNC region” (Fig. 1, A andC) (4.Blackwell T.K. Bowerman B. Priess J. Weintraub H. Science. 1994; 266: 621-628Crossref PubMed Scopus (137) Google Scholar, 6.Carroll A.S. Gilbert D.E. Liu X. Cheung J.W. Michnowicz J.E. Wagner G. Ellenberger T.E. Blackwell T.K. Genes Dev. 1997; 11: 2227-2238Crossref PubMed Scopus (37) Google Scholar, 7.Lo M.-C. Ha S. Pelczer I. Pal S. Walker S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8455-8460Crossref PubMed Scopus (15) Google Scholar, 8.Rupert P.B. Daughdrill G.W. Bowerman B. Matthews B.W. Nat. Struct. Biol. 1998; 5: 484-491Crossref PubMed Scopus (49) Google Scholar), which is otherwise found only in CNC proteins. An adjacent SKN-1 element that is lacking in CNC proteins, the NH2-terminal arm (Fig. 1, A and C), contributes additional binding affinity and specificity (6.Carroll A.S. Gilbert D.E. Liu X. Cheung J.W. Michnowicz J.E. Wagner G. Ellenberger T.E. Blackwell T.K. Genes Dev. 1997; 11: 2227-2238Crossref PubMed Scopus (37) Google Scholar, 9.Kophengnavong T. Carroll A.S. Blackwell T.K. Mol. Cell. Biol. 1999; 19: 3039-3050Crossref PubMed Scopus (19) Google Scholar). These similarities and differences pose the question of whether SKN-1- and CNC-related proteins simply bind DNA through related but divergent mechanisms or might share a closer functional relationship. Like SKN-1, many CNC proteins are involved in the development or function of endodermal or mesodermal cells. DrosophilaCNC is required for specification of pharyngeal segments (10.Mohler J. Mahaffey J.W. Deutsch E. Vani K. Development. 1995; 121: 237-247Crossref PubMed Google Scholar), and vertebrate p45NF-E2 is involved in hematopoiesis (11.Shivdasani R.A. Rosenblatt M.F. Zucker-Franklin D. Jackson C.W. Hunt P. Saris C.J. Orkin S.H. Cell. 1995; 81: 695-704Abstract Full Text PDF PubMed Scopus (614) Google Scholar). In mice, different knockouts of the Nrf1(NF-E2 relatedfactor-1; also LCRF-1/TC11) gene either cause a fetal liver microenvironment defect (12.Chan J.Y. Kwong M. Lu R. Chang J. Wang B. Yen T.S. Kan Y.W. EMBO J. 1998; 17: 1779-1787Crossref PubMed Scopus (215) Google Scholar) or appear to block cell-to-cell induction of the mesoderm, a function usually ascribed to endodermal cells (13.Farmer S.C. Sun C.W. Winnier G.E. Hogan B.L. Townes T.M. Genes Dev. 1997; 11: 786-798Crossref PubMed Scopus (98) Google Scholar). Both NRF1 and NRF2 directly induce expression of detoxification enzymes (14.Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3127) Google Scholar, 15.Venugopal R. Jaiswal A.K. Oncogene. 1998; 17: 3145-3156Crossref PubMed Scopus (483) Google Scholar, 16.Kwong M. Kan Y.W. Chan J.Y. J. Biol. Chem. 1999; 274: 37491-37498Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 17.Alam J. Stewart D. Touchard C. Boinapally S. Choi A.M. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1051) Google Scholar), a pathway that is markedly stimulated in the liver and intestine (14.Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3127) Google Scholar). Supporting the idea that some CNC protein functions might parallel those of SKN-1, neither they nor their dimerization partners, the Maf basic leucine zipper proteins (18.Blank V. Andrews N.C. Trends Biochem. Sci. 1997; 22: 437-441Abstract Full Text PDF PubMed Scopus (219) Google Scholar, 19.Motohashi H. Shavit J.A. Igarashi K. Yamamoto M. Engel J.D. Nucleic Acids Res. 1997; 25: 2953-2959Crossref PubMed Scopus (237) Google Scholar), appear to be encoded in the complete C. elegans genome (data not shown). Genetic evidence suggests that SKN-1 may interact functionally with CBP-1, the C. elegans ortholog of the p300/CBP transcription coactivators. (20.Shi Y. Mello C. Genes Dev. 1998; 12: 943-955Crossref PubMed Scopus (126) Google Scholar). p300/CBP proteins are metazoan histone acetyltransferases