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A Nuclear Antagonistic Mechanism of Inhibitory Smads in Transforming Growth Factor-β Signaling

2002; Elsevier BV; Volume: 277; Issue: 6 Linguagem: Inglês

10.1074/jbc.m105105200

1083-351X

Shuting Bai, Xu Cao,

Growth Hormone and Insulin-like Growth Factors

Inhibitory Smads (I-Smads), including Smad6 and Smad7, were initially characterized as cytoplasmic antagonists in the transforming growth factor-β signaling pathway. However, I-Smads are also localized in the nucleus. Previously, we have shown that Smad6 can function as a transcriptional co-repressor. In this study, we found both Smad6 and Smad7 interact with histone deacetylases (HDACs). Acetylation state of core histones plays a critical role in gene transcription regulation. An HDAC inhibitor, trichostatin A, released Smad6-mediated transcription repression. Moreover, class I HDACs (HDAC-1 and -3), not class II HDACs (HDAC-4, -5, and -6), were co-immunoprecipitated with Smad6. Endogenous HDAC-1 was also shown to interact with both Smad6 and Hoxc-8. Mapping of the interaction domain indicates Smad6 MH2 domain is mainly involved in recruiting HDAC-1. Most interestingly, Smad6 also binds to DNA through its MH1 domain, and the MH2 domain of Smad6 masks this binding activity, indicating that Smad6 MH1 and MH2 domains associate reciprocally and inhibit each other's function. Hoxc-8 induces Smad6 binding to DNA as a transcriptional complex. Our findings revealed that I-Smads act as antagonists in the nucleus by recruiting HDACs. Inhibitory Smads (I-Smads), including Smad6 and Smad7, were initially characterized as cytoplasmic antagonists in the transforming growth factor-β signaling pathway. However, I-Smads are also localized in the nucleus. Previously, we have shown that Smad6 can function as a transcriptional co-repressor. In this study, we found both Smad6 and Smad7 interact with histone deacetylases (HDACs). Acetylation state of core histones plays a critical role in gene transcription regulation. An HDAC inhibitor, trichostatin A, released Smad6-mediated transcription repression. Moreover, class I HDACs (HDAC-1 and -3), not class II HDACs (HDAC-4, -5, and -6), were co-immunoprecipitated with Smad6. Endogenous HDAC-1 was also shown to interact with both Smad6 and Hoxc-8. Mapping of the interaction domain indicates Smad6 MH2 domain is mainly involved in recruiting HDAC-1. Most interestingly, Smad6 also binds to DNA through its MH1 domain, and the MH2 domain of Smad6 masks this binding activity, indicating that Smad6 MH1 and MH2 domains associate reciprocally and inhibit each other's function. Hoxc-8 induces Smad6 binding to DNA as a transcriptional complex. Our findings revealed that I-Smads act as antagonists in the nucleus by recruiting HDACs. transforming growth factor-β bone morphogenetic protein osteopontin histone acetyltransferase histone deacetylase cAMP-responsive element-binding protein-binding protein glutathione S-transferase homeobox gene trichostatin A amino acid(s) inhibitory Smad receptor-regulated Smad common partner Smad hemagglutinin Transforming growth factor β (TGF-β)1 superfamily members, which include TGF-βs, activins, and bone morphogenetic proteins (BMPs), play a very important role during embryonic development and maintaining adult tissue homeostasis (1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar). TGF-β signaling is mediated by two transmembrane serine-threonine kinase receptors, type II and type I receptors (2Wrana J.L. Attisano L. Wieser R. Ventura F. Massague J. Nature. 1994; 370: 341-347Crossref PubMed Scopus (2094) Google Scholar). Upon ligand binding, the constitutively active type II receptors phosphorylate and activate type I receptors, leading to the propagation of signaling by recruiting and phosphorylating a group of specific proteins, Smads (3Massague J. Hata A. Liu F. Trends Cell Biol. 1997; 7: 187-192Abstract Full Text PDF PubMed Scopus (270) Google Scholar). Smads are pivotal intracellular nuclear effectors of TGF-β family members, which transduce the signal from the cell membrane to the nucleus (3Massague J. Hata A. Liu F. Trends Cell Biol. 1997; 7: 187-192Abstract Full Text PDF PubMed Scopus (270) Google Scholar). Smads contain two highly conserved domains: MH1 and MH2 domains (1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar). Commonly, MH1 domain binds to DNA, whereas MH2 domain is the protein-protein interaction and transactivation domain (1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar). These two domains interact reciprocally and inhibit each other's function (1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar,4Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3316) Google Scholar). Based on their function and sequence similarity, Smads are divided into three subgroups. 1) The receptor-regulated Smads (R-Smads) are the targets of the activated type I receptors. Smad1, Smad5, and Smad8 mediate BMP signaling (3Massague J. Hata A. Liu F. Trends Cell Biol. 1997; 7: 187-192Abstract Full Text PDF PubMed Scopus (270) Google Scholar), whereas Smad2 and Smad3 mediate TGF-β signaling (5Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J.-I. Heldin C.-H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (901) Google Scholar). 2) The common partner Smads (Co-Smads), Smad4 being the only one identified in mammals thus far, are shared by all of the R-Smads (1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar). 3) Inhibitory Smads (I-Smads), including Smad6 and Smad7, stably bind to activated type I receptors and block phosphorylation of R-Smads (6Imamura T. Takase M. Nishihara A. Oeda E. Hanai J.-I. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 7Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.-E. Heldin C.-H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1546) Google Scholar). Both TGF-β and BMP induce I-Smad expression, indicating their negative feedback function in TGF-β signaling (8Afrakhte M. Moren A. Jossan S. Itoh S. Sampath K. Westermark B. Heldin C.