Requirement of Histone Methyltransferase SMYD3 for Estrogen Receptor-mediated Transcription
2009; Elsevier BV; Volume: 284; Issue: 30 Linguagem: Inglês
10.1074/jbc.m109.021485
ISSN1083-351X
AutoresHyun‐Jung Kim, Kyu Heo, Jeong Hoon Kim, Kyunghwan Kim, Jongkyu Choi, Woojin An,
Tópico(s)Estrogen and related hormone effects
ResumoSMYD3 is a SET domain-containing protein with histone methyltransferase activity on histone H3–K4. Recent studies showed that SMYD3 is frequently overexpressed in different types of cancer cells, but how SMYD3 regulates the development and progression of these malignancies remains unknown. Here, we report the previously unrecognized role of SMYD3 in estrogen receptor (ER)-mediated transcription via its histone methyltransferase activity. We demonstrate that SMYD3 functions as a coactivator of ERα and potentiates ERα activity in response to ligand. SMYD3 directly interacts with the ligand binding domain of ER and is recruited to the proximal promoter regions of ER target genes upon gene induction. Importantly, our chromatin immunoprecipitation analyses provide compelling evidence that SMYD3 is responsible for the accumulation of di- and trimethylation of H3–K4 at the induced ER target genes. Furthermore, RNA interference-directed down-regulation of SMYD3 reveals that SMYD3 is required for ER-regulated gene transcription in estrogen signaling pathway. Thus, our results identify SMYD3 as a new coactivator for ER-mediated transcription, providing a possible link between SMYD3 overexpression and breast cancer. SMYD3 is a SET domain-containing protein with histone methyltransferase activity on histone H3–K4. Recent studies showed that SMYD3 is frequently overexpressed in different types of cancer cells, but how SMYD3 regulates the development and progression of these malignancies remains unknown. Here, we report the previously unrecognized role of SMYD3 in estrogen receptor (ER)-mediated transcription via its histone methyltransferase activity. We demonstrate that SMYD3 functions as a coactivator of ERα and potentiates ERα activity in response to ligand. SMYD3 directly interacts with the ligand binding domain of ER and is recruited to the proximal promoter regions of ER target genes upon gene induction. Importantly, our chromatin immunoprecipitation analyses provide compelling evidence that SMYD3 is responsible for the accumulation of di- and trimethylation of H3–K4 at the induced ER target genes. Furthermore, RNA interference-directed down-regulation of SMYD3 reveals that SMYD3 is required for ER-regulated gene transcription in estrogen signaling pathway. Thus, our results identify SMYD3 as a new coactivator for ER-mediated transcription, providing a possible link between SMYD3 overexpression and breast cancer. Estrogen receptor (ER) 3The abbreviations used are: ERestrogen receptorSMYD3SET and MYND domain containing protein 3AdoMetS-adenosylmethionineEREestrogen response elementHAhemagglutininGSTglutathione S-transferaseHMThistone methyltransferaseChIPchromatin immunoprecipitationshRNAshort hairpin RNANTD+DBDN-terminal and DNA binding domainLBDligand binding domainE2estradiol. 3The abbreviations used are: ERestrogen receptorSMYD3SET and MYND domain containing protein 3AdoMetS-adenosylmethionineEREestrogen response elementHAhemagglutininGSTglutathione S-transferaseHMThistone methyltransferaseChIPchromatin immunoprecipitationshRNAshort hairpin RNANTD+DBDN-terminal and DNA binding domainLBDligand binding domainE2estradiol. α is a member of the nuclear receptor superfamily and the primary biosensor for estrogen (1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6026) Google Scholar, 2McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1638) Google Scholar). Upon activation by estrogen, ER binds to specific DNA sequences called estrogen response elements (EREs) to induce expression of a number of target genes in specific organs, including the female reproductive organs, the central nervous system, and bone (1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6026) Google Scholar, 3Métivier R. Penot G. Hübner M.R. Reid G. Brand H. Kos M. Gannon F. Cell. 2003; 115: 751-763Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar, 4Tsai M.J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2678) Google Scholar). ER is comprised of several structural domains that are highly conserved in the various nuclear receptors: the N-terminal transcription activation domain, the DNA binding domain, the hinge region, and the C-terminal conserved ligand binding domain (5Enmark E. Gustafsson J.A. J. Intern. Med. 1999; 246: 133-138Crossref PubMed Scopus (302) Google Scholar, 6Godowski P.J. Picard D. Yamamoto K.R. Science. 1988; 241: 812-816Crossref PubMed Scopus (167) Google Scholar, 7Yamamoto K.R. Godowski P.J. Picard D. Cold Spring Harbor Symp. Quant. Biol. 1988; 53: 803-811Crossref PubMed Google Scholar). Like other nuclear receptors, the ER collaborates with a number of transcriptional cofactors to effectively modulate transcription of its target genes (3Métivier R. Penot G. Hübner M.R. Reid G. Brand H. Kos M. Gannon F. Cell. 2003; 115: 751-763Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar, 8Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1432) Google Scholar, 9McDonnell D.P. Norris J.D. Science. 2002; 296: 1642-1644Crossref PubMed Scopus (487) Google Scholar, 10Kraus W.L. Wong J. Eur. J. Biochem. 2002; 269: 2275-2283Crossref PubMed Scopus (64) Google Scholar). These cofactors appear to regulate the chromatin configuration in a highly specific manner by controlling nucleosomal rearrangement and histone modifications at the promoter (11McKenna N.J. O'Malley B.W. Endocrinology. 2002; 143: 2461-2465Crossref PubMed Scopus (279) Google Scholar, 12Daujat S. Bauer U.M. Shah V. Turner B. Berger S. Kouzarides T. Curr. Biol. 2002; 12: 2090-2097Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 13Imhof A. Wolffe A.P. Curr. Biol. 1998; 8: R422-R424Abstract Full Text Full Text PDF PubMed Google Scholar). This targeted alteration of chromatin structure allows the transcriptional machinery to access the chromatin DNA and form functional preinitiation complexes, thereby facilitating transcription initiation (14Kim M.Y. Hsiao S.J. Kraus W.L. EMBO J. 2001; 20: 6084-6094Crossref PubMed Scopus (90) Google Scholar, 15Becker P.B. Hörz W. Annu. Rev. Biochem. 