The Metastasis-associated Proteins 1 and 2 Form Distinct Protein Complexes with Histone Deacetylase Activity
2003; Elsevier BV; Volume: 278; Issue: 43 Linguagem: Inglês
10.1074/jbc.m302955200
ISSN1083-351X
Autores Tópico(s)Protein Degradation and Inhibitors
ResumoThe metastasis-associated protein MTA1 has been shown to express differentially to high levels in metastatic cells. MTA2, which is homologous to MTA1, is a component of the NuRD ATP-dependent chromatin remodeling and histone deacetylase complex. Here we report evidence that although both human MTA1 and MTA2 repress transcription specifically, are located in the nucleus, and contain associated histone deacetylase activity, they exist in two biochemically distinct protein complexes and may perform different functions pertaining to tumor metastasis. Specifically, both MTA1 and MTA2 complexes exert histone deacetylase activity. However, the MTA1 complex contained HDAC1/2, RbAp46/48, and MBD3, but not Sin3 or Mi2, two important components of the MTA2 complex. Moreover, the MTA2 complex is similar to the HDAC1 complex, suggesting a housekeeping role of the MTA2 complex. The MTA1 complex could be further separated, resulting in a core MTA1-HDAC complex, showing that the histone deacetylase activity and transcriptional repression activity were integral properties of the MTA1 complex. Finally, MTA1, unlike MTA2, did not interact with the pleotropic transcription factor YY1 or the immunophilin FKBP25. We suggest that MTA1 associates with a different set of transcription factors from MTA2 and that this property may contribute to the metastatic potential of cells overexpressing MTA1. We also report the finding of human MTA3, which is highly homologous to both MTA1 and MTA2. However, MTA3 does not repress transcription to a significant level and appears to have a diffused pattern of subcellular localization, suggesting a biological role distinct from that of the other two MTA proteins. The metastasis-associated protein MTA1 has been shown to express differentially to high levels in metastatic cells. MTA2, which is homologous to MTA1, is a component of the NuRD ATP-dependent chromatin remodeling and histone deacetylase complex. Here we report evidence that although both human MTA1 and MTA2 repress transcription specifically, are located in the nucleus, and contain associated histone deacetylase activity, they exist in two biochemically distinct protein complexes and may perform different functions pertaining to tumor metastasis. Specifically, both MTA1 and MTA2 complexes exert histone deacetylase activity. However, the MTA1 complex contained HDAC1/2, RbAp46/48, and MBD3, but not Sin3 or Mi2, two important components of the MTA2 complex. Moreover, the MTA2 complex is similar to the HDAC1 complex, suggesting a housekeeping role of the MTA2 complex. The MTA1 complex could be further separated, resulting in a core MTA1-HDAC complex, showing that the histone deacetylase activity and transcriptional repression activity were integral properties of the MTA1 complex. Finally, MTA1, unlike MTA2, did not interact with the pleotropic transcription factor YY1 or the immunophilin FKBP25. We suggest that MTA1 associates with a different set of transcription factors from MTA2 and that this property may contribute to the metastatic potential of cells overexpressing MTA1. We also report the finding of human MTA3, which is highly homologous to both MTA1 and MTA2. However, MTA3 does not repress transcription to a significant level and appears to have a diffused pattern of subcellular localization, suggesting a biological role distinct from that of the other two MTA proteins. Metastasis represents one of the fundamental differences between benign and malignant tumors and poses a major obstacle in effective cancer treatment. The metastasis-associated gene 1 (MTA1) is closely associated with cancer metastasis. The rat mta1 gene was first identified using differential cDNA library screening techniques in a mammary adenocarcinoma metastatic system (1Pencil S.D. Toh Y. Nicolson G.L. Breast Cancer Res. Treat. 