that are involved in developmental and inducible gene expression and that are recruited by numerous activators (21.Shikama N.X. Lyon J. La Thangue N.B. Trends Cell Biol. 1997; 7: 230-236Abstract Full Text PDF PubMed Scopus (424) Google Scholar), including the CNC protein p45NF-E2 (22.Cheng X. Reginato M.J. Andrews N.C. Lazar M.A. Mol. Cell. Biol. 1997; 17: 1407-1416Crossref PubMed Scopus (97) Google Scholar). C. elegans CBP-1 is required for specification of all non-neuronal embryonic developmental lineages (20.Shi Y. Mello C. Genes Dev. 1998; 12: 943-955Crossref PubMed Scopus (126) Google Scholar). Endodermal differentiation can be restored in embryos that lack either CBP-1 or SKN-1, however, by inhibition of histone deacetylases (20.Shi Y. Mello C. Genes Dev. 1998; 12: 943-955Crossref PubMed Scopus (126) Google Scholar). This suggests that in the endoderm, SKN-1 might recruit the CBP-1 histone acetyltransferase activity directly. It remains possible, nevertheless, that lack of histone deacetylases simply restores expression of downstream genes independently of possible histone acetyltransferase recruitment by SKN-1. We have addressed these issues by investigating how SKN-1 regulates transcription. C. elegans cell lines have not been developed, but given the conservation of the eukaryotic mRNA transcription machinery (23.Hampsey M. Microbiol. Mol. Biol. Rev. 1998; 62: 465-503Crossref PubMed Google Scholar), SKN-1 would be predicted to be active in mammalian cells. Using transfection assays, we show here that SKN-1 is a powerful activator of transcription that contains three transactivation domains. The functional core of one domain consists of a short sequence (the DIDLID element) (Fig. 1 B) that is specific to SKN-1-related proteins and to the NRF group of CNC proteins. The p300/CBP proteins appear to be direct cofactors of SKN-1, but not to be critical for the activity of the DIDLID element. The conservation and transcriptional function of the DIDLID element suggest that SKN-1 and the NRF CNC proteins share a common transcriptional protein target in addition to p300/CBP and, during evolution, may have maintained some parallel functions in endodermal cells. DISCUSSIONThe presence of a DNA-binding domain in SKN-1 (4.Blackwell T.K. Bowerman B. Priess J. Weintraub H. Science. 1994; 266: 621-628Crossref PubMed Scopus (137) Google Scholar) and its localization to nuclei (36.Bowerman B. Draper B.W. Mello C. Priess J. Cell. 1993; 74: 443-452Abstract Full Text PDF PubMed Scopus (196) Google Scholar) suggested previously that SKN-1 is likely to regulate transcription. We have shown here that SKN-1 is a potent activator of transcription when it binds its cognate site (Fig. 2). SKN-1 interacts with two regions of human p300 in vitro(Fig. 5 A), and p300/CBP proteins contribute to its activity (Fig. 5, B and C). Given the extensive similarity between p300/CBP proteins and their C. elegans ortholog CBP-1, these data support the model that SKN-1 may recruit CBP-1 directly to promoters as a cofactor in vivo (20.Shi Y. Mello C. Genes Dev. 1998; 12: 943-955Crossref PubMed Scopus (126) Google Scholar). Overexpression of p300 potentiated SKN-1 activation of domains B and C (Fig. 5 D), suggesting that it can be recruited directly or indirectly by them. Domain B was not inhibited by E1A mutants in which binding to either p300/CBP (p300mut) or PCAF (E55) in particular was impaired (Fig. 5 E), implicating each of these histone acetyltransferases in its activity. Domain C was partially inhibited by these E1A mutants (Fig. 5 E), however, suggesting either that it may require both histone acetyltransferases simultaneously or might act on an independent E1A target. The activity of domain A, which contains the DIDLID element (Fig. 1 A), was not enhanced significantly by p300 expression (Fig. 5 D), and was inhibited by E1A p300mut (Fig. 5E), suggests that it has a target that is distinct from p300/CBP.The conservation of the DIDLID element across evolution (Fig. 1,B and C) supports the model that SKN-1 and the CNC proteins evolved from a common precursor (4.Blackwell T.K. Bowerman B. Priess J. Weintraub H. Science. 