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Stimulation of NF-E2 DNA Binding by CREB-binding Protein (CBP)-mediated Acetylation

2001; Elsevier BV; Volume: 276; Issue: 14 Linguagem: Inglês

10.1074/jbc.m007846200

ISSN

1083-351X

Autores

Hsiao-Ling Hung, Alexander Y. Kim, Hong Wei, Carrie Rakowski, Gerd A. Blobel,

Tópico(s)

RNA Research and Splicing

Resumo

The hematopoietic transcription factor NF-E2 is an important regulator of erythroid and megakaryocytic gene expression. The transcription cofactor cAMP-response element-binding protein (CREB)-binding protein (CBP) has previously been implicated in mediating NF-E2 function. In this report, we examined the role of CBP, a coactivator with intrinsic acetyltransferase activity, in the regulation of NF-E2. We found that both the hematopoietic-specific subunit of NF-E2, p45, and the widely expressed small subunit, MafG, interact with CBP in vitro and in vivo. CBP acetylates MafG, but not p45, predominantly in the basic region of MafG. Immunoprecipitation experiments with anti-acetyl lysine antibodies demonstrate that MafG is acetylated in vivo in erythroid cells. Transfection experiments further show that CBP stimulates MafG acetylation in intact cells in an E1A-sensitive manner. Acetylation of MafG augments DNA binding activity of NF-E2, and mutations at the major acetylation sites markedly reduce DNA binding and transcriptional activation by NF-E2. Together, these results suggest that recruitment of CBP by NF-E2 to specific erythroid/megakaryocytic promoters might regulate transcription by at least two mechanisms involving both modification of chromatin structure and modulation of transcription factor activity. The hematopoietic transcription factor NF-E2 is an important regulator of erythroid and megakaryocytic gene expression. The transcription cofactor cAMP-response element-binding protein (CREB)-binding protein (CBP) has previously been implicated in mediating NF-E2 function. In this report, we examined the role of CBP, a coactivator with intrinsic acetyltransferase activity, in the regulation of NF-E2. We found that both the hematopoietic-specific subunit of NF-E2, p45, and the widely expressed small subunit, MafG, interact with CBP in vitro and in vivo. CBP acetylates MafG, but not p45, predominantly in the basic region of MafG. Immunoprecipitation experiments with anti-acetyl lysine antibodies demonstrate that MafG is acetylated in vivo in erythroid cells. Transfection experiments further show that CBP stimulates MafG acetylation in intact cells in an E1A-sensitive manner. Acetylation of MafG augments DNA binding activity of NF-E2, and mutations at the major acetylation sites markedly reduce DNA binding and transcriptional activation by NF-E2. Together, these results suggest that recruitment of CBP by NF-E2 to specific erythroid/megakaryocytic promoters might regulate transcription by at least two mechanisms involving both modification of chromatin structure and modulation of transcription factor activity. basic leucine zipper cap and collar cAMP-response element-binding protein (CREB)-binding protein locus control region hypersensitive site 2 erythroid Krüppel-like factor acetyltransferase glutathione S-transferase hemagglutinin acetyl lysine murine erythroid leukemia porphobilinogen deaminase amino acids The basic-zipper (bZip)1transcription factor NF-E2 plays a critical role in erythroid and megakaryocytic gene expression (for review see Ref. 1Andrews N.C. Int. J. Biochem. Cell Biol. 1998; 30: 429-432Crossref PubMed Scopus (72) Google Scholar). NF-E2 binds to an extended AP-1-like element, TGCTGA(G/C)TCA, which is found in the locus control regions (LCRs) of the α- and β-globin genes and in the promoters of several heme biosynthetic enzyme genes (for review see Refs. 2Motohashi H. Shavit J.A. Igarashi K. Yamamoto M. Engel J.D. Nucleic Acids Res. 1997; 25: 2953-2959Crossref PubMed Scopus (237) Google Scholar and 3Blank V. Andrews N.C. Trends Biochem. Sci. 1997; 22: 437-441Abstract Full Text PDF PubMed Scopus (219) Google