1993; 25: 165-174Crossref PubMed Scopus (91) Google Scholar, 2Toh Y. Pencil S.D. Nicolson G.L. J. Biol. Chem. 1994; 269: 22958-22963Abstract Full Text PDF PubMed Google Scholar, 3Toh Y. Pencil S.D. Nicolson G.L. Gene. 1995; 159: 97-104Crossref PubMed Scopus (101) Google Scholar). The expression levels of MTA1 are elevated in human metastatic breast cell lines (2Toh Y. Pencil S.D. Nicolson G.L. J. Biol. Chem. 1994; 269: 22958-22963Abstract Full Text PDF PubMed Google Scholar) and metastatic cancer tissues, such as breast, colorectal, gastric, and esophageal carcinomas (2Toh Y. Pencil S.D. Nicolson G.L. J. Biol. Chem. 1994; 269: 22958-22963Abstract Full Text PDF PubMed Google Scholar, 4Toh Y. Oki E. Oda S. Tokunaga E. Ohno S. Maehara Y. Nicolson G.L. Sugimachi K. Intl. J. Cancer. 1997; 74: 459-463Crossref PubMed Scopus (162) Google Scholar, 5Toh Y. Kuwano H. Mori M. Nicolson G.L. Sugimachi K. Br. J. Cancer. 1999; 79: 1723-1726Crossref PubMed Scopus (113) Google Scholar). Overexpression of MTA1 correlates with enhancement of the ability of human breast cancer cells to invade and to grow in an anchorage-independent manner (6Mazumdar A. Wang R.A. Mishra S.K. Adam L. Bagheri-Yarmand R. Mandal M. Vadlamudi R.K. Kumar R. Nat. Cell Biol. 2001; 3: 30-37Crossref PubMed Scopus (332) Google Scholar). In patients, colorectal and gastric carcinomas overexpressing MTA1 show deeper invasion and higher rates of penetration into lymph nodes (4Toh Y. Oki E. Oda S. Tokunaga E. Ohno S. Maehara Y. Nicolson G.L. Sugimachi K. Intl. J. Cancer. 1997; 74: 459-463Crossref PubMed Scopus (162) Google Scholar). In culture, administration of antisense phosphorothioate oligonucleotides specific for MTA1 inhibits the rapid growth of human breast cancer cells that express high levels of MTA1 compared with normal breast epithelial cells (7Nawa A. Nishimori K. Lin P. Maki Y. Moue K. Sawada H. Toh Y. Fumitaka K. Nicolson G.L. J. Cell. Biochem. 2000; 79: 202-212Crossref PubMed Scopus (73) Google Scholar). Although evidence shows that MTA1 is closely linked to cancer metastasis, it was unclear how MTA1 is involved in the metastatic process. Genetic studies in Caenorhabditis elegans suggest that MTA1 may function in embryonic patterning, determination of cell polarity, and cell migration (8Herman M.A. Ch'ng Q. Hettenbach S.M. Ratliff T.M. Kenyon C. Herman R.K. Development. 1999; 126: 1055-1064PubMed Google Scholar, 9Solari F. Bateman A. Ahringer J. Development. 1999; 126: 2483-2494Crossref PubMed Google Scholar). Recently, an MTA1 homologue, MTA2, was found to be part of an ATP-dependent chromatin-remodeling complex called NuRD (nucleosome remodeling histone deacetylation) (10Zhang Y. Ng H.H. Erdjument-Bromage H. Tempst P. Bird A. Reinberg D. Genes Dev. 1999; 13: 1924-1935Crossref PubMed Scopus (937) Google Scholar). Interestingly, NuRD also has histone deacetylase activity (11Zhang Y. LeRoy G. Seelig H.P. Lane W.S. Reinberg D. Cell. 1998; 95: 279-289Abstract Full Text Full Text PDF PubMed Scopus (695) Google Scholar). In eukaryotes, DNA is packaged into chromatin (12Kornberg R.D. Thomas J.O. Science. 1974; 184: 865-868Crossref PubMed Scopus (598) Google Scholar, 13Kornberg R.D. Science. 