2002; 71: 247-273Crossref PubMed Scopus (619) Google Scholar, 16Thomas M.C. Chiang C.M. Crit. Rev. Biochem. Mol. Biol. 2006; 41: 105-178Crossref PubMed Scopus (604) Google Scholar).Two major types of chromatin remodeling have been widely investigated for ER transcription. The remodeling activities include the ATP-dependent chromatin remodeling factors, which alter structure and position of nucleosomes at the promoters of ER target genes. These include proteins such as brahma-related BRG1 (also known as hBRG1 or hSNF2) and BRM, both of which are subunits of the mammalian homologue of the yeast SWI/SNF complex (17Yoshinaga S.K. Peterson C.L. Herskowitz I. Yamamoto K.R. Science. 1992; 258: 1598-1604Crossref PubMed Scopus (412) Google Scholar, 18Huang Z.Q. Li J. Sachs L.M. Cole P.A. Wong J. EMBO J. 2003; 22: 2146-2155Crossref PubMed Scopus (161) Google Scholar). The second class of remodeling factors includes a diverse group of single/multisubunit factors that effect post-translational modifications of the histone tails protruding from the surface of the nucleosome (17Yoshinaga S.K. Peterson C.L. Herskowitz I. Yamamoto K.R. Science. 1992; 258: 1598-1604Crossref PubMed Scopus (412) Google Scholar, 18Huang Z.Q. Li J. Sachs L.M. Cole P.A. Wong J. EMBO J. 2003; 22: 2146-2155Crossref PubMed Scopus (161) Google Scholar, 19Kobayashi Y. Kitamoto T. Masuhiro Y. Watanabe M. Kase T. Metzger D. Yanagisawa J. Kato S. J. Biol. Chem. 2000; 275: 15645-15651Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Among the well known histone-modifying factors acting in ER-mediated transcription are histone acetyltransferases, including p300/CBP and GCN5/PCAF, and histone methyltransferases, including the arginine methyltransferases CARM1 and PRMT1, as well as SET domain lysine methyltransferases such as G9a, RIZ1, NSD1, and MLL2 (10Kraus W.L. Wong J. Eur. J. Biochem. 2002; 269: 2275-2283Crossref PubMed Scopus (64) Google Scholar, 14Kim M.Y. Hsiao S.J. Kraus W.L. EMBO J. 2001; 20: 6084-6094Crossref PubMed Scopus (90) Google Scholar, 20Carling T. Kim K.C. Yang X.H. Gu J. Zhang X.K. Huang S. Mol. Cell. Biol. 2004; 24: 7032-7042Crossref PubMed Scopus (60) Google Scholar, 21Rayasam G.V. Wendling O. Angrand P.O. Mark M. Niederreither K. Song L. Lerouge T. Hager G.L. Chambon P. Losson R. EMBO J. 2003; 22: 3153-3163Crossref PubMed Scopus (270) Google Scholar, 22Lee D.Y. Northrop J.P. Kuo M.H. Stallcup M.R. J. Biol. Chem. 2006; 281: 8476-8485Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). These remodeling factors are recruited to the promoter proximal region of the ER target genes (23Dubik D. Dembinski T.C. Shiu R.P. Cancer Res. 1987; 47: 6517-6521PubMed Google Scholar, 24Jakowlew S.B. Breathnach R. Jeltsch J.M. Masiakowski P. Chambon P. Nucleic Acids Res. 1984; 12: 2861-2878Crossref PubMed Scopus (270) Google Scholar, 25Rochefort H. Capony F. Garcia M. Morisset M. Touïtou I. Vignon F. Prog. Clin. Biol. Res. 1986; 204: 125-140PubMed Google Scholar, 26Foster J.S. Wimalasena J. Mol. Endocrinol. 1996; 10: 488-498Crossref PubMed Scopus (204) Google Scholar) and facilitate either remodeling or removal of the underlying nucleosome, thereby increasing the accessibility of promoter regions to the transcription machinery.Recent studies identified that SMYD3 possesses histone methyltransferase activity responsible for catalyzing methylation of histone H3 at K4 (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar). SMYD3 contains a SET domain, which is crucial for HMT activity, and an MYND-type zinc-finger domain (zf-MYND) domain, which is common to developmental proteins (24Jakowlew S.B. Breathnach R. Jeltsch J.M. Masiakowski P. Chambon P. Nucleic Acids Res. 1984; 12: 2861-2878Crossref PubMed Scopus (270) Google Scholar). Interestingly, misregulation of H3 methylation events upon overexpression of SMYD3 has been shown to correlate with the development and progression of colorectal and hepatocellular carcinoma (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar). In addition to its role in growth of cancer cells, a possible role of SMYD3 in transcription has been supported by its interaction with RNA polymerase II (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar). In fact, a microarray analysis of SMYD3-transfected cells has revealed that a large number of genes were up-regulated >3-fold in the SMYD3-overexpressing cells compared with those in the normal cells (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar). Of special relevance to the present study is that the overexpressed levels of SMYD3 have been observed in breast cancer tissues as well as breast cancer cell lines with associated effects on cancer growth (28Hamamoto R. Silva F.P. Tsuge M. Nishidate T. Katagiri T. Nakamura Y. Furukawa Y. Cancer Sci. 2006; 97: 113-118Crossref PubMed Scopus (232) Google Scholar). ER serves as a sequence-specific transcription factor to regulate a cascade of gene targets whose products mediate the initiation, development, and metastasis of breast cancers. Thus, these results support the idea that SMYD3 might play a functional role in the transactivation of ER-mediated gene transcription in breast cancer cells.As a starting point for the study of transcriptional processes regulated by SMYD3, we checked a possible role of SMYD3 in the ER signaling process. From molecular and cellular studies, we have obtained evidence indicating that SMYD3 is critically involved in ligand-activated, ER-mediated transcription, by methylating histone H3–K4 at the ERE in the promoter regions of target genes. The function of SMYD3 in ER-mediated transcription requires its direct interaction with ER, which in turn allows its recruitment to promoter regions of ER target genes. Down-regulation of SMYD3 expression and concomitant reduction of H3–K4 methylation substantially repressed expression of ER target