1994; 266: 621-628Crossref PubMed Scopus (137) Google Scholar). This conservation is particularly striking because the DIDLID motif is separate from the DNA-binding domain and was maintained despite divergences in how these proteins bind DNA (Fig. 1 C). Point mutants in the DIDLID element dramatically decreased SKN-1-driven transcription in mammalian cells (Fig. 4 A), supporting the idea that it mediates a highly specific interaction that is common to these proteins. In apparent contrast to our findings, an NRF2 fragment that contained the DIDLID element appeared to lack transcriptional activity in Gal4 fusion assays (29.Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2711) Google Scholar). This particular NRF2 fragment also included the inhibitory domain that can retain NRF2 in the cytoplasm (29.Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2711) Google Scholar), however, suggesting that in this context, the DIDLID element might have been masked or not present in the nucleus.Our findings suggest that SKN-1 and CNC proteins may have preserved some parallel functions in the endoderm and mesoderm. Also supporting this idea, in all of these proteins, hydrophobic residues on the CNC region surface are conserved that are not predicted to influence folding or DNA binding, but instead form a pocket, suggesting a common protein-protein interaction (8.Rupert P.B. Daughdrill G.W. Bowerman B. Matthews B.W. Nat. Struct. Biol. 1998; 5: 484-491Crossref PubMed Scopus (49) Google Scholar). The CNC protein most analogous to SKN-1 may be NRF1, which contains DIDLID (Fig. 1, B andC) and appears to be involved in endodermal and mesodermal differentiation and regulation of detoxification genes (13.Farmer S.C. Sun C.W. Winnier G.E. Hogan B.L. Townes T.M. Genes Dev. 1997; 11: 786-798Crossref PubMed Scopus (98) Google Scholar, 15.Venugopal R. Jaiswal A.K. Oncogene. 1998; 17: 3145-3156Crossref PubMed Scopus (483) Google Scholar, 16.Kwong M. Kan Y.W. Chan J.Y. J. Biol. Chem. 1999; 274: 37491-37498Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). These similarities suggest a particularly intriguing possibility, that the little understood requirement for SKN-1 to maintain the viability of differentiated intestinal cells (5.Bowerman B. Eaton B.A. Priess J.R. Cell. 1992; 68: 1061-1075Abstract Full Text PDF PubMed Scopus (298) Google Scholar) might involve functions that parallel the role of the NRF1 and NRF2 proteins in antioxidant responses. The presence of a DNA-binding domain in SKN-1 (4.Blackwell T.K. Bowerman B. Priess J. Weintraub H. Science. 1994; 266: 621-628Crossref PubMed Scopus (137) Google Scholar) and its localization to nuclei (36.Bowerman B. Draper B.W. Mello C. Priess J. Cell. 1993; 74: 443-452Abstract Full Text PDF PubMed Scopus (196) Google Scholar) suggested previously that SKN-1 is likely to regulate transcription. We have shown here that SKN-1 is a potent activator of transcription when it binds its cognate site (Fig. 2). SKN-1 interacts with two regions of human p300 in vitro(Fig. 5 A), and p300/CBP proteins contribute to its activity (Fig. 5, B and C). Given the extensive similarity between p300/CBP proteins and their C. elegans ortholog CBP-1, these data support the model that SKN-1 may recruit CBP-1 directly to promoters as a cofactor in vivo (20.Shi Y. Mello C. Genes Dev. 1998; 12: 943-955Crossref PubMed Scopus (126) Google Scholar). Overexpression of p300 potentiated SKN-1 activation of domains B and C (Fig. 