Scholar)). NF-E2 binding sites in the DNase I hypersensitive site 2 (HS2) of the β-globin LCR are essential for its enhancer activity (4Ney P.A. Sorrentino B.P. Lowrey C.H. Nienhuis A.W. Nucleic Acids Res. 1990; 18: 6011-6017Crossref PubMed Scopus (113) Google Scholar, 5Ney P.A. Sorrentino B.P. McDonagh K. Nienhuis A.W. Genes Dev. 1990; 4: 993-1006Crossref PubMed Scopus (209) Google Scholar, 6Talbot D. Grosveld F. EMBO J. 1991; 10: 1391-1398Crossref PubMed Scopus (180) Google Scholar).NF-E2 is a heterodimer consisting of a hematopoietic-specific subunit p45, which is a member of the cap and collar (CNC) family, and a more widely expressed small subunit, which is a member of the small Maf protein family (MafG, MafK, and MafF) (for review see Refs. 2Motohashi H. Shavit J.A. Igarashi K. Yamamoto M. Engel J.D. Nucleic Acids Res. 1997; 25: 2953-2959Crossref PubMed Scopus (237) Google Scholar and 3Blank V. Andrews N.C. Trends Biochem. Sci. 1997; 22: 437-441Abstract Full Text PDF PubMed Scopus (219) Google Scholar)). MafG and MafK are the predominant small Maf molecules in erythroid cells and megakaryocytes (7Shavit J.A. Motohashi H. Onodera K. Akasaka J.-E. Yamamoto M. Engel J.D. Genes Dev. 1998; 12: 2164-2174Crossref PubMed Scopus (92) Google Scholar). p45 and a small Maf protein dimerize through their leucine zipper domains to generate a composite DNA binding domain that consists of the basic regions of both molecules. Other members of the CNC family, including Nrf1 (8Chan J.Y. Han X.L. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11371-11375Crossref PubMed Scopus (293) Google Scholar), Nrf2 (9Moi P. Chan K. Asunis I. Cao A. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9926-9930Crossref PubMed Scopus (1191) Google Scholar), Nrf3 (10Kobayashi A. Ito E. Toki T. Kogame K. Takahashi S. Igarashi K. Hayashi N. Yamamoto M. J. Biol. Chem. 1999; 274: 6443-6452Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), Bach1, and Bach2 (11Oyake T. Itoh K. Motohashi H. Hayashi N. Hoshino H. Nishizawa M. Yamamoto M. Igarashi K. Mol. Cell. Biol. 1996; 16: 6083-6095Crossref PubMed Scopus (516) Google Scholar) can also dimerize with small Maf proteins. Despite the high levels of p45 expression in erythroid cells, mice that are null for p45 displayed a surprisingly mild defect in globin gene expression, suggesting that other members of the CNC protein family can substitute for p45 function in vivo(12Shivdasani R.A. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8690-8694Crossref PubMed Scopus (182) Google Scholar).The N terminus of p45 contains an activation domain that is important for the biological activity of p45 (13Kotkow K.J. Orkin S.H. Mol. Cell. Biol. 1995; 15: 4640-4647Crossref PubMed Scopus (148) Google Scholar, 14Bean T.L. Ney P.A. Nucleic Acids Res. 1997; 25: 2509-2515Crossref PubMed Scopus (41) Google Scholar). Several molecules interact with this domain and are candidate mediators of p45 activity. These include TAFII130 (a component of the TFIID complex) (15Amrolia P.J. Ramamurthy L. Saluja D. Tanese N. Jane S.M. Cunningham J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10051-10056Crossref PubMed Scopus (52) Google Scholar), cAMP-response element-binding protein (CREB)-binding protein (CBP) (16Cheng X. Reginato M.J. Andrews N.C. Lazar M.A. Mol. Cell. Biol. 1997; 17: 1407-1416Crossref PubMed Scopus (97) Google Scholar), and several ubiquitin ligases (17Gavva N.R. Gavva R. Ermekova K. Sudol M. Shen C.J. J. Biol. Chem. 1997; 272: 24105-24108Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 18Mosser E.A. Kasanov J.D. Forsberg E.C. Kay B.K. Ney P.A. Bresnick E.H. Biochemistry. 1998; 37: 13686-13695Crossref PubMed Scopus (60) Google Scholar). The small Maf proteins lack a typical activation domain and are believed to activate transcription as heterodimers with members of the CNC family of proteins. Small Maf proteins can also form homodimers and repress transcription (19Igarashi K. Kataoka K. Itoh K. Hayashi N. Nishizawa M. Yamamoto M. Nature. 