genes, revealing a major role for SMYD3 as regulator of ER-mediated target gene transcription.DISCUSSIONIn this study, we investigated a possible role of SMYD3 histone methyltransferase in activating ER target genes. The present data demonstrate that (i) SMYD3 physically interacts with ER both in vitro and in vivo, (ii) SMYD3 acts as a transcriptional coactivator of ER that enhances ER-mediated transcription, (iii) SMYD3-imparted transactivation correlates with SMYD3 recruitment and H3–K4 methylation at ER target genes, and (iv) SMYD3 knockdown significantly reduces the ligand-induced expression of ER target genes. These results reveal an essential role of SMYD3 in modulating ER-mediated transcription and provide an example of epigenetic regulation of ER function.The ability of ER to activate transcription requires the repeated cycling of various coregulators onto its target gene promoters in the presence of continuous stimulation by estrogen (3Métivier R. Penot G. Hübner M.R. Reid G. Brand H. Kos M. Gannon F. Cell. 2003; 115: 751-763Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar, 8Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1432) Google Scholar). Currently, at least two mechanistic models have been proposed to describe the function of these coregulators. First, they transmit the signal of ligand-induced ER conformational change to the basal transcription machinery (31Rachez C. Lemon B.D. Suldan Z. Bromleigh V. Gamble M. Näär A.M. Erdjument-Bromage H. Tempst P. Freedman L.P. Nature. 1999; 398: 824-828Crossref PubMed Scopus (629) Google Scholar). Second, they are associated with targeted chromatin remodeling by ER (32Belandia B. Parker M.G. Cell. 2003; 114: 277-280Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Recent biochemical and genetic studies support that methylation of histone H3 at K4 is characteristic to gene activation, and removal of this modification is involved in transcriptional repression (24Jakowlew S.B. Breathnach R. Jeltsch J.M. Masiakowski P. Chambon P. Nucleic Acids Res. 1984; 12: 2861-2878Crossref PubMed Scopus (270) Google Scholar, 33Lachner M. Jenuwein T. Curr. Opin. Cell Biol. 2002; 14: 286-298Crossref PubMed Scopus (688) Google Scholar, 34Rudolph T. Yonezawa M. Lein S. Heidrich K. Kubicek S. Schäfer C. Phalke S. Walther M. Schmidt A. Jenuwein T. Reuter G. Mol. Cell. 2007; 26: 103-115Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The data presented here demonstrate that SMYD3, through its HMT activity, plays a significant role in dictating the transcriptional activity of ER. That the effects of SMYD3 were found to be dependent upon the ability to interact with ER LBD implies that SMYD3 is a functionally important component of estrogen-stimulated ER transcription. Combined with the observation that shRNA-induced silencing of SMYD gene inhibits ER target gene expression, these results argue strongly in favor of SMYD3 as an integral component of the ER response. Although our analyses have been restricted to ER-dependent function of SMYD3, previous studies indicated that activation of other nuclear receptors also involves H3–K4 methylation (35Guccione E. Bassi C. Casadio F. Martinato F. Cesaroni M. Schuchlautz H. Lüscher B. Amati B. Nature. 2007; 449: 933-937Crossref PubMed Scopus (347) Google Scholar, 36Dreijerink K.M. Mulder K.W. Winkler G.S. Höppener J.W. Lips C.J. Timmers H.T. Cancer Res. 2006; 66: 4929-4935Crossref PubMed Scopus (154) Google Scholar). As such, elucidation of a possible role of SMYD3 in the promoter-localized H3–K4 methylation and the consequent activation of transcription at other nuclear receptor target genes is an important issue that warrants further investigation.Many SET domain-containing proteins with HMT activity harbor two conserved amino acid sequence called NHSC and EEL motifs (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar, 30Huang J. Perez-Burgos L. Placek B.J. Sengupta R. Richter M. Dorsey J.A. Kubicek S. Opravil S. Jenuwein T. Berger S.L. Nature. 2006; 444: 629-632Crossref PubMed Scopus (474) Google Scholar). SMYD3 also possesses these motifs within its SET domain, and they have been shown to be critical for SMYD3 enzymatic activity in HMT reaction (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar). Our studies performed with SMYD3 mutants demonstrated that, although the NHSC and EEL motifs within SET domain are essential for SMYD3 HMT activity, these motifs are dispensable for SMYD3 binding to ER (Fig. 3E). Importantly, when these deletion mutants were checked in ER luciferase reporter assays, they failed to show coactivator function (Fig. 3, A–D). A simple interpretation of these results is that HMT activity of SMYD3 is required for its action on the transactivation of reporter gene following estrogen stimulation. These characteristics ascribed to NHSC and EEL motifs fit very well with the generic properties previously assigned to the NHSC/EEL motif-containing SET domains in regulating and mediating the enzymatic activity of HMT proteins (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar, 30Huang J. Perez-Burgos L. Placek B.J. Sengupta R. Richter M. Dorsey J.A. Kubicek S. Opravil S. Jenuwein T. Berger S.L. Nature. 2006; 444: 629-632Crossref PubMed Scopus (474) Google Scholar). These results also suggest that NHSC and EEL motifs of SMYD3 could provide the molecular target for regulation of H3–K4 methylation-dependent transcriptional responses by ER. More thorough domain mapping and mutagenesis experiments will be required to provide further insights into the ER-SMYD3 interactions, which facilitate SMYD3 recruitment and H3–K4 methylation in ER-mediated transcription.We have shown that ER promoter occupancy upon E2 treatment coincides with promoter recruitment of SMYD3 and appearance of di-/trimethyl H3–K4 (Fig. 4, A–C). This similar timing of ER occupancy and SMYD3 recruitment strongly supporting that H3–K4 methylation per se endows coactivator properties of SMYD3 in regulating ER-mediated transcription. In further support of a regulatory role of SMYD3-mediated H3–K4 methylation, SMYD3 depletion showed a significant effect on the level of di-/trimethylation, but not monomethylation, of H3–K4 at the promoter of pS2 gene upon E2 treatment (Fig. 6). However, it is also possible that SMYD3 augments the activities of other transcription components at the initial stage of gene induction. In fact, a recent study demonstrated that SMYD3 interacts with an RNA helicase to form a complex with RNA polymerase II (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar). Hence, SMYD3 could act as a “bridge” protein that mediates functional interaction between ER and RNA polymerase II to coordinate the tightly integrated processes of chromatin remodeling and transcription in ER-driven transcription (31Rachez C. Lemon B.D. Suldan Z. Bromleigh V. Gamble M. Näär A.M. Erdjument-Bromage H. Tempst P. Freedman L.P. Nature. 