-H. Heldin N.-E. ten Dijke P. Biochem. Biophys. Res. Commun. 1998; 249: 505-511Crossref PubMed Scopus (297) Google Scholar,9Takase M. Immamura T. Sampath T.K. Takeda K. Ichijo H. Miyazono K. Massahiro K. Biochem. Biophys. Res. Commun. 1998; 244: 26-29Crossref PubMed Scopus (132) Google Scholar).Smad6 preferably inhibits BMP signaling, whereas Smad7 is more a general inhibitor (10Itoh S. Itoh F. Goumans M. ten Dijke P. Eur. J. Biochem. 2000; 267: 6954-6967Crossref PubMed Scopus (454) Google Scholar). Smad6 and Smad7 are expressed at the earliest stage during embryo development and highly expressed in the developing cardiovascular system, eyes, bones, and other tissues (11Yamada M. Szendro P.I. Prokscha A. Schwartz R. Eichele G. Dev. Biol. 1999; 215: 48-61Crossref PubMed Scopus (72) Google Scholar, 12Luukko K. Ylikorkala A. Makela T.P. Mech. Dev. 2001; 101: 209-212Crossref PubMed Scopus (41) Google Scholar). The expression of Smad6 overlaps BMP-2, -4, and -7 expression, which orchestrates BMP-mediated cardiac development (11Yamada M. Szendro P.I. Prokscha A. Schwartz R. Eichele G. Dev. Biol. 1999; 215: 48-61Crossref PubMed Scopus (72) Google Scholar). Aortic ossification and elevated blood pressure were reported in viable Smad6 mutants (13Galvin K.M. Donovan M.J. Lynch C.A. Meyer R.I. Paul R.J. Lorenz J.N. Fairchild-Huntress V. Dixon K.L. Dunmore J.H. Gimbrone Jr., M.A Falb D. Huszar D. Nat. Genet. 2000; 24: 171-174Crossref PubMed Scopus (396) Google Scholar). BMP induces the ventral mesoderm formation (14Hogan B.L.M. Genes Dev. 1996; 10: 1580-1594Crossref PubMed Scopus (1713) Google Scholar, 15Kawabata M. Imamura T. Miyazono K. Cytokine Growth Factor Rev. 1998; 9: 49-61Crossref PubMed Scopus (448) Google Scholar), and the overlapped expression of Smad6 and BMP indicates that Smad6 is a key protein in balancing the function of BMP during embryo development. Smad6 inhibits BMP-induced osteoblast differentiation (16Fujii M. Takeda K. Imamura T. Aoki H. Sampath T.K. Enomoto S. Kawabata M. Kato M. Ichijo H. Miyazono K. Mol. Biol. Cell. 1999; 10: 3801-3813Crossref PubMed Scopus (368) Google Scholar). It is also reported that Smad6 inhibits adipogenesis (17Choy L. Skillington J. Derynck R. J. Cell Biol. 2001; 149: 667-682Crossref Scopus (239) Google Scholar). Smad7, not Smad6, was identified as inhibiting TGF-β function during embryonic vasculogenesis (18Zwijsen A. van Rooijen M.A. Goumans M.J. Dewulf N. Bosman E.A. ten Dijke P. Mummery C.L. Huylebroeck D. Dev. Dyn. 2000; 218: 663-670Crossref PubMed Scopus (21) Google Scholar) and lung development, injury, and repair (19Nakao A. Fujii M. Matsumura R. Kumano K. Saito Y. Miyazono K. Iwamoto I. J. Clin. Invest. 1999; 104: 5-11Crossref PubMed Scopus (384) Google Scholar, 20Zhao J. Shi W. Chen H. Warburton D. J. Biol. Chem. 2000; 275: 23992-23997Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar).The biological activities of Smads are closely associated to their cellular localization. R-Smads are located in the cytoplasm in the absence of signaling. Upon ligand stimulation, they are phosphorylated at their extreme carboxyl end SSXS motif and recruit the common partner Smad4 into the nucleus, where they act as transcriptional activators (21Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (475) Google Scholar). I-Smads are located in both the cytosol and the nucleus (22Itoh S. Landstrom M. Hermasson A. Itoh F. Heldin C.-H. Heldin N.-E. ten Dijke P. J. Biol. Chem. 1998; 273: 29195-29201Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 23Nakayama T. Gardner H. Berg L.K. Christian J.L. Genes Cells. 1998; 3: 387-394Crossref PubMed Scopus (66) Google Scholar, 24Sakou T. Onishi T. Yamamoto T. Nagamine T. Sampath T.K. ten Dijke P. J. Bone Miner. Res. 1999; 14: 1145-1152Crossref PubMed Scopus (128) Google Scholar, 25Bai S. Shi X. Yang X. Cao X. J. Biol. Chem. 2000; 275: 8267-8270Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Smad6 cellular distribution is not affected by TGF-β or BMP treatment (24Sakou T. Onishi T. Yamamoto T. Nagamine T. Sampath T.K. ten Dijke P. J. Bone Miner. Res. 1999; 14: 1145-1152Crossref PubMed Scopus (128) Google Scholar, 25Bai S. Shi X. Yang X. Cao X. J. Biol. Chem. 2000; 275: 8267-8270Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) whereas TGF-β induces nuclear export of Smad7 (22Itoh S. Landstrom M. Hermasson A. Itoh F. Heldin C.-H. Heldin N.-E. ten Dijke P. J. Biol. Chem. 1998; 273: 29195-29201Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). The inhibitory mechanisms of I-Smads have been characterized in the cytoplasm. I-Smads interact with the activated type I receptors, which then block the phosphorylation of the R-Smads (6Imamura T. Takase M. Nishihara A. Oeda E. Hanai J.-I. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 7Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.-E. Heldin C.-H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1546) Google Scholar). Smad6 was also demonstrated to interact with the phosphorylated Smad1 in the cytoplasm, competing with Smad4 to form an inactive Smad1-Smad6 complex (26Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (577) Google Scholar). We have shown that, in the nucleus, Smad6 acts as a transcriptional co-repressor on osteopontin promoter by interacting with Hoxc-8 (25Bai S. Shi X. Yang X. Cao X. J. Biol. Chem. 2000; 275: 8267-8270Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), a transcriptional repressor in BMP signaling (27Shi X. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 28Wan M. Shi X. Feng X. Cao X. J. Biol. Chem. 2001; 276: 10119-10125Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The mechanism of Smad6 repressive function in the nucleus remains unclear.Signaling to chromatin through histone modification is demonstrated as a major step for regulating target gene transcription (29Workman J.L. Kingston R.E. Annu. Rev. Biochem. 