1994; 367: 568-572Crossref PubMed Scopus (396) Google Scholar).CBP and its close relative p300 serve as coactivators for a large and diverse set of nuclear factors (20Shikama N. Lyon J. LaThangue N.B. Trends Cell Biol. 1997; 7: 230-236Abstract Full Text PDF PubMed Scopus (424) Google Scholar, 21Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar). CBP and p300 possess intrinsic histone acetyltransferase (AT) activity (21Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar, 22Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2368) Google Scholar, 23Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1523) Google Scholar). Histone acetylation is associated with a relaxed chromatin configuration, suggesting that coactivators act in part through modifying chromatin structure. Consistent with this idea, at the chicken β-globin gene locus, the area of general DNaseI sensitivity coincides well with the region of elevated histone acetylation (24Hebbes T.R. Clayton A.L. Thorne A.W. Crane-Robinson C. EMBO J. 1994; 13: 1823-1830Crossref PubMed Scopus (481) Google Scholar). Interestingly, hyper-acetylation of histones H3 and H4 was observed at the human β-globin LCR and at the transcribed β-globin gene when compared with the acetylation status of the inactive β-like globin genes (25Schubeler D. Franxastel C. Cimbora D.M. Reik A. Martin D.I.K. Groudine M. Genes Dev. 2000; 14: 940-950PubMed Google Scholar, 26Forsberg E.C. Downs K.M. Christensen H.M. Im H. Nuzzi P.A. H B.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14494-14499Crossref PubMed Scopus (210) Google Scholar). Together, these findings suggest that erythroid transcription factors might recruit histone-modifying enzymes such as CBP/p300 to the LCR and globin gene locus, thereby altering chromatin structure (27Blobel G.A. Blood. 2000; 95: 745-755Crossref PubMed Google Scholar). This idea is supported by our observation that E1A-mediated inactivation of CBP/p300 in erythroid cells leads to a block in cell differentiation and globin gene induction (28Blobel G.A. Nakajima T. Eckner R. Montminy M. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2061-2066Crossref PubMed Scopus (313) Google Scholar). CBP binds to several hematopoietic-restricted transcription factors involved in globin gene expression and enhances their activity, including GATA-1 (28Blobel G.A. Nakajima T. Eckner R. Montminy M. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2061-2066Crossref PubMed Scopus (313) Google Scholar) and the erythroid Krüppel-like factor EKLF (29Zhang W. Bieker J.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9855-9860Crossref PubMed Scopus (326) Google Scholar).A new layer of complexity in the function of acetyltransferases emerged with the discovery that CBP/p300 also acetylates a variety of transcription factors. Acetylation can alter transcriptional activity through several mechanisms. For example, acetylation of the tumor suppressor protein p53 strongly increases its affinity for DNA (30Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2152) Google Scholar, 31Sakaguchi K. Herrera J.E. Saito S. Miki T. Bustin M. Vassilev A. Anderson C.W. Appella E. Genes Dev. 1998; 12: 2831-2841Crossref PubMed Scopus (1013) Google Scholar, 32Liu L. Scolnick D.M. Trievel R.C. Zhang H.B. Marmorstein R. Halazonetis T.D. Berger S.L. Mol. Cell. Biol. 1999; 19: 1202-1209Crossref PubMed Scopus (648) Google Scholar). Both GATA-1 and EKLF are also acetylated by CBP (29Zhang W. Bieker J.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9855-9860Crossref PubMed Scopus (326) Google Scholar, 33Boyes J. Byfield P. Nakatani Y. Ogryzko V. Nature. 1998; 396: 594-598Crossref PubMed Scopus (632) Google Scholar, 34Hung H.-L. Lau J. Kim A.Y. Weiss M.J. Blobel G.A. Mol. Cell. Biol. 1999; 19: 3496-3505Crossref PubMed Scopus (218) Google Scholar). Mutations in the acetylation sites in GATA-1 compromise its function in erythroid cells, suggesting that GATA-1 acetylation is biologically relevant (34Hung H.