1999; 398: 824-828Crossref PubMed Scopus (629) Google Scholar, 37Torchia J. Glass C. Rosenfeld M.G. Curr. Opin. Cell Biol. 1998; 10: 373-383Crossref PubMed Scopus (509) Google Scholar). Furthermore, increasing evidence suggests that coactivator proteins such as p300, PRMT1, and CARM1 act together as potential regulators of ER-induced transcription (2McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1638) Google Scholar, 12Daujat S. Bauer U.M. Shah V. Turner B. Berger S. Kouzarides T. Curr. Biol. 2002; 12: 2090-2097Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 18Huang Z.Q. Li J. Sachs L.M. Cole P.A. Wong J. EMBO J. 2003; 22: 2146-2155Crossref PubMed Scopus (161) Google Scholar, 38Chen D. Huang S.M. Stallcup M.R. J. Biol. Chem. 2000; 275: 40810-40816Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 39Spencer T.E. Jenster G. Burcin M.M. Allis C.D. Zhou J. Mizzen C.A. McKenna N.J. Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Nature. 1997; 389: 194-198Crossref PubMed Scopus (1054) Google Scholar). Thus, it is possible that a distinct group of coactivators play a crucial role in orchestrating ER-mediated transcription. In this respect, it will be interesting to determine the relative contribution of SMYD3, together with other coactivators that associate with ER target gene expression.In conclusion, the analysis of SMYD3 described here establishes its role in ER-mediated transcription as a coactivator. H3–K4 methylation appears to be critical for SMYD3 function in ER transcription. Identifying cofactors that influence ER has been important in the development of effective therapies and prevention of breast cancer. Because our studies provide clues as to the requirement of SMYD3 HMT activity for its function, the development of HMT inhibitors may be of therapeutic benefit in modulating SMYD3 action in ER transcription. Estrogen receptor (ER) 3The abbreviations used are: ERestrogen receptorSMYD3SET and MYND domain containing protein 3AdoMetS-adenosylmethionineEREestrogen response elementHAhemagglutininGSTglutathione S-transferaseHMThistone methyltransferaseChIPchromatin immunoprecipitationshRNAshort hairpin RNANTD+DBDN-terminal and DNA binding domainLBDligand binding domainE2estradiol. 3The abbreviations used are: ERestrogen receptorSMYD3SET and MYND domain containing protein 3AdoMetS-adenosylmethionineEREestrogen response elementHAhemagglutininGSTglutathione S-transferaseHMThistone methyltransferaseChIPchromatin immunoprecipitationshRNAshort hairpin RNANTD+DBDN-terminal and DNA binding domainLBDligand binding domainE2estradiol. α is a member of the nuclear receptor superfamily and the primary biosensor for estrogen (1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6026) Google Scholar, 2McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1638) Google Scholar). Upon activation by estrogen, ER binds to specific DNA sequences called estrogen response elements (EREs) to induce expression of a number of target genes in specific organs, including the female reproductive organs, the central nervous system, and bone (1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6026) Google Scholar, 3Métivier R. Penot G. Hübner M.R. Reid G. Brand H. Kos M. Gannon F. Cell. 2003; 115: 751-763Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar, 4Tsai M.J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2678) Google Scholar). ER is comprised of several structural domains that are highly conserved in the various nuclear receptors: the N-terminal transcription activation domain, the DNA binding domain, the hinge region, and the C-terminal conserved ligand binding domain (5Enmark E. Gustafsson J.A. J. Intern. Med. 1999; 246: 133-138Crossref PubMed Scopus (302) Google Scholar, 6Godowski P.J. Picard D. Yamamoto K.R. Science. 1988; 241: 812-816Crossref PubMed Scopus (167) Google Scholar, 7Yamamoto K.R. Godowski P.J. Picard D. Cold Spring Harbor Symp. Quant. Biol. 1988; 53: 803-811Crossref PubMed Google Scholar). Like other nuclear receptors, the ER collaborates with a number of transcriptional cofactors to effectively modulate transcription of its target genes (3Métivier R. Penot G. Hübner M.R. Reid G. Brand H. Kos M. Gannon F. Cell. 2003; 115: 751-763Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar, 8Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1432) Google Scholar, 9McDonnell D.P. Norris J.D. Science. 2002; 296: 1642-1644Crossref PubMed Scopus (487) Google Scholar, 10Kraus W.L. Wong J. Eur. J. Biochem. 2002; 269: 2275-2283Crossref PubMed Scopus (64) Google Scholar). These cofactors appear to regulate the chromatin configuration in a highly specific manner by controlling nucleosomal rearrangement and histone modifications at the promoter (11McKenna N.J. O'Malley B.W. Endocrinology. 2002; 143: 2461-2465Crossref PubMed Scopus (279) Google Scholar, 12Daujat S. Bauer U.M. Shah V. Turner B. Berger S. Kouzarides T. Curr. Biol. 2002; 12: 2090-2097Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 13Imhof A. Wolffe A.P. Curr. Biol. 1998; 8: R422-R424Abstract Full Text Full Text PDF PubMed Google Scholar). This targeted alteration of chromatin structure allows the transcriptional machinery to access the chromatin DNA and form functional preinitiation complexes, thereby facilitating transcription initiation (14Kim M.Y. Hsiao S.J. Kraus W.L. EMBO J. 2001; 20: 6084-6094Crossref PubMed Scopus (90) Google Scholar, 15Becker P.B. Hörz W. Annu. Rev. Biochem. 