1998; 67: 545-579Crossref PubMed Scopus (959) Google Scholar). One of the modifications of histones is acetylation, where specific lysine residues are functional targets for histone acetyltransferases (HATs) and histone deacetylases (HDACs) (30Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2367) Google Scholar, 31Kuo M.-H. Allis C.D. BioEssays. 1998; 20: 615-626Crossref PubMed Scopus (1054) Google Scholar, 32Mannervik M. Nibu Y. Zhang H. Levine M. Science. 1999; 284: 606-609Crossref PubMed Scopus (192) Google Scholar). The acetylation of chromosomal histones loosens the structure of the target gene promoter and results in increased accessibility of the chromatin for transcription factors (30Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2367) Google Scholar). Conversely, hypoacetylation of a gene regulatory region is strongly related to silencing gene expression (30Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2367) Google Scholar). Several transcription repressors and co-repressors have been demonstrated to recruit HDACs to specific genes for silencing gene expression (33Burke L.J. Baniahmad A. FASEB J. 2000; 14: 1876-1888Crossref PubMed Scopus (170) Google Scholar). For example, TGIF, a Smad transcriptional co-repressor, was identified as interacting with HDAC-1 and inhibiting TGF-β-induced gene transcription (34Wotton D. Lo R.S. Lee S. Massague J. Cell. 1999; 97: 29-39Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar). In addition to HDAC-1, eight other HDACs have been identified thus far (35Ng H.H. Bird A. Trends Biochem. Sci. 2000; 25: 121-126Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 36Zhou X. marks P.A. Rifkind R.A. Richon V.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10572-10577Crossref PubMed Scopus (203) Google Scholar).Given that Smad6 displays similar activity as TGIF and other transcriptional repressors, all of which appear to recruit different HDAC complexes, we considered whether histone deacetylase activity might extend to Smad6 and affect its transcriptional activity in the nucleus. To address this issue, we characterized the mechanism of Smad6-mediated gene transcription. We demonstrated that an HDAC activity inhibitor, trichostatin A (TSA), rescued the repressive function of Smad6 in BMP signaling, indicating that HDACs engaged in the inhibitory effect of Smad6 in the nucleus. Furthermore, Smad6 was shown to interact with HDAC-1 and -3 in an immunoprecipitation assay. HDAC activity was detected in HDAC assay with an immunoprecipitated Smad6 protein complex. These data suggest that Smad6 represses gene transcription by recruiting class I HDACs and modifying chromatin conformation.DISCUSSIONI-Smads are antagonistic mediators in TGF-β signaling (1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar). Both Smad6 and Smad7 expressions are induced by TGF-β or BMPs as negative feedback responses (8Afrakhte M. Moren A. Jossan S. Itoh S. Sampath K. Westermark B. Heldin C.-H. Heldin N.-E. ten Dijke P. Biochem. Biophys. Res. Commun. 1998; 249: 505-511Crossref PubMed Scopus (297) Google Scholar, 9Takase M. Immamura T. Sampath T.K. Takeda K. Ichijo H. Miyazono K. Massahiro K. Biochem. Biophys. Res. Commun. 1998; 244: 26-29Crossref PubMed Scopus (132) Google Scholar). I-Smad interacts with the activated type I receptors, blocking the phosphorylation of R-Smads (6Imamura T. Takase M. Nishihara A. Oeda E. Hanai J.-I. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 7Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.-E. Heldin C.-H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1546) Google Scholar). Particularly, Smad6 competes with Smad4 to form an inactive complex with phosphorylated Smad1 (26Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (577) Google Scholar). However, antagonistic function of I-Smads is not well characterized in the nucleus. I-Smads are localized in both the cytosol and the nucleus, and TGF-β or BMP stimulation does not change the cellular distribution of Smad6. Initially, we reported that Smad6 functions as a transcriptional co-repressor through interacting with Hoxc-8 (25Bai S. Shi X. Yang X. Cao X. J. Biol. Chem. 2000; 275: 8267-8270Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In this report, we demonstrated that both Smad6 and Smad7 were co-immunoprecipitated with HDAC-1, which is normally recruited by transcriptional repressors or co-repressors in inhibition of gene transcription. Importantly, we also find that Smad6 is a DNA-binding protein as a partner of Smad6/Hoxc-8 complex. Like other Smads, Smad6 binds to DNA through its MH1 domain, whereas Smad6 MH2 domain recruits HDAC-1. These findings revealed an important functional aspect of I-Smads in the nucleus.Chromatin modification plays a critical role in regulating gene transcription. Acetylation is one of the major modification processes. HATs acetylate lysine residues on histone N-tails, leading to loosening of the chromatin structure and increasing the accessibility of transcriptional factors to the chromosome, whereas HDACs mediate silencing of gene transcription. R-Smads translocate to the nucleus upon BMP or TGF-β stimulation, where they function as transcriptional activators through interacting and recruiting p300/CBP to specific gene promoters (34Wotton D. Lo R.S. Lee S. Massague J. Cell. 1999; 97: 29-39Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar, 41Pearson K.L. Hunter T. Janknecht R. Biochim. Biophys. Acta. 1999; 1489: 354-364Crossref PubMed Scopus (45) Google Scholar). TGIF, a Smad co-repressor in the TGF-β signaling pathway, interacts with HDAC-1 and inhibits target gene expression (34Wotton D. Lo R.S. Lee S. Massague J. Cell. 1999; 97: 29-39Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar). HDACs inhibit gene transcription by tightening the chromosome structure. TSA, an HDAC inhibitor, rescued Smad6- and Hoxc-8-mediated transcription repression. Interaction of Smad6 and Smad7 with HDAC-1 and recruitment of endogenous HDAC activity clearly indicate the nuclear function of I-Smads as transcription repressors or co-repressors. Smad7 does not interact with Hox