1974; 184: 868-871Crossref PubMed Scopus (1694) Google Scholar). Formation of closed chromatin precludes the access of transcription factors to DNA and inevitably leads to transcriptional repression (14Knezetic J.A. Luse D.S. Cell. 1986; 45: 95-104Abstract Full Text PDF PubMed Scopus (209) Google Scholar, 15Lorch Y. LaPointe J.W. Kornberg R.D. Cell. 1987; 49: 203-210Abstract Full Text PDF PubMed Scopus (394) Google Scholar). Dynamic changes in the chromatin structure, therefore, lead to either transcriptional activation or transcriptional repression. Alteration of the chromatin structure utilizing energy derived from ATP hydrolysis is called "chromatin remodeling" and is one of the two transcriptional regulatory mechanisms at the chromatin level (reviewed in Refs. 16Kingston R.E. Narlikar G.J. Genes Dev. 1999; 13: 2339-2352Crossref PubMed Scopus (610) Google Scholar and 17Urnov F.D. Wolffe A.P. Oncogene. 2001; 20: 2991-3006Crossref PubMed Scopus (171) Google Scholar). The other mechanism involves covalent modifications of nucleosomes, the basic units of chromatin. Nucleosomes are formed by DNA wrapping around a histone octamer, which composes of two copies of four histone proteins, H2A, H2B, H3, and H4 (12Kornberg R.D. Thomas J.O. Science. 1974; 184: 865-868Crossref PubMed Scopus (598) Google Scholar, 18Kelley R.I. Biochem. Biophys. Res. Commun. 1973; 54: 1588-1594Crossref PubMed Scopus (58) Google Scholar, 19Roark D.E. Geoghegan T.E. Keller G.H. Biochem. Biophys. Res. Commun. 1974; 59: 542-547Crossref PubMed Scopus (88) Google Scholar). The N termini of core histones are exposed and unstructured (20Luger K. Mader A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (7094) Google Scholar); these exposed tails are thought to contact neighboring core histones as well as DNA and are critical in mediating higher order structures of chromatin. As a result, covalent modifications of nucleosomes also lead to changes in the chromatin structure. One of the most well studied nucleosomal modification is acetylation and deacetylation of histone proteins at the unstructured N-terminal tails (reviewed in Ref. 16Kingston R.E. Narlikar G.J. Genes Dev. 1999; 13: 2339-2352Crossref PubMed Scopus (610) Google Scholar). The addition of acetyl groups to the N-terminal tails of histones is catalyzed by histone acetyltransferases and the hydrolysis of the acetylated histone tails by HDACs. 1The abbreviations used are: HDAC, histone deacetylase; PBS, phosphate-buffered saline; GST, glutathione S-transferase; GFP, green fluorescent protein. Acetylation of histone proteins opens up the chromatin structure and leads to transcriptional activation (21Hong L. Schroth G.P. Matthews H.R. Yau P. Bradbury E.M. J. Biol. Chem. 1993; 268: 305-314Abstract Full Text PDF PubMed Google Scholar). Conversely, deacetylation of histone proteins condenses the chromatin structure and is associated with transcriptional repression (reviewed in Ref. 22Cress W.D. Seto E. J. Cell. Physiol. 2000; 184: 1-16Crossref PubMed Scopus (582) Google Scholar). The finding that MTA2 is found in a protein complex containing both ATP-dependent chromatin-remodeling activity and histone deacetylase activity suggests that the molecular mechanism of tumor metastasis might involve aberrant regulations of the chromatin structure. Shortly after the discovery of the NuRD complex, MTA1 was found to be a component of another protein complex with histone deacetylase activity (23Xue Y. Wong J. Moreno G.T. Young M.K. Cote J. Wang W. Mol. Cell. 1998; 2: 851-861Abstract Full Text Full Text PDF PubMed Scopus (805) Google Scholar). Furthermore, evidence shows that MTA1 may repress estrogen receptor-mediated transcription by recruiting HDACs, leading to loss of estradiol responsiveness and progression of breast cancer to more invasive phenotypes (6Mazumdar A. Wang R.A. Mishra S.K. Adam L. Bagheri-Yarmand R. Mandal M. Vadlamudi R.K. Kumar R. Nat. Cell Biol. 2001; 3: 30-37Crossref