2002; 71: 247-273Crossref PubMed Scopus (619) Google Scholar, 16Thomas M.C. Chiang C.M. Crit. Rev. Biochem. Mol. Biol. 2006; 41: 105-178Crossref PubMed Scopus (604) Google Scholar). estrogen receptor SET and MYND domain containing protein 3 S-adenosylmethionine estrogen response element hemagglutinin glutathione S-transferase histone methyltransferase chromatin immunoprecipitation short hairpin RNA N-terminal and DNA binding domain ligand binding domain estradiol. estrogen receptor SET and MYND domain containing protein 3 S-adenosylmethionine estrogen response element hemagglutinin glutathione S-transferase histone methyltransferase chromatin immunoprecipitation short hairpin RNA N-terminal and DNA binding domain ligand binding domain estradiol. Two major types of chromatin remodeling have been widely investigated for ER transcription. The remodeling activities include the ATP-dependent chromatin remodeling factors, which alter structure and position of nucleosomes at the promoters of ER target genes. These include proteins such as brahma-related BRG1 (also known as hBRG1 or hSNF2) and BRM, both of which are subunits of the mammalian homologue of the yeast SWI/SNF complex (17Yoshinaga S.K. Peterson C.L. Herskowitz I. Yamamoto K.R. Science. 1992; 258: 1598-1604Crossref PubMed Scopus (412) Google Scholar, 18Huang Z.Q. Li J. Sachs L.M. Cole P.A. Wong J. EMBO J. 2003; 22: 2146-2155Crossref PubMed Scopus (161) Google Scholar). The second class of remodeling factors includes a diverse group of single/multisubunit factors that effect post-translational modifications of the histone tails protruding from the surface of the nucleosome (17Yoshinaga S.K. Peterson C.L. Herskowitz I. Yamamoto K.R. Science. 1992; 258: 1598-1604Crossref PubMed Scopus (412) Google Scholar, 18Huang Z.Q. Li J. Sachs L.M. Cole P.A. Wong J. EMBO J. 2003; 22: 2146-2155Crossref PubMed Scopus (161) Google Scholar, 19Kobayashi Y. Kitamoto T. Masuhiro Y. Watanabe M. Kase T. Metzger D. Yanagisawa J. Kato S. J. Biol. Chem. 2000; 275: 15645-15651Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Among the well known histone-modifying factors acting in ER-mediated transcription are histone acetyltransferases, including p300/CBP and GCN5/PCAF, and histone methyltransferases, including the arginine methyltransferases CARM1 and PRMT1, as well as SET domain lysine methyltransferases such as G9a, RIZ1, NSD1, and MLL2 (10Kraus W.L. Wong J. Eur. J. Biochem. 2002; 269: 2275-2283Crossref PubMed Scopus (64) Google Scholar, 14Kim M.Y. Hsiao S.J. Kraus W.L. EMBO J. 2001; 20: 6084-6094Crossref PubMed Scopus (90) Google Scholar, 20Carling T. Kim K.C. Yang X.H. Gu J. Zhang X.K. Huang S. Mol. Cell. Biol. 2004; 24: 7032-7042Crossref PubMed Scopus (60) Google Scholar, 21Rayasam G.V. Wendling O. Angrand P.O. Mark M. Niederreither K. Song L. Lerouge T. Hager G.L. Chambon P. Losson R. EMBO J. 2003; 22: 3153-3163Crossref PubMed Scopus (270) Google Scholar, 22Lee D.Y. Northrop J.P. Kuo M.H. Stallcup M.R. J. Biol. Chem. 2006; 281: 8476-8485Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). These remodeling factors are recruited to the promoter proximal region of the ER target genes (23Dubik D. Dembinski T.C. Shiu R.P. Cancer Res. 1987; 47: 6517-6521PubMed Google Scholar, 24Jakowlew S.B. Breathnach R. Jeltsch J.M. Masiakowski P. Chambon P. Nucleic Acids Res. 1984; 12: 2861-2878Crossref PubMed Scopus (270) Google Scholar, 25Rochefort H. Capony F. Garcia M. Morisset M. Touïtou I. Vignon F. Prog. Clin. Biol. Res. 1986; 204: 125-140PubMed Google Scholar, 26Foster J.S. Wimalasena J. Mol. Endocrinol. 1996; 10: 488-498Crossref PubMed Scopus (204) Google Scholar) and facilitate either remodeling or removal of the underlying nucleosome, thereby increasing the accessibility of promoter regions to the transcription machinery. Recent studies identified that SMYD3 possesses histone methyltransferase activity responsible for catalyzing methylation of histone H3 at K4 (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar). SMYD3 contains a SET domain, which is crucial for HMT activity, and an MYND-type zinc-finger domain (zf-MYND) domain, which is common to developmental proteins (24Jakowlew S.B. Breathnach R. Jeltsch J.M. Masiakowski P. Chambon P. Nucleic Acids Res. 1984; 12: 2861-2878Crossref PubMed Scopus (270) Google Scholar). Interestingly, misregulation of H3 methylation events upon overexpression of SMYD3 has been shown to correlate with the development and progression of colorectal and hepatocellular carcinoma (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar). In addition to its role in growth of cancer cells, a possible role of SMYD3 in transcription has been supported by its interaction with RNA polymerase II (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar). In fact, a microarray analysis of SMYD3-transfected cells has revealed that a large number of genes were up-regulated >3-fold in the SMYD3-overexpressing cells compared with those in the normal cells (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar). Of special relevance to the present study is that the overexpressed levels of SMYD3 have been observed in breast cancer tissues as well as breast cancer cell lines with associated effects on cancer growth (28Hamamoto R. Silva F.P. Tsuge M. Nishidate T. Katagiri T. Nakamura Y. Furukawa Y. Cancer Sci. 2006; 97: 113-118Crossref PubMed Scopus (232) Google Scholar). ER serves as a sequence-specific transcription factor to regulate a cascade of gene targets whose products mediate the initiation, development, and metastasis of breast cancers. Thus, these results support the idea that SMYD3 might play a functional role in the transactivation of ER-mediated gene transcription in breast cancer cells. As a starting point for the study of transcriptional processes regulated by SMYD3, we checked a possible role of SMYD3 in the ER signaling process. From molecular and cellular studies, we have obtained