proteins (25Bai S. Shi X. Yang X. Cao X. J. Biol. Chem. 2000; 275: 8267-8270Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Therefore, Smad7 was not shown as a transcriptional repressor or co-repressor. However, the interaction of Smad7 with HDAC-1 suggests its potential function as a transcriptional repressor or co-repressor in different transcription mechanisms because other cytokines such as TNF-α induce Smad7 expression (49Bitzer M. von Gersdoeff G. Liang D. Dominguez-Rosales A. Beg A.A. Rojkind M. Bottinger E.P. Genes Dev. 2000; 14: 187-197PubMed Google Scholar). Thus, Smad7 could also mediate cross-talk between TGF-β and other signaling pathways (49Bitzer M. von Gersdoeff G. Liang D. Dominguez-Rosales A. Beg A.A. Rojkind M. Bottinger E.P. Genes Dev. 2000; 14: 187-197PubMed Google Scholar, 50Lallemand F. Mazars A. Prunier C. Bertrand F. Kornprost M. Gallea S. Roman-Roman S. Cherqui G. Atfi A. Oncogene. 2001; 20: 879-884Crossref PubMed Scopus (96) Google Scholar). The detailed mechanism of Smad7 in the nucleus remains to be characterized.The finding of Smad6 binding to DNA suggests that all of the Smads are DNA binding proteins. I-Smads contain both MH1 and MH2 domains, which are necessary for full activities of Xenopus Smad6 and Smad7 (51Nakayama T. Berg L.K. Christian J.L. Mech. Dev. 2001; 100: 251-262Crossref PubMed Scopus (29) Google Scholar). The MH2 domain is highly conserved with other Smads, but the MH1 domain is distinct (44Topper J. Cai J. Qiu Y. Anderson K. Xu Y.-Y. Deeds J. Feeley R. Gimeno C. Woolf E. Tayber O. Mays G. Sampson B. Schoen F. Gimbrone Jr., M. Falb D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9314-9319Crossref PubMed Scopus (289) Google Scholar). MH1 domains of R-Smads and Smad4 bind DNA Smad box (GTCT) (41Pearson K.L. Hunter T. Janknecht R. Biochim. Biophys. Acta. 1999; 1489: 354-364Crossref PubMed Scopus (45) Google Scholar, 42Zawel L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar, 43Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1573) Google Scholar). Like other Smads, Smad6 binds to DNA also through its MH1 domain. The full length of Smad6 does not bind to DNA, whereas, in the presence of Hoxc-8, Smad6 binds to DNA and forms a heterodimer. Importantly, the MH1 domain of Smad6 is required for the Hoxc-8-Smad6 complex formation although it is the MH2 domain of Smad6 that interacts with Hoxc-8. It appears that the MH2 domain of Smad6 masks its MH1 domain DNA binding activity, and that the MH1 and MH2 domains of Smad6 interact reciprocally and inhibit each other's function. Indeed, Smad6 MH1 DNA binding activity decreased when the length of Smad6 truncated MH1 domain extended to MH2 domain. This observation suggests that Hoxc-8 induces Smad6 conformation change, in which Smad6 MH1 domain becomes available to bind to DNA. Phosphorylation of Smad6 could be another way to change its conformation for DNA binding activity, which could also serve as a cross-talk with other signaling pathways.Taken together, we demonstrated a specific interaction of I-Smads with HDACs. As a model, we describe a transcription repression mechanism of Smad6. In response to BMP or TGF-β stimulation, Smad6 interacts with Hoxc-8 and binds to DNA as a heterodimer, which inhibits Smad1-induced gene transcription by recruiting HDACs. Importantly, Smad6 is also a DNA-binding protein. Like other Smads, Smad6 binds to DNA through its MH1 domain, and Smad6 MH2 domain interacts with HDAC-1 and Hoxc-8. Consistent with the observation of I-Smads cellular distribution, our data indicate that I-Smads can function as transcription repressors or co-repressors in the nucleus as antagonistic feedback loop of the TGF-β signaling pathway. Transforming growth factor β (TGF-β)1 superfamily members, which include TGF-βs, activins, and bone morphogenetic proteins (BMPs), play a very important role during embryonic development and maintaining adult tissue homeostasis (1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar). TGF-β signaling is mediated by two transmembrane serine-threonine kinase receptors, type II and type I receptors (2Wrana J.L. Attisano L. Wieser R. Ventura F. Massague J. Nature. 1994; 370: 341-347Crossref PubMed Scopus (2094) Google Scholar). Upon ligand binding, the constitutively active type II receptors phosphorylate and activate type I receptors, leading to the propagation of signaling by recruiting and phosphorylating a group of specific proteins, Smads (3Massague J. Hata A. Liu F. Trends Cell Biol. 1997; 7: 187-192Abstract Full Text PDF PubMed Scopus (270) Google Scholar). Smads are pivotal intracellular nuclear effectors of TGF-β family members, which transduce the signal from the cell membrane to the nucleus (3Massague J. Hata A. Liu F. Trends Cell Biol. 1997; 7: 187-192Abstract Full Text PDF PubMed Scopus (270) Google Scholar). Smads contain two highly conserved domains: MH1 and MH2 domains (1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar). Commonly, MH1 domain binds to DNA, whereas MH2 domain is the protein-protein interaction and transactivation domain (1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar). These two domains interact reciprocally and inhibit each other's function (1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar,4Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3316) Google Scholar). Based on their function and sequence similarity, Smads are divided into three subgroups. 1) The receptor-regulated Smads (R-Smads) are the targets of the activated type I receptors. Smad1, Smad5, and Smad8 mediate BMP signaling (3Massague J. Hata A. Liu F. Trends Cell Biol. 1997; 7: 187-192Abstract Full Text PDF PubMed Scopus (270) Google Scholar), whereas Smad2 and Smad3 mediate TGF-β signaling (5Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J.-I. Heldin C.-H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (901) Google Scholar). 2) The common partner Smads (Co-Smads), Smad4 being the only one identified in mammals thus far, are shared by all of the R-Smads (1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar). 