-L. Lau J. Kim A.Y. Weiss M.J. Blobel G.A. Mol. Cell. Biol. 1999; 19: 3496-3505Crossref PubMed Scopus (218) Google Scholar).Recent studies implicated CBP/p300 in the regulation of NF-E2 activity by showing that E1A, which inhibits CBP/p300 function, reduced the enhancer activity of HS2 and that NF-E2 was an important target of E1A-mediated inhibition (35Forsberg E.C. Johnson K. Zaboikina T.N. Mosser E.A. Bresnick E.H. J. Biol. Chem. 1999; 274: 26850-26859Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In addition, glutathioneS-transferase (GST) pull-down experiments showed that p45 binds CBP in vitro (16Cheng X. Reginato M.J. Andrews N.C. Lazar M.A. Mol. Cell. Biol. 1997; 17: 1407-1416Crossref PubMed Scopus (97) Google Scholar).Here we report that both subunits of NF-E2 interact with CBP in vitro. In addition, NF-E2 can recruit CBP to a DNA template containing NF-E2 binding sites. Immunoprecipitation experiments demonstrate in vivo association between MafG and CBP in erythroid cells. CBP acetylates MafG predominantly in the basic region, thereby stimulating DNA binding of NF-E2. Mutations at the major acetylation sites reduce DNA binding and transcriptional activation by NF-E2. Thus, recruitment of CBP by NF-E2 might serve two functions, regulation of chromatin structure and transcription factor activity.DISCUSSIONIn this report we demonstrate that both subunits of NF-E2 interact with CBP. We further show that MafG and CBP associate in erythroid cells. CBP and acetylates MafG in vitro and in vivo mainly in the basic region, thereby increasing DNA binding and transcriptional activation of NF-E2.Our observation that both subunits of NF-E2 bind to CBP suggests that the complex might be stabilized by multiple protein contacts in vivo. It is possible that by binding to both subunits, CBP might stimulate heterodimer formation, leading to enhanced DNA binding independent of protein acetylation. Recruitment of CBP to the β-globin LCR might be aided further by the presence of additional erythroid transcription factors that interact with CBP. Both GATA-1 and EKLF fulfill these criteria since GATA-1 and EKLF binding sites are important elements at the LCR, and both factors have been shown to bind CBP (28Blobel G.A. Nakajima T. Eckner R. Montminy M. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2061-2066Crossref PubMed Scopus (313) Google Scholar, 29Zhang W. Bieker J.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9855-9860Crossref PubMed Scopus (326) Google Scholar). GATA-1 and EKLF are also acetylated by CBP (29Zhang W. Bieker J.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9855-9860Crossref PubMed Scopus (326) Google Scholar, 33Boyes J. Byfield P. Nakatani Y. Ogryzko V. Nature. 1998; 396: 594-598Crossref PubMed Scopus (632) Google Scholar, 34Hung H.-L. Lau J. Kim A.Y. Weiss M.J. Blobel G.A. Mol. Cell. Biol. 1999; 19: 3496-3505Crossref PubMed Scopus (218) Google Scholar), indicating that a common theme underlies the regulation of structurally diverse nuclear factors involved in globin gene expression.In agreement with a requirement of CBP for LCR function, interference with CBP/p300 activity leads to a complete block in globin gene expression and erythroid differentiation (28Blobel G.A. Nakajima T. Eckner R. Montminy M. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2061-2066Crossref PubMed Scopus (313) Google Scholar). In the context of HS2, NF-E2 binding sites are the predominant E1A-sensitivecis-acting elements, implicating a functional link between NF-E2 and CBP (35Forsberg E.C. Johnson K. Zaboikina T.N. Mosser E.A. Bresnick E.H. J. Biol. Chem. 1999; 274: 26850-26859Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Therefore, it is possible that the effects of E1A on globin gene expression are the combined result of inhibition of GATA-1, EKLF, and NF-E2 function.In vitro acetylation of MafG by CBP occurs predominantly at a fragment in the basic region containing four lysine residues. Further deletion analysis showed that each of two lysine pairs (residues 53 and 60, and 71 and 76, respectively) contributes to MafG acetylation. Although all four lysines account for the majority of MafG acetylation when assayed alone or in a complex with p45, the relative contribution of each single lysine to the total acetylation of MafG remains to be determined. All four sites are conserved among all small Maf proteins and across all species examined (44Onodera K. Shavit J.A. Motohashi H. Katsuoka F. Akasaka J.