PubMed Scopus (332) Google Scholar). However, despite the high homology between MTA1 and MTA2, MTA2 has not been shown to involve in metastasis or cancer formation. The level of MTA2 remains constant while MTA1 protein level is up-regulated by heregulin-β1 in breast cancer cells (6Mazumdar A. Wang R.A. Mishra S.K. Adam L. Bagheri-Yarmand R. Mandal M. Vadlamudi R.K. Kumar R. Nat. Cell Biol. 2001; 3: 30-37Crossref PubMed Scopus (332) Google Scholar). The findings that the homologous proteins MTA1 and MTA2 are both present in HDAC-containing complexes but only MTA1 has been concretely linked to cancer metastasis pose a conundrum. To further understand the biological functions of MTA1 and MTA2, we used a variety of assays to probe the differences and similarities between the MTA proteins. We also found a third member of the human MTA family, MTA3. We found that both MTA1 and MTA2 mediate transcriptional repression, but MTA3 does not appear to repress transcription to a significant level. In addition, MTA1 and MTA2 form distinct protein complexes, both containing histone deacetylase activity. Our results suggest that although the highly homologous N-terminal regions of the MTA proteins are important in forming protein complexes with histone deacetylases, the divergent C termini are likely to be critical in modulating the histone deacetylase activity associated with the MTA complexes and possibly in the downstream biological events, including the metastatic process. Plasmids—Gal4-MTA1 was expressed from pM1-MTA1, Gal4-MTA2 from pM2-MTA2, and Gal4-MTA3 from pM2-MTA3. pM1-MTA1, pM2-MTA2, and pM2-MTA3 were constructed by inserting the full-length MTA cDNAs in frame with the Gal4 DNA-binding domain in pM1 (24Sadowski I. Bell B. Broad P. Hollis M. Gene (Amst.). 1992; 118: 137-141Crossref PubMed Scopus (201) Google Scholar) or pM2 (24Sadowski I. Bell B. Broad P. Hollis M. Gene (Amst.). 1992; 118: 137-141Crossref PubMed Scopus (201) Google Scholar). Gal4-MTA1* was made by restriction enzyme digestion and religation of pM1-MTA1. pcDNA3-MTA1, which contained full-length MTA1 in pcDNA3 (Invitrogen), was used to express MTA1 without the Gal4 DNA-binding domain. GFP fusion constructs of MTA1, MTA2, and MTA3 were generated by inserting the full-length MTA cDNAs in frame with and C-terminal to the GFP open reading frame in the pEGFP vector (Clontech). GFPHDAC1, which expressed an N-terminal fusion of GFP to HDAC1, was made by inserting HDAC1 cDNA into pEGFP. FLAG-tagged MTA1 (FLAG-MTA1) was expressed from pME18S-MTA1, which was generated by inserting the MTA1 cDNA into pME18S (25Laherty C.D. Yang W.M. Sun J.M. Davie J.R. Seto E. Eisenman R.N. Cell. 1997; 89: 349-356Abstract Full Text Full Text PDF PubMed Scopus (856) Google Scholar). FLAG-MTA1* was made by restriction enzyme digestion and religation of pME18S-MTA1. FLAG-MTA2 expression plasmid has been described (10Zhang Y. Ng H.H. Erdjument-Bromage H. Tempst P. Bird A. Reinberg D. Genes Dev. 1999; 13: 1924-1935Crossref PubMed Scopus (937) Google Scholar). pGEM7Zf3X-MTA1 and pGEM7Zf3X-MTA2, which generated in vitro translated MTA1 and MTA2 proteins, were made by subcloning full-length MTA1 and MTA2 into the pGEM7Zf3X vector (26Yao Y.L. Yang W.M. Seto E. Mol. Cell. Biol. 2001; 21: 5979-5991Crossref PubMed Scopus (370) Google Scholar). Expression plasmids for the following proteins or promoter-reporters have been previously described: GST-p53 (27Huibregtse J.M. Scheffner M. Howley P.M. EMBO J. 1991; 10: 4129-4135Crossref PubMed Scopus (710) Google Scholar), GST-FKBP12 (28Yang W.M. Inouye C.J. Seto E. J. Biol. Chem. 1995; 270: 15187-15193Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), GSTFKBP25 (29Yang W.M. Yao Y.L. Seto E. EMBO J. 2001; 20: 4814-4825Crossref PubMed Scopus (90) Google Scholar), GST-YY1 (26Yao Y.L. Yang W.M. Seto E. Mol. Cell. Biol. 2001; 21: 5979-5991Crossref PubMed Scopus (370) Google Scholar), G5TKLuc and TKLuc (29Yang W.M. Yao Y.L. Seto E. EMBO J. 2001; 20: 4814-4825Crossref PubMed Scopus (90) Google Scholar), and HDAC1-FLAG (25Laherty C.D. Yang W.M. Sun J.M. Davie J.R. Seto E. Eisenman R.N. Cell. 1997; 89: 349-356Abstract Full Text Full Text PDF PubMed Scopus (856) Google Scholar). GST by itself was expressed from pGSTag (30Ron D. Dressler H. BioTechniques. 