3) Inhibitory Smads (I-Smads), including Smad6 and Smad7, stably bind to activated type I receptors and block phosphorylation of R-Smads (6Imamura T. Takase M. Nishihara A. Oeda E. Hanai J.-I. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 7Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.-E. Heldin C.-H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1546) Google Scholar). Both TGF-β and BMP induce I-Smad expression, indicating their negative feedback function in TGF-β signaling (8Afrakhte M. Moren A. Jossan S. Itoh S. Sampath K. Westermark B. Heldin C.-H. Heldin N.-E. ten Dijke P. Biochem. Biophys. Res. Commun. 1998; 249: 505-511Crossref PubMed Scopus (297) Google Scholar,9Takase M. Immamura T. Sampath T.K. Takeda K. Ichijo H. Miyazono K. Massahiro K. Biochem. Biophys. Res. Commun. 1998; 244: 26-29Crossref PubMed Scopus (132) Google Scholar). Smad6 preferably inhibits BMP signaling, whereas Smad7 is more a general inhibitor (10Itoh S. Itoh F. Goumans M. ten Dijke P. Eur. J. Biochem. 2000; 267: 6954-6967Crossref PubMed Scopus (454) Google Scholar). Smad6 and Smad7 are expressed at the earliest stage during embryo development and highly expressed in the developing cardiovascular system, eyes, bones, and other tissues (11Yamada M. Szendro P.I. Prokscha A. Schwartz R. Eichele G. Dev. Biol. 1999; 215: 48-61Crossref PubMed Scopus (72) Google Scholar, 12Luukko K. Ylikorkala A. Makela T.P. Mech. Dev. 2001; 101: 209-212Crossref PubMed Scopus (41) Google Scholar). The expression of Smad6 overlaps BMP-2, -4, and -7 expression, which orchestrates BMP-mediated cardiac development (11Yamada M. Szendro P.I. Prokscha A. Schwartz R. Eichele G. Dev. Biol. 1999; 215: 48-61Crossref PubMed Scopus (72) Google Scholar). Aortic ossification and elevated blood pressure were reported in viable Smad6 mutants (13Galvin K.M. Donovan M.J. Lynch C.A. Meyer R.I. Paul R.J. Lorenz J.N. Fairchild-Huntress V. Dixon K.L. Dunmore J.H. Gimbrone Jr., M.A Falb D. Huszar D. Nat. Genet. 2000; 24: 171-174Crossref PubMed Scopus (396) Google Scholar). BMP induces the ventral mesoderm formation (14Hogan B.L.M. Genes Dev. 1996; 10: 1580-1594Crossref PubMed Scopus (1713) Google Scholar, 15Kawabata M. Imamura T. Miyazono K. Cytokine Growth Factor Rev. 1998; 9: 49-61Crossref PubMed Scopus (448) Google Scholar), and the overlapped expression of Smad6 and BMP indicates that Smad6 is a key protein in balancing the function of BMP during embryo development. Smad6 inhibits BMP-induced osteoblast differentiation (16Fujii M. Takeda K. Imamura T. Aoki H. Sampath T.K. Enomoto S. Kawabata M. Kato M. Ichijo H. Miyazono K. Mol. Biol. Cell. 1999; 10: 3801-3813Crossref PubMed Scopus (368) Google Scholar). It is also reported that Smad6 inhibits adipogenesis (17Choy L. Skillington J. Derynck R. J. Cell Biol. 2001; 149: 667-682Crossref Scopus (239) Google Scholar). Smad7, not Smad6, was identified as inhibiting TGF-β function during embryonic vasculogenesis (18Zwijsen A. van Rooijen M.A. Goumans M.J. Dewulf N. Bosman E.A. ten Dijke P. Mummery C.L. Huylebroeck D. Dev. Dyn. 2000; 218: 663-670Crossref PubMed Scopus (21) Google Scholar) and lung development, injury, and repair (19Nakao A. Fujii M. Matsumura R. Kumano K. Saito Y. Miyazono K. Iwamoto I. J. Clin. Invest. 1999; 104: 5-11Crossref PubMed Scopus (384) Google Scholar, 20Zhao J. Shi W. Chen H. Warburton D. J. Biol. Chem. 2000; 275: 23992-23997Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The biological activities of Smads are closely associated to their cellular localization. R-Smads are located in the cytoplasm in the absence of signaling. Upon ligand stimulation, they are phosphorylated at their extreme carboxyl end SSXS motif and recruit the common partner Smad4 into the nucleus, where they act as transcriptional activators (21Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (475) Google Scholar). I-Smads are located in both the cytosol and the nucleus (22Itoh S. Landstrom M. Hermasson A. Itoh F. Heldin C.-H. Heldin N.-E. ten Dijke P. J. Biol. Chem. 1998; 273: 29195-29201Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 23Nakayama T. Gardner H. Berg L.K. Christian J.L. Genes Cells. 1998; 3: 387-394Crossref PubMed Scopus (66) Google Scholar, 24Sakou T. Onishi T. Yamamoto T. Nagamine T. Sampath T.K. ten Dijke P. J. Bone Miner. Res. 1999; 14: 1145-1152Crossref PubMed Scopus (128) Google Scholar, 25Bai S. Shi X. Yang X. Cao X. J. Biol. Chem. 2000; 275: 8267-8270Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Smad6 cellular distribution is not affected by TGF-β or BMP treatment (24Sakou T. Onishi T. Yamamoto T. Nagamine T. Sampath T.K. ten Dijke P. J. Bone Miner. Res. 1999; 14: 1145-1152Crossref PubMed Scopus (128) Google Scholar, 25Bai S. Shi X. Yang X. Cao X. J. Biol. Chem. 2000; 275: 8267-8270Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) whereas TGF-β induces nuclear export of Smad7 (22Itoh S. Landstrom M. Hermasson A. Itoh F. Heldin C.-H. Heldin N.-E. ten Dijke P. J. Biol. Chem. 1998; 273: 29195-29201Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). The inhibitory mechanisms of I-Smads have been characterized in the cytoplasm. I-Smads interact with the activated type I receptors, which then block the phosphorylation of the R-Smads (6Imamura T. Takase M. Nishihara A. Oeda E. Hanai J.-I. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 7Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.-E. Heldin C.