-E. Engel J.D. Yamamoto Y. J. Biol. Chem. 1999; 274: 21162-21169Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), suggesting that they are functionally important. Minor acetylation was also observed at the N terminus and the leucine zipper. The functional significance of these acetylation sites is unknown. In contrast, in vivoacetylation as determined by anti-AK immunoprecipitation experiments showed virtually no acetylation outside the basic region (Fig.5 C). This suggests that the basic region is the major acetylation site in vivo. However, it is possible that the anti-AK antibodies used may have selectivity toward the acetylated residues in the basic region.Although the molecular consequences of GATA-1 and EKLF acetylation are not yet established (29Zhang W. Bieker J.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9855-9860Crossref PubMed Scopus (326) Google Scholar, 33Boyes J. Byfield P. Nakatani Y. Ogryzko V. Nature. 1998; 396: 594-598Crossref PubMed Scopus (632) Google Scholar, 34Hung H.-L. Lau J. Kim A.Y. Weiss M.J. Blobel G.A. Mol. Cell. Biol. 1999; 19: 3496-3505Crossref PubMed Scopus (218) Google Scholar), this work shows that acetylation by CBP augments DNA binding of NF-E2. Although acetylation of MafG might increase DNA binding by multiple mechanisms, the observation that acetylation occurs predominantly in the basic region suggests that it might directly increase the affinity of MafG for DNA. Although the residues in MafG that contact DNA have not been determined, the crystal structure of the yeast bZip protein GCN4 complexed with DNA provides some insight into how bZip proteins contact DNA (45Ellenberger T.E. Brandl C.J. Struhl K. Harrison S.C. Cell. 1992; 71: 1223-1237Abstract Full Text PDF PubMed Scopus (813) Google Scholar). In GCN4, the underlined residues of theNTEAARRSR motif in the basic region contact the central 7 base pairs in the GCN4 binding site. Two of the four major acetylation sites in MafG (aa 60 and aa 71, inbold) directly flank theKNXXYAXXCRYK core motif (Fig. 4 B), supporting a possible role for acetylation in the formation of DNA contacts. However, it remains possible that acetylation might stimulate DNA binding by triggering allosteric changes in NF-E2, similar to what has been described for p53 (30Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2152) Google Scholar). Some acetylation of MafG was also observed in the leucine zipper domain, suggesting that this might stimulate heterodimerization with p45, thereby indirectly increasing DNA binding. However, two observations suggest that this is unlikely. First, in vitro protein binding studies failed to yield any significant differences in dimerization of acetylated and nonacetylated MafG with p45. Second, acetylation in the zipper domain both in vitro and in vivo is minimal when compared with the acetylation observed in the basic region. Although an acetylation-induced increase in DNA binding was observed on two distinct NF-E2 elements derived from the PBGD promoter and human HS2, respectively, it remains possible that acetylation might lead to subtle changes in DNA binding site preferences between variant NF-E2 sites. Comparable increases in DNA binding upon acetylation have been observed in several transcription factors (21Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar). One can envision at least two scenarios regarding the order of events. First, DNA binding of NF-E2 might occur before recruitment of CBP and NF-E2 acetylation. In this case, acetylation might stabilize the NF-E2 complex on DNA. Second, CBP and NF-E2 might be bound to each other in solution before DNA binding. In this event, acetylation might increase the rate of