1992; 13: 866-869PubMed Google Scholar). Cell Culture, Transfection, and Luciferase Assay—HeLa cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and penicillin/streptomycin. 1 × 106 HeLa cells were seeded into 60-mm-diameter tissue culture dishes. Sixteen hours later, 0.5 μg of pRL-TK, 5 μg of the MTA construct (with or without Gal4 fusion), and 5 μg of either G5TKLuc or TKLuc were transfected into HeLa cells using the calcium phosphate co-precipitation method (31Graham F.L. van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6508) Google Scholar). Forty-eight hours after transfection, cells were harvested and a luciferase assay performed using the dual luciferase assay system (Promega). Fluorescence Microscopy—HeLa cells were seeded on chamber slides and grown for 18 h. 5 μg of expression plasmids for various GFP fusion proteins were transfected into cells using the calcium phosphate co-precipitation method (31Graham F.L. van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6508) Google Scholar). Forty-eight hours later, cells were washed with ice-cold PBS, fixed with 4% paraformaldehyde, rinsed with cold PBS again, and dried. One drop of anti-fade mounting medium with 4′,6′-diamidino-2-phenylindole (Vector) was added to the cells before coverslips were applied. Fluorescence images were observed and analyzed under a fluorescence microscope. Immunoprecipitation, Western Blot Analysis, and Histone Deacetylase Assay—Immunoprecipitation of FLAG-MTA1, FLAG-MTA1*, and FLAG-MTA2 was carried out using anti-FLAG M2 affinity gel (Sigma) following the manufacturer's suggestions. Western blot analyses were performed using standard protocols (32Harlow E. Lane D. Using Antibodies: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1999: 490-505Google Scholar). Histone deacetylase assay was performed using immunoprecipitated MTA proteins or the eluted fractions of the FLAG fusion protein complexes as the source of the enzyme and a labeled peptide corresponding to residues 2-24 of histone H4 as substrate following a previously published procedure except that incubation was performed at room temperature overnight (33Taunton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1561) Google Scholar). Trichostatin A (400 nm final concentration) (Sigma) was added to the FLAG-MTA1 immunoprecipitate 30 min prior to the addition of the H4 substrate peptide in one of the reactions. Protein Complex Purification—Anti-FLAG immunoaffinity columns were prepared using anti-FLAG M2 affinity gel (Sigma) following the manufacturer's suggestions. Approximately for every 6 × 107 HeLa cells, 5 μg of the plasmids expressing FLAG fusion proteins were transfected using the calcium phosphate co-precipitation method (31Graham F.L. van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6508) Google Scholar). Forty-eight hours after transfection, cells were harvested by scraping. Cells were subsequently lysed by adding PBS plus 0.1% Nonidet P-40 and then briefly sonicating. Cell lysate obtained from about 4.8 × 109 cells was applied to an equilibrated FLAG column of 1-ml bed volume to allow for adsorption of the protein complex to the column resin. After binding, the column was washed with cold PBS