-H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1546) Google Scholar). Smad6 was also demonstrated to interact with the phosphorylated Smad1 in the cytoplasm, competing with Smad4 to form an inactive Smad1-Smad6 complex (26Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (577) Google Scholar). We have shown that, in the nucleus, Smad6 acts as a transcriptional co-repressor on osteopontin promoter by interacting with Hoxc-8 (25Bai S. Shi X. Yang X. Cao X. J. Biol. Chem. 2000; 275: 8267-8270Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), a transcriptional repressor in BMP signaling (27Shi X. Yang X. Chen D. Chang Z. Cao X. J. Biol. Chem. 1999; 274: 13711-13717Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 28Wan M. Shi X. Feng X. Cao X. J. Biol. Chem. 2001; 276: 10119-10125Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The mechanism of Smad6 repressive function in the nucleus remains unclear. Signaling to chromatin through histone modification is demonstrated as a major step for regulating target gene transcription (29Workman J.L. Kingston R.E. Annu. Rev. Biochem. 1998; 67: 545-579Crossref PubMed Scopus (959) Google Scholar). One of the modifications of histones is acetylation, where specific lysine residues are functional targets for histone acetyltransferases (HATs) and histone deacetylases (HDACs) (30Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2367) Google Scholar, 31Kuo M.-H. Allis C.D. BioEssays. 1998; 20: 615-626Crossref PubMed Scopus (1054) Google Scholar, 32Mannervik M. Nibu Y. Zhang H. Levine M. Science. 1999; 284: 606-609Crossref PubMed Scopus (192) Google Scholar). The acetylation of chromosomal histones loosens the structure of the target gene promoter and results in increased accessibility of the chromatin for transcription factors (30Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2367) Google Scholar). Conversely, hypoacetylation of a gene regulatory region is strongly related to silencing gene expression (30Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2367) Google Scholar). Several transcription repressors and co-repressors have been demonstrated to recruit HDACs to specific genes for silencing gene expression (33Burke L.J. Baniahmad A. FASEB J. 2000; 14: 1876-1888Crossref PubMed Scopus (170) Google Scholar). For example, TGIF, a Smad transcriptional co-repressor, was identified as interacting with HDAC-1 and inhibiting TGF-β-induced gene transcription (34Wotton D. Lo R.S. Lee S. Massague J. Cell. 1999; 97: 29-39Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar). In addition to HDAC-1, eight other HDACs have been identified thus far (35Ng H.H. Bird A. Trends Biochem. Sci. 2000; 25: 121-126Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 36Zhou X. marks P.A. Rifkind R.A. Richon V.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10572-10577Crossref PubMed Scopus (203) Google Scholar). Given that Smad6 displays similar activity as TGIF and other transcriptional repressors, all of which appear to recruit different HDAC complexes, we considered whether histone deacetylase activity might extend to Smad6 and affect its transcriptional activity in the nucleus. To address this issue, we characterized the mechanism of Smad6-mediated gene transcription. We demonstrated that an HDAC activity inhibitor, trichostatin A (TSA), rescued the repressive function of Smad6 in BMP signaling, indicating that HDACs engaged in the inhibitory effect of Smad6 in the nucleus. Furthermore, Smad6 was shown to interact with HDAC-1 and -3 in an immunoprecipitation assay. HDAC activity was detected in HDAC assay with an immunoprecipitated Smad6 protein complex. These data suggest that Smad6 represses gene transcription by recruiting class I HDACs and modifying chromatin conformation. DISCUSSIONI-Smads are antagonistic mediators in TGF-β signaling (1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar). Both Smad6 and Smad7 expressions are induced by TGF-β or BMPs as negative feedback responses (8Afrakhte M. Moren A. Jossan S. Itoh S. Sampath K. Westermark B. Heldin C.-H. Heldin N.-E. ten Dijke P. Biochem. Biophys. Res. Commun. 1998; 249: 505-511Crossref PubMed Scopus (297) Google Scholar, 9Takase M. Immamura T. Sampath T.K. Takeda K. Ichijo H. Miyazono K. Massahiro K. Biochem. Biophys. Res. Commun. 1998; 244: 26-29Crossref PubMed Scopus (132) Google Scholar). I-Smad interacts with the activated type I receptors, blocking the phosphorylation of R-Smads (6Imamura T. Takase M. Nishihara A. Oeda E. Hanai J.-I. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 7Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.-E. Heldin C.-H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1546) Google Scholar). Particularly, Smad6 competes with Smad4 to form an inactive complex with phosphorylated Smad1 (26Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (577) Google Scholar). However, antagonistic function of I-Smads is not well characterized in the nucleus. I-Smads are localized in both the cytosol and the nucleus, and TGF-β or BMP stimulation does not change the cellular distribution of Smad6. Initially, we reported that Smad6 functions as a transcriptional co-repressor through interacting with Hoxc-8 (25Bai S. Shi X. Yang X. Cao X. J. Biol. Chem. 2000; 275: 8267-8270Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In this report, we demonstrated that both Smad6 and Smad7 were co-immunoprecipitated with HDAC-1, which is normally recruited by transcriptional repressors or co-repressors in inhibition of gene transcription. Importantly, we also find that Smad6 is a DNA-binding protein as a partner of Smad6/Hoxc-8 complex. Like other Smads, Smad6 binds to DNA through its MH1 domain, whereas Smad6 MH2 domain recruits HDAC-1. These findings revealed an important functional aspect of I-Smads in the nucleus.Chromatin modification plays a critical role in regulating gene transcription. Acetylation is one of the major modification processes. HATs acetylate lysine residues on histone N-tails, leading to loosening of the chromatin structure and increasing the accessibility of transcriptional factors to the chromosome, whereas HDACs mediate silencing of gene transcription. R-Smads translocate to the nucleus upon BMP or TGF-β stimulation, where they function as transcriptional activators through interacting and recruiting p300/CBP to specific gene promoters (34Wotton D. Lo R.S. Lee S. Massague J. Cell. 1999; 97: 29-39Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar, 41Pearson K.L. Hunter T. Janknecht R. Biochim. Biophys. Acta. 1999; 