association of NF-E2 with its cognate binding element. Our observation that CBP can bind and acetylate NF-E2 in solution in the absence of DNA is consistent with the latter possibility.We showed that acetylation-defective NF-E2 (NF-E2-4A) has diminished transcriptional activity in transient reporter gene assays, further suggesting that acetylation of NF-E2 is important for its function. However, NF-E2-4A still retained significant activity, especially at higher amounts of transfected DNA. This suggests that the reduced affinity of NF-E2-4A for DNA can be overcome by increased protein concentrations.The interaction between NF-E2 and CBP suggests that CBP regulates transcription by modulating chromatin structure as well as transcription factor activity. Although protein acetylation is an attractive mechanism by which CBP acts at the LCR or other erythroid or megakaryocytic genes, additional mechanisms have to be considered as well. For example, since CBP also contacts certain components of the basal transcription machinery (for review see Refs. 20Shikama N. Lyon J. LaThangue N.B. Trends Cell Biol. 1997; 7: 230-236Abstract Full Text PDF PubMed Scopus (424) Google Scholar and 21Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar), it might mediate enhancer activity of the LCR in an acetylation-independent manner, for example by bridging to the globin gene core promoters. Furthermore, NF-E2 has been shown to associate with a chromatin remodeling activity (46Armstrong J.A. Emerson B.M. Mol. Cell. Biol. 1996; 16: 5634-5644Crossref PubMed Google Scholar, 47Gong Q. McDowell J.C. Dean A. Mol. Cell. Biol. 1996; 16: 6055-6064Crossref PubMed Google Scholar), suggesting that CBP-independent activities might contribute to NF-E2 function. Analogously, EKLF associates with E-RC1, an SNF/SWI-related protein complex with ATP-dependent chromatin-remodeling activity (48Armstrong J.A. Bieker J.J. Emerson B.M. Cell. 1998; 95: 93-104Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). It is possible that ATP-dependent remodeling complexes and AT complexes act in different promoter contexts. Alternatively, it is conceivable that ATP-dependent and AT-containing complexes act sequentially at the same genes, similar to what has been described for the yeast HO gene (49Cosma M.P. Tanaka T. Nasmyth K. Cell. 1999; 97: 299-311Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar, 50Krebs J.E. Kuo M.-H. Allis C.D. Peterson C.L. Genes Dev. 1999; 13: 1412-1421Crossref PubMed Scopus (250) Google Scholar). The basic-zipper (bZip)1transcription factor NF-E2 plays a critical role in erythroid and megakaryocytic gene expression (for review see Ref. 1Andrews N.C. Int. J. Biochem. Cell Biol. 1998; 30: 429-432Crossref PubMed Scopus (72) Google Scholar). NF-E2 binds to an extended AP-1-like element, TGCTGA(G/C)TCA, which is found in the locus control regions (LCRs) of the α- and β-globin genes and in the promoters of several heme biosynthetic enzyme genes (for review see Refs. 2Motohashi H. Shavit J.A. Igarashi K. Yamamoto M. Engel J.D. Nucleic Acids Res. 1997; 25: 2953-2959Crossref PubMed Scopus (237) Google Scholar and 3Blank V. Andrews N.C. Trends Biochem. Sci. 1997; 22: 437-441Abstract Full Text PDF PubMed Scopus (219) Google Scholar)). NF-E2 binding sites in the DNase I hypersensitive site 2 (HS2) of the β-globin LCR are essential for its enhancer activity (4Ney P.A. Sorrentino B.P. Lowrey C.H. Nienhuis A.W. Nucleic Acids Res. 1990; 18: 6011-6017Crossref PubMed Scopus (113) Google Scholar, 5Ney P.A. Sorrentino B.P. McDonagh K. Nienhuis A.W. Genes Dev. 1990; 4: 993-1006Crossref PubMed Scopus (209) Google Scholar, 6Talbot D. Grosveld F. EMBO J. 1991; 10: 1391-1398Crossref PubMed Scopus (180) Google Scholar). NF-E2 is a heterodimer consisting of a hematopoietic-specific subunit p45, which is a member of the cap and collar (CNC) family, and a more widely expressed small subunit, which is a member of the small Maf protein family (MafG, MafK, and MafF) (for review see Refs. 2Motohashi H. Shavit J.A. Igarashi K. Yamamoto M. Engel J.D. Nucleic Acids Res. 1997; 25: 2953-2959Crossref PubMed Scopus (237) Google Scholar and 3Blank V. Andrews N.C. Trends Biochem. Sci. 1997; 22: 437-441Abstract Full Text PDF PubMed Scopus (219) Google Scholar)). MafG and MafK are the predominant small Maf molecules in erythroid cells and megakaryocytes (7Shavit J.A. Motohashi H. Onodera K. Akasaka J.