plus 0.1% Nonidet P-40. FLAG peptide (Sigma) was applied to the column as described by the manufacturer to elute the FLAG protein complex. Fractions of 1 bed volume were collected. For the purification of the MTA1-HDAC complex, anti-HDAC2 antibody (25Laherty C.D. Yang W.M. Sun J.M. Davie J.R. Seto E. Eisenman R.N. Cell. 1997; 89: 349-356Abstract Full Text Full Text PDF PubMed Scopus (856) Google Scholar) was used to pack an immunoaffinity column following a previously published method (34Zhang Y. Sun Z.W. Iratni R. Erdjument-Bromage H. Tempst P. Hampsey M. Reinberg D. Mol. Cell. 1998; 1: 1021-1031Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Eluted FLAG-MTA1 complex was applied to the anti-HDAC2 column and allowed for adsorption. After extensive washing, the MTA1-HDAC complex was eluted with excess HDAC2 peptide that contained the epitope of the anti-HDAC2 antibody (25Laherty C.D. Yang W.M. Sun J.M. Davie J.R. Seto E. Eisenman R.N. Cell. 1997; 89: 349-356Abstract Full Text Full Text PDF PubMed Scopus (856) Google Scholar). In Vitro Protein-Protein Interaction Assay—35S-Labeled MTA1 was generated from pGEM7Zf3X-MTA1 using T7 RNA polymerase and the TNT Reticulocyte Lysate System (Promega). 35S-labeled MTA2 was generated using the same method from pGEM7Zf3X-MTA2. GST, GST-p53, GST-FKBP12, GST-FKBP25, and GST-YY1 were expressed in Escherichia coli strain DH5α and captured onto glutathione-agarose beads (Sigma). In vitro translated MTA1 or MTA2 (5 μl) was mixed with the beads in the presence of PBS plus 0.2% Nonidet P-40 at 4 °C for 1 h. Beads were washed extensively in PBS plus 0.2% Nonidet P-40. Bound proteins were eluted by boiling in Laemmli sample buffer, separated by SDS-PAGE, and detected by Coomassie Blue staining and autoradiography. MTA1 and MTA2 Are Highly Homologous in the N Terminus but Divergent in the C Terminus—To further understand the potential functions of MTA1 and MTA2 and to probe the functional differences between them, we began by analyzing the primary structure of the MTA proteins. Human MTA2 is 65% identical to human MTA1, with the highest homology concentrated in the N-terminal half of the proteins. The C-terminal half of the MTA proteins is divergent; data base searches (EMBL) show that in MTA1 there are a GATA zinc finger domain, a bipartite nuclear localization signal embedded in a myb DNA-binding domain, and an Src homology 3 binding domain (Fig. 1A). MTA2 also contains a GATA zinc finger domain and a nuclear localization signal, but there is no C-terminal myb domain or Src homology 3 binding domain in MTA2. It has not been shown that MTA1 possesses DNA binding activity; therefore, the consequence of the additional myb DNA-binding domain in MTA1 is unclear. However, the functional differences between MTA1 and MTA2, if any, might relate to the differences in the C-terminal parts of the proteins. Contrary to the C-terminal half, the N-terminal half of the MTA proteins is highly similar and can be organized into four functional domains. At the extreme N terminus is a BAH (bromo-adjacent homology) domain, followed by an ELM2 (Egl-27 and MTA1 homology 2) domain (InterPro, European Bioinformatics Institute). Next is a conserved leucine zipper. C-terminal to the leucine zipper is a SANT domain related to the myb DNA-binding domain. The BAH domain has been identified in a number of transcriptional regulators including DNA cytosine-5 methyltransferase (35Nicolas R.H. Goodwin G.H. Gene (Amst.). 