1489: 354-364Crossref PubMed Scopus (45) Google Scholar). TGIF, a Smad co-repressor in the TGF-β signaling pathway, interacts with HDAC-1 and inhibits target gene expression (34Wotton D. Lo R.S. Lee S. Massague J. Cell. 1999; 97: 29-39Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar). HDACs inhibit gene transcription by tightening the chromosome structure. TSA, an HDAC inhibitor, rescued Smad6- and Hoxc-8-mediated transcription repression. Interaction of Smad6 and Smad7 with HDAC-1 and recruitment of endogenous HDAC activity clearly indicate the nuclear function of I-Smads as transcription repressors or co-repressors. Smad7 does not interact with Hox proteins (25Bai S. Shi X. Yang X. Cao X. J. Biol. Chem. 2000; 275: 8267-8270Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Therefore, Smad7 was not shown as a transcriptional repressor or co-repressor. However, the interaction of Smad7 with HDAC-1 suggests its potential function as a transcriptional repressor or co-repressor in different transcription mechanisms because other cytokines such as TNF-α induce Smad7 expression (49Bitzer M. von Gersdoeff G. Liang D. Dominguez-Rosales A. Beg A.A. Rojkind M. Bottinger E.P. Genes Dev. 2000; 14: 187-197PubMed Google Scholar). Thus, Smad7 could also mediate cross-talk between TGF-β and other signaling pathways (49Bitzer M. von Gersdoeff G. Liang D. Dominguez-Rosales A. Beg A.A. Rojkind M. Bottinger E.P. Genes Dev. 2000; 14: 187-197PubMed Google Scholar, 50Lallemand F. Mazars A. Prunier C. Bertrand F. Kornprost M. Gallea S. Roman-Roman S. Cherqui G. Atfi A. Oncogene. 2001; 20: 879-884Crossref PubMed Scopus (96) Google Scholar). The detailed mechanism of Smad7 in the nucleus remains to be characterized.The finding of Smad6 binding to DNA suggests that all of the Smads are DNA binding proteins. I-Smads contain both MH1 and MH2 domains, which are necessary for full activities of Xenopus Smad6 and Smad7 (51Nakayama T. Berg L.K. Christian J.L. Mech. Dev. 2001; 100: 251-262Crossref PubMed Scopus (29) Google Scholar). The MH2 domain is highly conserved with other Smads, but the MH1 domain is distinct (44Topper J. Cai J. Qiu Y. Anderson K. Xu Y.-Y. Deeds J. Feeley R. Gimeno C. Woolf E. Tayber O. Mays G. Sampson B. Schoen F. Gimbrone Jr., M. Falb D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9314-9319Crossref PubMed Scopus (289) Google Scholar). MH1 domains of R-Smads and Smad4 bind DNA Smad box (GTCT) (41Pearson K.L. Hunter T. Janknecht R. Biochim. Biophys. Acta. 1999; 1489: 354-364Crossref PubMed Scopus (45) Google Scholar, 42Zawel L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar, 43Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1573) Google Scholar). Like other Smads, Smad6 binds to DNA also through its MH1 domain. The full length of Smad6 does not bind to DNA, whereas, in the presence of Hoxc-8, Smad6 binds to DNA and forms a heterodimer. Importantly, the MH1 domain of Smad6 is required for the Hoxc-8-Smad6 complex formation although it is the MH2 domain of Smad6 that interacts with Hoxc-8. It appears that the MH2 domain of Smad6 masks its MH1 domain DNA binding activity, and that the MH1 and MH2 domains of Smad6 interact reciprocally and inhibit each other's function. Indeed, Smad6 MH1 DNA binding activity decreased when the length of Smad6 truncated MH1 domain extended to MH2 domain. This observation suggests that Hoxc-8 induces Smad6 conformation change, in which Smad6 MH1 domain becomes available to bind to DNA. Phosphorylation of Smad6 could be another way to change its conformation for DNA binding activity, which could also serve as a cross-talk with other signaling pathways.Taken together, we demonstrated a specific interaction of I-Smads with HDACs. As a model, we describe a transcription repression mechanism of Smad6. In response to BMP or TGF-β stimulation, Smad6 interacts with Hoxc-8 and binds to DNA as a heterodimer, which inhibits Smad1-induced gene transcription by recruiting HDACs. Importantly, Smad6 is also a DNA-binding protein. Like other Smads, Smad6 binds to DNA through its MH1 domain, and Smad6 MH2 domain interacts with HDAC-1 and Hoxc-8. Consistent with the observation of I-Smads cellular distribution, our data indicate that I-Smads can function as transcription repressors or co-repressors in the nucleus as antagonistic feedback loop of the TGF-β signaling pathway. I-Smads are antagonistic mediators in TGF-β signaling (1Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar). Both Smad6 and Smad7 expressions are induced by TGF-β or BMPs as negative feedback responses (8Afrakhte M. Moren A. Jossan S. Itoh S. Sampath K. Westermark B. Heldin C.-H. Heldin N.-E. ten Dijke P. Biochem. Biophys. Res. Commun. 1998; 249: 505-511Crossref PubMed Scopus (297) Google Scholar, 9Takase M. Immamura T. Sampath T.K. Takeda K. Ichijo H. Miyazono K. Massahiro K. Biochem. Biophys. Res. Commun. 1998; 244: 26-29Crossref PubMed Scopus (132) Google Scholar). I-Smad interacts with the activated type I receptors, blocking the phosphorylation of R-Smads (6Imamura T. Takase M. Nishihara A. Oeda E. Hanai J.-I. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 7Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.-E. Heldin C.