evidence indicating that SMYD3 is critically involved in ligand-activated, ER-mediated transcription, by methylating histone H3–K4 at the ERE in the promoter regions of target genes. The function of SMYD3 in ER-mediated transcription requires its direct interaction with ER, which in turn allows its recruitment to promoter regions of ER target genes. Down-regulation of SMYD3 expression and concomitant reduction of H3–K4 methylation substantially repressed expression of ER target genes, revealing a major role for SMYD3 as regulator of ER-mediated target gene transcription. DISCUSSIONIn this study, we investigated a possible role of SMYD3 histone methyltransferase in activating ER target genes. The present data demonstrate that (i) SMYD3 physically interacts with ER both in vitro and in vivo, (ii) SMYD3 acts as a transcriptional coactivator of ER that enhances ER-mediated transcription, (iii) SMYD3-imparted transactivation correlates with SMYD3 recruitment and H3–K4 methylation at ER target genes, and (iv) SMYD3 knockdown significantly reduces the ligand-induced expression of ER target genes. These results reveal an essential role of SMYD3 in modulating ER-mediated transcription and provide an example of epigenetic regulation of ER function.The ability of ER to activate transcription requires the repeated cycling of various coregulators onto its target gene promoters in the presence of continuous stimulation by estrogen (3Métivier R. Penot G. Hübner M.R. Reid G. Brand H. Kos M. Gannon F. Cell. 2003; 115: 751-763Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar, 8Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1432) Google Scholar). Currently, at least two mechanistic models have been proposed to describe the function of these coregulators. First, they transmit the signal of ligand-induced ER conformational change to the basal transcription machinery (31Rachez C. Lemon B.D. Suldan Z. Bromleigh V. Gamble M. Näär A.M. Erdjument-Bromage H. Tempst P. Freedman L.P. Nature. 1999; 398: 824-828Crossref PubMed Scopus (629) Google Scholar). Second, they are associated with targeted chromatin remodeling by ER (32Belandia B. Parker M.G. Cell. 2003; 114: 277-280Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Recent biochemical and genetic studies support that methylation of histone H3 at K4 is characteristic to gene activation, and removal of this modification is involved in transcriptional repression (24Jakowlew S.B. Breathnach R. Jeltsch J.M. Masiakowski P. Chambon P. Nucleic Acids Res. 1984; 12: 2861-2878Crossref PubMed Scopus (270) Google Scholar, 33Lachner M. Jenuwein T. Curr. Opin. Cell Biol. 2002; 14: 286-298Crossref PubMed Scopus (688) Google Scholar, 34Rudolph T. Yonezawa M. Lein S. Heidrich K. Kubicek S. Schäfer C. Phalke S. Walther M. Schmidt A. Jenuwein T. Reuter G. Mol. Cell. 2007; 26: 103-115Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The data presented here demonstrate that SMYD3, through its HMT activity, plays a significant role in dictating the transcriptional activity of ER. That the effects of SMYD3 were found to be dependent upon the ability to interact with ER LBD implies that SMYD3 is a functionally important component of estrogen-stimulated ER transcription. Combined with the observation that shRNA-induced silencing of SMYD gene inhibits ER target gene expression, these results argue strongly in favor of SMYD3 as an integral component of the ER response. Although our analyses have been restricted to ER-dependent function of SMYD3, previous studies indicated that activation of other nuclear receptors also involves H3–K4 methylation (35Guccione E. Bassi C. Casadio F. Martinato F. Cesaroni M. Schuchlautz H. Lüscher B. Amati B. Nature. 2007; 449: 933-937Crossref PubMed Scopus (347) Google Scholar, 36Dreijerink K.M. Mulder K.W. Winkler G.S. Höppener J.W. Lips C.J. Timmers H.T. Cancer Res. 2006; 66: 4929-4935Crossref PubMed Scopus (154) Google Scholar). As such, elucidation of a possible role of SMYD3 in the promoter-localized H3–K4 methylation and the consequent activation of transcription at other nuclear receptor target genes is an important issue that warrants further investigation.Many SET domain-containing proteins with HMT activity harbor two conserved amino acid sequence called NHSC and EEL motifs (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar, 30Huang J. Perez-Burgos L. Placek B.J. Sengupta R. Richter M. Dorsey J.A. Kubicek S. Opravil S. Jenuwein T. Berger S.L. Nature. 2006; 444: 629-632Crossref PubMed Scopus (474) Google Scholar). SMYD3 also possesses these motifs within its SET domain, and they have been shown to be critical for SMYD3 enzymatic activity in HMT reaction (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar). Our studies performed with SMYD3 mutants demonstrated that, although the NHSC and EEL motifs within SET domain are essential for SMYD3 HMT activity, these motifs are dispensable for SMYD3 binding to ER (Fig. 3E). Importantly, when these deletion mutants were checked in ER luciferase reporter assays, they failed to show coactivator function (Fig. 3, A–D). A simple interpretation of these results is that HMT activity of SMYD3 is required for its action on the transactivation of reporter gene following estrogen stimulation. These characteristics ascribed to NHSC and EEL motifs fit very well with the generic properties previously assigned to the NHSC/EEL motif-containing SET domains in regulating and mediating the enzymatic activity of HMT proteins (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar, 30Huang J. Perez-Burgos L. Placek B.J. Sengupta R. Richter M. Dorsey J.A. Kubicek S. Opravil S. Jenuwein T. Berger S.L. Nature. 