5 D), suggesting that it can be recruited directly or indirectly by them. Domain B was not inhibited by E1A mutants in which binding to either p300/CBP (p300mut) or PCAF (E55) in particular was impaired (Fig. 5 E), implicating each of these histone acetyltransferases in its activity. Domain C was partially inhibited by these E1A mutants (Fig. 5 E), however, suggesting either that it may require both histone acetyltransferases simultaneously or might act on an independent E1A target. The activity of domain A, which contains the DIDLID element (Fig. 1 A), was not enhanced significantly by p300 expression (Fig. 5 D), and was inhibited by E1A p300mut (Fig. 5E), suggests that it has a target that is distinct from p300/CBP. The conservation of the DIDLID element across evolution (Fig. 1,B and C) supports the model that SKN-1 and the CNC proteins evolved from a common precursor (4.Blackwell T.K. Bowerman B. Priess J. Weintraub H. Science. 1994; 266: 621-628Crossref PubMed Scopus (137) Google Scholar). This conservation is particularly striking because the DIDLID motif is separate from the DNA-binding domain and was maintained despite divergences in how these proteins bind DNA (Fig. 1 C). Point mutants in the DIDLID element dramatically decreased SKN-1-driven transcription in mammalian cells (Fig. 4 A), supporting the idea that it mediates a highly specific interaction that is common to these proteins. In apparent contrast to our findings, an NRF2 fragment that contained the DIDLID element appeared to lack transcriptional activity in Gal4 fusion assays (29.Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2711) Google Scholar). This particular NRF2 fragment also included the inhibitory domain that can retain NRF2 in the cytoplasm (29.Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2711) Google Scholar), however, suggesting that in this context, the DIDLID element might have been masked or not present in the nucleus. Our findings suggest that SKN-1 and CNC proteins may have preserved some parallel functions in the endoderm and mesoderm. Also supporting this idea, in all of these proteins, hydrophobic residues on the CNC region surface are conserved that are not predicted to influence folding or DNA binding, but instead form a pocket, suggesting a common protein-protein interaction (8.Rupert P.B. Daughdrill G.W. Bowerman B. Matthews B.W. Nat. Struct. Biol. 1998; 5: 484-491Crossref PubMed Scopus (49) Google Scholar). The CNC protein most analogous to SKN-1 may be NRF1, which contains DIDLID (Fig. 1, B andC) and appears to be involved in endodermal and mesodermal differentiation and regulation of detoxification genes (13.Farmer S.C. Sun C.W. Winnier G.E. Hogan B.L. Townes T.M. Genes Dev. 1997; 11: 786-798Crossref PubMed Scopus (98) Google Scholar, 15.Venugopal R. Jaiswal A.K. Oncogene. 1998; 17: 3145-3156Crossref PubMed Scopus (483) Google Scholar, 16.Kwong M. Kan Y.W. Chan J.Y. J. Biol. Chem. 1999; 274: 37491-37498Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). These similarities suggest a particularly intriguing possibility, that the little understood requirement for SKN-1 to maintain the viability of differentiated intestinal cells (5.Bowerman B. Eaton B.A. Priess J.R. Cell. 1992; 68: 1061-1075Abstract Full Text PDF PubMed Scopus (298) Google Scholar) might involve functions that parallel the role of the NRF1 and NRF2 proteins in antioxidant responses. We thank Martin Victor and Blackwell laboratory members for critically reading the manuscript." @default.
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• W2122520123 title "A Conserved Transcription Motif Suggesting Functional Parallels between Caenorhabditis elegans SKN-1 and Cap'n'Collar-related Basic Leucine Zipper Proteins" @default.
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