-H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1546) Google Scholar). Particularly, Smad6 competes with Smad4 to form an inactive complex with phosphorylated Smad1 (26Hata A. Lagna G. Massague J. Hemmati-Brivanlou A. Genes Dev. 1998; 12: 186-197Crossref PubMed Scopus (577) Google Scholar). However, antagonistic function of I-Smads is not well characterized in the nucleus. I-Smads are localized in both the cytosol and the nucleus, and TGF-β or BMP stimulation does not change the cellular distribution of Smad6. Initially, we reported that Smad6 functions as a transcriptional co-repressor through interacting with Hoxc-8 (25Bai S. Shi X. Yang X. Cao X. J. Biol. Chem. 2000; 275: 8267-8270Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In this report, we demonstrated that both Smad6 and Smad7 were co-immunoprecipitated with HDAC-1, which is normally recruited by transcriptional repressors or co-repressors in inhibition of gene transcription. Importantly, we also find that Smad6 is a DNA-binding protein as a partner of Smad6/Hoxc-8 complex. Like other Smads, Smad6 binds to DNA through its MH1 domain, whereas Smad6 MH2 domain recruits HDAC-1. These findings revealed an important functional aspect of I-Smads in the nucleus. Chromatin modification plays a critical role in regulating gene transcription. Acetylation is one of the major modification processes. HATs acetylate lysine residues on histone N-tails, leading to loosening of the chromatin structure and increasing the accessibility of transcriptional factors to the chromosome, whereas HDACs mediate silencing of gene transcription. R-Smads translocate to the nucleus upon BMP or TGF-β stimulation, where they function as transcriptional activators through interacting and recruiting p300/CBP to specific gene promoters (34Wotton D. Lo R.S. Lee S. Massague J. Cell. 1999; 97: 29-39Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar, 41Pearson K.L. Hunter T. Janknecht R. Biochim. Biophys. Acta. 1999; 1489: 354-364Crossref PubMed Scopus (45) Google Scholar). TGIF, a Smad co-repressor in the TGF-β signaling pathway, interacts with HDAC-1 and inhibits target gene expression (34Wotton D. Lo R.S. Lee S. Massague J. Cell. 1999; 97: 29-39Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar). HDACs inhibit gene transcription by tightening the chromosome structure. TSA, an HDAC inhibitor, rescued Smad6- and Hoxc-8-mediated transcription repression. Interaction of Smad6 and Smad7 with HDAC-1 and recruitment of endogenous HDAC activity clearly indicate the nuclear function of I-Smads as transcription repressors or co-repressors. Smad7 does not interact with Hox proteins (25Bai S. Shi X. Yang X. Cao X. J. Biol. Chem. 2000; 275: 8267-8270Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Therefore, Smad7 was not shown as a transcriptional repressor or co-repressor. However, the interaction of Smad7 with HDAC-1 suggests its potential function as a transcriptional repressor or co-repressor in different transcription mechanisms because other cytokines such as TNF-α induce Smad7 expression (49Bitzer M. von Gersdoeff G. Liang D. Dominguez-Rosales A. Beg A.A. Rojkind M. Bottinger E.P. Genes Dev. 2000; 14: 187-197PubMed Google Scholar). Thus, Smad7 could also mediate cross-talk between TGF-β and other signaling pathways (49Bitzer M. von Gersdoeff G. Liang D. Dominguez-Rosales A. Beg A.A. Rojkind M. Bottinger E.P. Genes Dev. 2000; 14: 187-197PubMed Google Scholar, 50Lallemand F. Mazars A. Prunier C. Bertrand F. Kornprost M. Gallea S. Roman-Roman S. Cherqui G. Atfi A. Oncogene. 2001; 20: 879-884Crossref PubMed Scopus (96) Google Scholar). The detailed mechanism of Smad7 in the nucleus remains to be characterized. The finding of Smad6 binding to DNA suggests that all of the Smads are DNA binding proteins. I-Smads contain both MH1 and MH2 domains, which are necessary for full activities of Xenopus Smad6 and Smad7 (51Nakayama T. Berg L.K. Christian J.L. Mech. Dev. 2001; 100: 251-262Crossref PubMed Scopus (29) Google Scholar). The MH2 domain is highly conserved with other Smads, but the MH1 domain is distinct (44Topper J. Cai J. Qiu Y. Anderson K. Xu Y.-Y. Deeds J. Feeley R. Gimeno C. Woolf E. Tayber O. Mays G. Sampson B. Schoen F. Gimbrone Jr., M. Falb D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9314-9319Crossref PubMed Scopus (289) Google Scholar). MH1 domains of R-Smads and Smad4 bind DNA Smad box (GTCT) (41Pearson K.L. Hunter T. Janknecht R. Biochim. Biophys. Acta. 1999; 1489: 354-364Crossref PubMed Scopus (45) Google Scholar, 42Zawel L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar, 43Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.-M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1573) Google Scholar). Like other Smads, Smad6 binds to DNA also through its MH1 domain. The full length of Smad6 does not bind to DNA, whereas, in the presence of Hoxc-8, Smad6 binds to DNA and forms a heterodimer. Importantly, the MH1 domain of Smad6 is required for the Hoxc-8-Smad6 complex formation although it is the MH2 domain of Smad6 that interacts with Hoxc-8. It appears that the MH2 domain of Smad6 masks its MH1 domain DNA binding activity, and that the MH1 and MH2 domains of Smad6 interact reciprocally and inhibit each other's function. Indeed, Smad6 MH1 DNA binding activity decreased when the length of Smad6 truncated MH1 domain extended to MH2 domain. This observation suggests that Hoxc-8 induces Smad6 conformation change, in which Smad6 MH1 domain becomes available to bind to DNA. Phosphorylation of Smad6 could be another way to change its conformation for DNA binding activity, which could also serve as a cross-talk with other signaling pathways. Taken together, we demonstrated a specific interaction of I-Smads with HDACs. As a model, we describe a transcription repression mechanism of Smad6. In response to BMP or TGF-β stimulation, Smad6 interacts with Hoxc-8 and binds to DNA as a heterodimer, which inhibits Smad1-induced gene transcription by recruiting HDACs. Importantly, Smad6 is also a DNA-binding protein. Like other Smads, Smad6 binds to DNA through its MH1 domain, and Smad6 MH2 domain interacts with HDAC-1 and Hoxc-8. Consistent with the observation of I-Smads cellular distribution, our data indicate that I-Smads can function as transcription repressors or co-repressors in the nucleus as antagonistic feedback loop of the TGF-β signaling pathway. We are grateful to Dr. S. L. Schreiber for kindly providing the HDAC-1, HDAC-4, HDAC-5, and HDAC-6 expression vectors; Dr. M. S. Featherstone for HDAC-3 expression plasmid; Dr. T. Kouzarides for GST-CBP (aa 1099–1758) cDNA clone; Dr. Ali H. Brivanlou for Smad6 cDNA; and Dr. Peter ten Dijke for Smad7 cDNA expression vector. We thank Janice Walker for proofreading of the manuscript and Yalei Wu for assistance with graphics formatting.