2006; 444: 629-632Crossref PubMed Scopus (474) Google Scholar). These results also suggest that NHSC and EEL motifs of SMYD3 could provide the molecular target for regulation of H3–K4 methylation-dependent transcriptional responses by ER. More thorough domain mapping and mutagenesis experiments will be required to provide further insights into the ER-SMYD3 interactions, which facilitate SMYD3 recruitment and H3–K4 methylation in ER-mediated transcription.We have shown that ER promoter occupancy upon E2 treatment coincides with promoter recruitment of SMYD3 and appearance of di-/trimethyl H3–K4 (Fig. 4, A–C). This similar timing of ER occupancy and SMYD3 recruitment strongly supporting that H3–K4 methylation per se endows coactivator properties of SMYD3 in regulating ER-mediated transcription. In further support of a regulatory role of SMYD3-mediated H3–K4 methylation, SMYD3 depletion showed a significant effect on the level of di-/trimethylation, but not monomethylation, of H3–K4 at the promoter of pS2 gene upon E2 treatment (Fig. 6). However, it is also possible that SMYD3 augments the activities of other transcription components at the initial stage of gene induction. In fact, a recent study demonstrated that SMYD3 interacts with an RNA helicase to form a complex with RNA polymerase II (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar). Hence, SMYD3 could act as a “bridge” protein that mediates functional interaction between ER and RNA polymerase II to coordinate the tightly integrated processes of chromatin remodeling and transcription in ER-driven transcription (31Rachez C. Lemon B.D. Suldan Z. Bromleigh V. Gamble M. Näär A.M. Erdjument-Bromage H. Tempst P. Freedman L.P. Nature. 1999; 398: 824-828Crossref PubMed Scopus (629) Google Scholar, 37Torchia J. Glass C. Rosenfeld M.G. Curr. Opin. Cell Biol. 1998; 10: 373-383Crossref PubMed Scopus (509) Google Scholar). Furthermore, increasing evidence suggests that coactivator proteins such as p300, PRMT1, and CARM1 act together as potential regulators of ER-induced transcription (2McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1638) Google Scholar, 12Daujat S. Bauer U.M. Shah V. Turner B. Berger S. Kouzarides T. Curr. Biol. 2002; 12: 2090-2097Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 18Huang Z.Q. Li J. Sachs L.M. Cole P.A. Wong J. EMBO J. 2003; 22: 2146-2155Crossref PubMed Scopus (161) Google Scholar, 38Chen D. Huang S.M. Stallcup M.R. J. Biol. Chem. 2000; 275: 40810-40816Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 39Spencer T.E. Jenster G. Burcin M.M. Allis C.D. Zhou J. Mizzen C.A. McKenna N.J. Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Nature. 1997; 389: 194-198Crossref PubMed Scopus (1054) Google Scholar). Thus, it is possible that a distinct group of coactivators play a crucial role in orchestrating ER-mediated transcription. In this respect, it will be interesting to determine the relative contribution of SMYD3, together with other coactivators that associate with ER target gene expression.In conclusion, the analysis of SMYD3 described here establishes its role in ER-mediated transcription as a coactivator. H3–K4 methylation appears to be critical for SMYD3 function in ER transcription. Identifying cofactors that influence ER has been important in the development of effective therapies and prevention of breast cancer. Because our studies provide clues as to the requirement of SMYD3 HMT activity for its function, the development of HMT inhibitors may be of therapeutic benefit in modulating SMYD3 action in ER transcription. In this study, we investigated a possible role of SMYD3 histone methyltransferase in activating ER target genes. The present data demonstrate that (i) SMYD3 physically interacts with ER both in vitro and in vivo, (ii) SMYD3 acts as a transcriptional coactivator of ER that enhances ER-mediated transcription, (iii) SMYD3-imparted transactivation correlates with SMYD3 recruitment and H3–K4 methylation at ER target genes, and (iv) SMYD3 knockdown significantly reduces the ligand-induced expression of ER target genes. These results reveal an essential role of SMYD3 in modulating ER-mediated transcription and provide an example of epigenetic regulation of ER function. The ability of ER to activate transcription requires the repeated cycling of various coregulators onto its target gene promoters in the presence of continuous stimulation by estrogen (3Métivier R. Penot G. Hübner M.R. Reid G. Brand H. Kos M. Gannon F. Cell. 2003; 115: 751-763Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar, 8Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1432) Google Scholar). Currently, at least two mechanistic models have been proposed to describe the function of these coregulators. First, they transmit the signal of ligand-induced ER conformational change to the basal transcription machinery (31Rachez C. Lemon B.D. Suldan Z. Bromleigh V. Gamble M. Näär A.M. Erdjument-Bromage H. Tempst P. Freedman L.P. Nature. 1999; 398: 824-828Crossref PubMed Scopus (629) Google Scholar). Second, they are associated with targeted chromatin remodeling by ER (32Belandia B. Parker M.G. Cell. 2003; 114: 277-280Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Recent biochemical and genetic studies support that methylation of histone H3 at K4 is characteristic to gene activation, and removal of this modification is involved in transcriptional repression (24Jakowlew S.B. Breathnach R. Jeltsch J.M. Masiakowski P. Chambon P. Nucleic Acids Res. 1984; 12: 2861-2878Crossref PubMed Scopus (270) Google Scholar, 33Lachner M. Jenuwein T. Curr. Opin. Cell Biol. 2002; 14: 286-298Crossref PubMed Scopus (688) Google Scholar, 34Rudolph T. Yonezawa M. Lein S. Heidrich K. Kubicek S. Schäfer C. Phalke S. Walther M. Schmidt A. Jenuwein T. Reuter G. Mol. Cell. 2007; 26: 103-115Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The data presented here demonstrate that SMYD3, through its HMT activity, plays a significant role in dictating the transcriptional activity of ER. That the effects of SMYD3 were found to be dependent upon the ability to interact with ER LBD implies that SMYD3 is a functionally important component of estrogen-stimulated ER transcription. Combined with the observation that shRNA-induced silencing of SMYD gene inhibits ER target gene expression, these results argue strongly in favor of SMYD3 as an integral component of the ER response. Although our analyses have been restricted to ER-dependent function of SMYD3, previous studies indicated that activation of other nuclear receptors also involves H3–K4 methylation (35Guccione E. Bassi C. Casadio F. Martinato F. Cesaroni M. Schuchlautz H. Lüscher B. Amati B. Nature. 