1996; 175: 233-240Crossref PubMed Scopus (39) Google Scholar, 36Callebaut I. Courvalin J.C. Mornon J.P. FEBS Lett. 1999; 446: 189-193Crossref PubMed Scopus (166) Google Scholar) and the Orc1 (origin recognition complex 1) protein (36Callebaut I. Courvalin J.C. Mornon J.P. FEBS Lett. 1999; 446: 189-193Crossref PubMed Scopus (166) Google Scholar). It has been suggested that the BAH domain may serve as a protein-protein interaction module linking DNA methylation, replication, and transcriptional regulation together (36Callebaut I. Courvalin J.C. Mornon J.P. FEBS Lett. 1999; 446: 189-193Crossref PubMed Scopus (166) Google Scholar, 37Goodwin G.H. Nicolas R.H. Gene (Amst.). 2001; 268: 1-7Crossref PubMed Scopus (43) Google Scholar). The ELM2 domain was named after the C. elegans homologue of MTA1, Egl-27, and MTA1. Although many unidentified proteins in C. elegans, Drosophila, Xenopus laevis, Arabidopsis thaliana, and humans also contain the unique ELM2 domain (Fig. 1B), it is unclear what function this domain might have. However, CoREST, a specific co-repressor for REST/NRSF (RE1 silencing transcription factor/neural restrictive factor) required for the regulation of neuron-specific gene expression (38Andres M.E. Burger C. Peral-Rubio M.J. Battaglioli E. Anderson M.E. Grimes J. Dallman J. Ballas N. Mandel G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9873-9878Crossref PubMed Scopus (379) Google Scholar), also contains the ELM2 domain (Fig. 1B). Interestingly, CoREST shares another domain, the SANT (SWI3/ADA2/NCoR/TFIIIB) domain, with MTA1 and MTA2. SANT domains are present in a variety of transcriptional regulators, including SWI3 of the yeast SWI/SNF transcriptional activation complex; ADA2 of the ADA activation complex; and NCoR and SMRT, two co-repressors mediating inducible repression by steroid hormone receptors (reviewed in Ref. 39Aasland R. Stewart A.F. Gibson T. Trends Biochem. Sci. 1996; 21: 87-88Abstract Full Text PDF PubMed Scopus (295) Google Scholar). It has been suggested that SANT domains are important in protein complex assembly in a recent study documenting the purification of stable protein complexes containing HDAC1 and HDAC2 (40Guenther M.G. Barak O. Lazar M.A. Mol. Cell. Biol. 2001; 21: 6091-6101Crossref PubMed Scopus (498) Google Scholar, 41You A. Tong J.K. Grozinger C.M. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1454-1458Crossref PubMed Scopus (399) Google Scholar). The juxtaposition of the SANT domain and the leucine zipper in the MTA proteins suggests the presence of a functional module in the formation of protein complexes. In summary, domain structures of MTA1 and MTA2 suggest that the MTA proteins are transcriptional regulators capable of forming protein complexes. The unique C-terminal regions of MTA1 and MTA2 might confer additional regulatory roles to the differences in their biological functions. Human MTA3—Through homology search, we identified a human cDNA clone that is highly homologous to MTA1 and MTA2 (clone KIAA1266; Kazuda DNA Research Institute). This clone was confirmed by dideoxyribonucleotide sequencing to contain a full-length open reading frame, and we named this protein human MTA3 (Fig. 1C). MTA3 appeared to share the highest homology with MTA1 and MTA2 in the N terminus (Fig. 1C). It is likely that MTA1, MTA2, and MTA3 represent members of the same gene family, and they might possess distinct or overlapping functions. MTA1 and MTA2 Repress Transcription, whereas MTA3 Has Minimal Repression Activity—Chimeric proteins of MTA1, MTA2, and MTA3 fused to the Gal4 DNA-binding domain were constructed to test whether they could repress transcription from a promoter containing Gal4-binding sites (Fig. 2A). Gal4-MTA1, Gal4-MTA2, or Gal4-MTA3 was co-transfected into HeLa cells with a reporter plasmid, G5TKLuc, which contained five Gal4-binding sites within the thymidine kinase promoter driving the luciferase reporter gene. As shown in Fig. 2B, whereas Gal4-MTA1 repressed the target promoter more than 10-fold (compare