-E. Yamamoto M. Engel J.D. Genes Dev. 1998; 12: 2164-2174Crossref PubMed Scopus (92) Google Scholar). p45 and a small Maf protein dimerize through their leucine zipper domains to generate a composite DNA binding domain that consists of the basic regions of both molecules. Other members of the CNC family, including Nrf1 (8Chan J.Y. Han X.L. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11371-11375Crossref PubMed Scopus (293) Google Scholar), Nrf2 (9Moi P. Chan K. Asunis I. Cao A. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9926-9930Crossref PubMed Scopus (1191) Google Scholar), Nrf3 (10Kobayashi A. Ito E. Toki T. Kogame K. Takahashi S. Igarashi K. Hayashi N. Yamamoto M. J. Biol. Chem. 1999; 274: 6443-6452Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), Bach1, and Bach2 (11Oyake T. Itoh K. Motohashi H. Hayashi N. Hoshino H. Nishizawa M. Yamamoto M. Igarashi K. Mol. Cell. Biol. 1996; 16: 6083-6095Crossref PubMed Scopus (516) Google Scholar) can also dimerize with small Maf proteins. Despite the high levels of p45 expression in erythroid cells, mice that are null for p45 displayed a surprisingly mild defect in globin gene expression, suggesting that other members of the CNC protein family can substitute for p45 function in vivo(12Shivdasani R.A. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8690-8694Crossref PubMed Scopus (182) Google Scholar). The N terminus of p45 contains an activation domain that is important for the biological activity of p45 (13Kotkow K.J. Orkin S.H. Mol. Cell. Biol. 1995; 15: 4640-4647Crossref PubMed Scopus (148) Google Scholar, 14Bean T.L. Ney P.A. Nucleic Acids Res. 1997; 25: 2509-2515Crossref PubMed Scopus (41) Google Scholar). Several molecules interact with this domain and are candidate mediators of p45 activity. These include TAFII130 (a component of the TFIID complex) (15Amrolia P.J. Ramamurthy L. Saluja D. Tanese N. Jane S.M. Cunningham J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10051-10056Crossref PubMed Scopus (52) Google Scholar), cAMP-response element-binding protein (CREB)-binding protein (CBP) (16Cheng X. Reginato M.J. Andrews N.C. Lazar M.A. Mol. Cell. Biol. 1997; 17: 1407-1416Crossref PubMed Scopus (97) Google Scholar), and several ubiquitin ligases (17Gavva N.R. Gavva R. Ermekova K. Sudol M. Shen C.J. J. Biol. Chem. 1997; 272: 24105-24108Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 18Mosser E.A. Kasanov J.D. Forsberg E.C. Kay B.K. Ney P.A. Bresnick E.H. Biochemistry. 1998; 37: 13686-13695Crossref PubMed Scopus (60) Google Scholar). The small Maf proteins lack a typical activation domain and are believed to activate transcription as heterodimers with members of the CNC family of proteins. Small Maf proteins can also form homodimers and repress transcription (19Igarashi K. Kataoka K. Itoh K. Hayashi N. Nishizawa M. Yamamoto M. Nature. 1994; 367: 568-572Crossref PubMed Scopus (396) Google Scholar). CBP and its close relative p300 serve as coactivators for a large and diverse set of nuclear factors (20Shikama N. Lyon J. LaThangue N.B. Trends Cell Biol. 1997; 7: 230-236Abstract Full Text PDF PubMed Scopus (424) Google Scholar, 21Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar). CBP and p300 possess intrinsic histone acetyltransferase (AT) activity (21Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar, 22Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2368) Google Scholar, 23Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1523) Google Scholar). Histone acetylation is associated with a relaxed chromatin configuration, suggesting that coactivators act in part through modifying chromatin structure. 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