2007; 449: 933-937Crossref PubMed Scopus (347) Google Scholar, 36Dreijerink K.M. Mulder K.W. Winkler G.S. Höppener J.W. Lips C.J. Timmers H.T. Cancer Res. 2006; 66: 4929-4935Crossref PubMed Scopus (154) Google Scholar). As such, elucidation of a possible role of SMYD3 in the promoter-localized H3–K4 methylation and the consequent activation of transcription at other nuclear receptor target genes is an important issue that warrants further investigation. Many SET domain-containing proteins with HMT activity harbor two conserved amino acid sequence called NHSC and EEL motifs (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar, 30Huang J. Perez-Burgos L. Placek B.J. Sengupta R. Richter M. Dorsey J.A. Kubicek S. Opravil S. Jenuwein T. Berger S.L. Nature. 2006; 444: 629-632Crossref PubMed Scopus (474) Google Scholar). SMYD3 also possesses these motifs within its SET domain, and they have been shown to be critical for SMYD3 enzymatic activity in HMT reaction (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar). Our studies performed with SMYD3 mutants demonstrated that, although the NHSC and EEL motifs within SET domain are essential for SMYD3 HMT activity, these motifs are dispensable for SMYD3 binding to ER (Fig. 3E). Importantly, when these deletion mutants were checked in ER luciferase reporter assays, they failed to show coactivator function (Fig. 3, A–D). A simple interpretation of these results is that HMT activity of SMYD3 is required for its action on the transactivation of reporter gene following estrogen stimulation. These characteristics ascribed to NHSC and EEL motifs fit very well with the generic properties previously assigned to the NHSC/EEL motif-containing SET domains in regulating and mediating the enzymatic activity of HMT proteins (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar, 30Huang J. Perez-Burgos L. Placek B.J. Sengupta R. Richter M. Dorsey J.A. Kubicek S. Opravil S. Jenuwein T. Berger S.L. Nature. 2006; 444: 629-632Crossref PubMed Scopus (474) Google Scholar). These results also suggest that NHSC and EEL motifs of SMYD3 could provide the molecular target for regulation of H3–K4 methylation-dependent transcriptional responses by ER. More thorough domain mapping and mutagenesis experiments will be required to provide further insights into the ER-SMYD3 interactions, which facilitate SMYD3 recruitment and H3–K4 methylation in ER-mediated transcription. We have shown that ER promoter occupancy upon E2 treatment coincides with promoter recruitment of SMYD3 and appearance of di-/trimethyl H3–K4 (Fig. 4, A–C). This similar timing of ER occupancy and SMYD3 recruitment strongly supporting that H3–K4 methylation per se endows coactivator properties of SMYD3 in regulating ER-mediated transcription. In further support of a regulatory role of SMYD3-mediated H3–K4 methylation, SMYD3 depletion showed a significant effect on the level of di-/trimethylation, but not monomethylation, of H3–K4 at the promoter of pS2 gene upon E2 treatment (Fig. 6). However, it is also possible that SMYD3 augments the activities of other transcription components at the initial stage of gene induction. In fact, a recent study demonstrated that SMYD3 interacts with an RNA helicase to form a complex with RNA polymerase II (27Hamamoto R. Furukawa Y. Morita M. Iimura Y. Silva F.P. Li M. Yagyu R. Nakamura Y. Nat. Cell Biol. 2004; 6: 731-740Crossref PubMed Scopus (615) Google Scholar). Hence, SMYD3 could act as a “bridge” protein that mediates functional interaction between ER and RNA polymerase II to coordinate the tightly integrated processes of chromatin remodeling and transcription in ER-driven transcription (31Rachez C. Lemon B.D. Suldan Z. Bromleigh V. Gamble M. Näär A.M. Erdjument-Bromage H. Tempst P. Freedman L.P. Nature. 1999; 398: 824-828Crossref PubMed Scopus (629) Google Scholar, 37Torchia J. Glass C. Rosenfeld M.G. Curr. Opin. Cell Biol. 1998; 10: 373-383Crossref PubMed Scopus (509) Google Scholar). Furthermore, increasing evidence suggests that coactivator proteins such as p300, PRMT1, and CARM1 act together as potential regulators of ER-induced transcription (2McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1638) Google Scholar, 12Daujat S. Bauer U.M. Shah V. Turner B. Berger S. Kouzarides T. Curr. Biol. 2002; 12: 2090-2097Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 18Huang Z.Q. Li J. Sachs L.M. Cole P.A. Wong J. EMBO J. 2003; 22: 2146-2155Crossref PubMed Scopus (161) Google Scholar, 38Chen D. Huang S.M. Stallcup M.R. J. Biol. Chem. 2000; 275: 40810-40816Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 39Spencer T.E. Jenster G. Burcin M.M. Allis C.D. Zhou J. Mizzen C.A. McKenna N.J. Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Nature. 1997; 389: 194-198Crossref PubMed Scopus (1054) Google Scholar). Thus, it is possible that a distinct group of coactivators play a crucial role in orchestrating ER-mediated transcription. In this respect, it will be interesting to determine the relative contribution of SMYD3, together with other coactivators that associate with ER target gene expression. In conclusion, the analysis of SMYD3 described here establishes its role in ER-mediated transcription as a coactivator. H3–K4 methylation appears to be critical for SMYD3 function in ER transcription. Identifying cofactors that influence ER has been important in the development of effective therapies and prevention of breast cancer. Because our studies provide clues as to the requirement of SMYD3 HMT activity for its function, the development of HMT inhibitors may be of therapeutic benefit in modulating SMYD3 action in ER transcription. We thank C. M. Chiang for ER baculovirus. We are also grateful to M. R. Stallcup for antibodies and primers for reverse transcription-PCR and ChIP. We acknowledge M. R. Stallcup for critical reading of the manuscript.
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