lane 4 with lane 3), it had no significant effect on an identical promoter with no Gal4-binding site (lanes 1 and 2). Similarly, Gal4-MTA2 also repressed transcription from the promoter containing Gal4-binding sites (lane 5). Furthermore, overexpression of MTA1 alone did not repress the thymidine kinase promoter with the Gal4-binding sites, strongly suggesting that the repression is specifically from Gal4-MTA1 (lane 8). Moreover, a carboxyl-terminally truncated form of MTA1 (Gal4-MTA1*, containing amino acids 1-387; lane 6) lost most of the transcriptional repression activity. Most significantly, the transcriptional repression activity of MTA3 appeared to be significantly lower than that of MTA1 or MTA2 (lane 7). These results demonstrate that MTA1 and MTA2 are able to repress transcription when recruited to a target promoter. More importantly, the reduced level of transcriptional repression activity observed for MTA3 and a C-terminally truncated form of MTA1 suggests that the C terminus of the MTA proteins has regulatory roles pertaining to the biological functions of the MTA family. MTA1 and MTA2, but Not MTA3, Are Located in the Nucleus—Transcriptional regulation takes place in the nucleus. To confirm the functional significance of the MTA proteins with respect to transcriptional repression, the subcellular localization of the MTA proteins was analyzed by direct fluorescence. GFP-MTA1, GFP-MTA2, and GFP-MTA3 fusion constructs were transfected into HeLa cells. After 48 h, transfected cells were fixed, and the localization of fluorescence was observed under a fluorescence microscope. As shown in Fig. 3, GFPMTA1 and GFP-MTA2 were clearly localized to the nucleus compared with 4′,6′-diamidino-2-phenylindole (DAPI) counterstaining for DNA. GFP was nonspecifically localized to both the nucleus and the cytoplasm, and GFP-HDAC1 was localized to the nucleus as previously reported (29Yang W.M. Yao Y.L. Seto E. EMBO J. 2001; 20: 4814-4825Crossref PubMed Scopus (90) Google Scholar). GFP-MTA3, however, appeared to have a diffused distribution and failed to localize to a specific subcellular locale (Fig. 3o). This result, together with the finding that MTA3 lacked significant repression activity, suggests that the major function of MTA3 may not involve transcriptional regulation. MTA1 and MTA2 Contain Histone Deacetylase Activity—Because both MTA1 and MTA2 have been found in protein complexes containing histone deacetylase activities, it is very possible that MTA1 and MTA2 themselves are tightly associated with histone deacetylase activity. To test this hypothesis, a construct that can express FLAG-tagged MTA1 protein was made and transfected into HeLa cells. After transfected cells were harvested, immunoprecipitation experiments were carried out using anti-FLAG antibody on the cell extracts. Precipitated MTA1 was subsequently examined for the histone deacetylase activity with a 3H-labeled H4 peptide as substrate. As shown in Fig. 4A, MTA1 indeed contained a significant level of histone deacetylase activity, but the cells transfected with FLAG vector alone did not. Moreover, MTA1* (containing amino acids 1-387), which lost much of the transcriptional repression activity compared with full-length MTA1 (Fig. 2B), retained only minimal enzyme activity, which further demonstrated that MTA1 indeed specifically contained histone deacetylase activity. Similarly, MTA2 also contained histone deacetylase activity. To avoid the possibility that the FLAG-tagged MTA1 mutant protein was not well expressed, the expression level of each construct was confirmed by Western blot analysis with the anti-FLAG antibody (Fig. 4B). The result from these experiments shows that MTA1 and MTA2 contain associated his
Referência(s)