Mediator Subunit MED28 (Magicin) Is a Repressor of Smooth Muscle Cell Differentiation

Abstract

Magicin, a protein that we isolated earlier as an interactor of the neurofibromatosis 2 protein merlin, was independently identified as MED28, a subunit of the mammalian Mediator complex. Mediator complex is an evolutionarily conserved transcriptional cofactor, which plays an essential role in positive and negative gene regulation. Distinct Mediator subunit composition is thought to contribute to gene regulation specificity based on the interaction of specific subunits with subsets of transcription factors. Here we report that down-regulation of Med28 expression in NIH3T3 cells results in a significant induction of several genes associated with smooth muscle cell (SMC) differentiation. Conversely, overexpression of MED28 represses expression of SMC genes, in concordance with our knockdown data. More importantly, multipotent mesenchymal-derived murine precursors can transdifferentiate into SMCs when Med28 is down-regulated. Our data also show that Med28 functions as a negative regulator of SMC differentiation in concert with other Mediator subunits including Med6, Med8, and Med18 within the Mediator head module. Our results provide strong evidence that MED28 may function as a scaffolding protein by maintaining the stability of a submodule within the head module and that components of this submodule act together in a gene regulatory program to suppress SMC differentiation. The results presented here demonstrate for the first time that the mammalian Mediator subunit MED28 functions as a repressor of SMC differentiation, which could have implications for disorders associated with abnormalities in SMC growth and differentiation, including atherosclerosis, asthma, hypertension, and smooth muscle tumors. Magicin, a protein that we isolated earlier as an interactor of the neurofibromatosis 2 protein merlin, was independently identified as MED28, a subunit of the mammalian Mediator complex. Mediator complex is an evolutionarily conserved transcriptional cofactor, which plays an essential role in positive and negative gene regulation. Distinct Mediator subunit composition is thought to contribute to gene regulation specificity based on the interaction of specific subunits with subsets of transcription factors. Here we report that down-regulation of Med28 expression in NIH3T3 cells results in a significant induction of several genes associated with smooth muscle cell (SMC) differentiation. Conversely, overexpression of MED28 represses expression of SMC genes, in concordance with our knockdown data. More importantly, multipotent mesenchymal-derived murine precursors can transdifferentiate into SMCs when Med28 is down-regulated. Our data also show that Med28 functions as a negative regulator of SMC differentiation in concert with other Mediator subunits including Med6, Med8, and Med18 within the Mediator head module. Our results provide strong evidence that MED28 may function as a scaffolding protein by maintaining the stability of a submodule within the head module and that components of this submodule act together in a gene regulatory program to suppress SMC differentiation. The results presented here demonstrate for the first time that the mammalian Mediator subunit MED28 functions as a repressor of SMC differentiation, which could have implications for disorders associated with abnormalities in SMC growth and differentiation, including atherosclerosis, asthma, hypertension, and smooth muscle tumors. We previously identified a novel protein, which we named magicin, as a specific binding partner for the cytoskeletal neurofibromatosis 2 tumor suppressor protein merlin. These two proteins interact in vitro and in vivo and colocalize beneath the plasma membrane. Magicin is a ∼24-kDa protein expressed in many cell lines and tissues. Similar to merlin, magicin associates with the actin cytoskeleton as determined by cofractionation, immunofluorescence, and electron microscopy. Our results suggest a role for magicin in receptor-mediated signaling at the cell surface and its possible involvement in the regulation of cytoskeletal reorganization (1Wiederhold T. Kim M.F. James M. Neujahr R. Smith N. Murthy A. Hartwig J. Gusella J.F. Ramesh V. Oncogene. 2004; 23: 8815-8825Crossref PubMed Scopus (58) Google Scholar). Our recent work also documents that magicin associates with Src-family kinases such as Fyn, Src, and Lck and is phosphorylated by Lck in the T lymphocyte leukemia Jurkat cell line upon CD3 stimulation (2Lee M.F. Kim R.L. Beyer K.S. Gusella J.F. Ramesh V. Biochem. Biophys. Res. Commun. 2006; 348: 826-831Crossref PubMed Scopus (13) Google Scholar). Magicin was independently identified as a differentially expressed gene in endothelial cells and named endothelial-derived gene (EG-1) (3Liu C. Kim L. Shao Z.M. Beatty P. Sartippour M. Lane T.F. Barsky S.H. Livingston E. Nguyen M. Biochem. Biophys. Res. Commun. 2002; 290: 602-612Crossref PubMed Scopus (32) Google Scholar). EG-1 expression is elevated in several tumors, and overexpression of EG-1 increases cell proliferation (4Lu M. Kim L. Maul R.S. Sartippour M.R. Norris A. Whitelegge J. Rao J.Y. Brooks M.N. Cancer Res. 2005; 65: 6159-6166Crossref PubMed Scopus (21) Google Scholar, 5Zhang L. Kim R.S. Rao J. Apple S. Seligson D. Sartippour M. Rubio R. Brooks M.N. Clin. Cancer Res. 2004; 10: 3504-3508Crossref PubMed Scopus (22) Google Scholar). Surprisingly, and more importantly, magicin was also identified as a subunit (MED28) within the mammalian Mediator complex, which functions in the nucleus to regulate transcription (6Brower C.S. Kim S. Tomomori-Sato C. Kamura T. Pause A. Stearman R. Klausner R.D. Malik S. Lane W.S. Sorokina I. Roeder R.G. Conaway J.W. Conaway R.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10353-10358Crossref PubMed Scopus (73) Google Scholar, 7Sato S. Kim C. Parmely T.J. Florens L. Zybailov B. Swanson S.K. Banks C.A. Jin J. Cai Y. Washburn M.P. Conaway J.W. Conaway R.C. Mol. Cell. 2004; 14: 685-691Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). The Mediator complex, originally identified in yeast, was subsequently isolated in mammalian cells comprising 30 subunits. Mediator is an important component of the RNA polymerase II transcriptional machinery and plays a crucial role in the activation and repression of eukaryotic mRNA synthesis (8Conaway R.C. Kim S. Tomomori-Sato C. Yao T. Conaway J.W. Trends Biochem. Sci. 2005; 30: 250-255Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 9Malik S. Kim R.G. Trends Biochem. Sci. 2005; 30: 256-263Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 10Kornberg R.D. Trends Biochem. Sci. 2005; 30: 235-239Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). Thus magicin, which we will hereafter refer to as MED28, may serve as a multifaceted adaptor/scaffolding protein to relay cellular signals to the cytoskeleton and from the cytoskeleton to the nucleus. Mediator exists in at least two forms, differing mainly by the presence or absence of a negative regulatory submodule consisting of cyclin-dependent kinase 8 (CDK8), 3The abbreviations used are: CDK8, cyclin-dependent kinase 8; SMC, smooth muscle cell; SM-MHC, smooth muscle-myosin heavy chain; SM-αActin, smooth muscle-αActin; SM-γActin, smooth muscle-γActin; CRP1, cysteinrich protein 1; RNAi, RNA interference; siRNA, small interfering RNA; GFP, green fluorescent protein; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; h, human; KD, knockdown. cyclin C, MED12, and MED13 (9Malik S. Kim R.G. Trends Biochem. Sci. 2005; 30: 256-263Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 11Dotson M.R. Kim C.X. Roeder R.G. Myers L.C. Gustafsson C.M. Jiang Y.W. Li Y. Kornberg R.D. Asturias F.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14307-14310Crossref PubMed Scopus (154) Google Scholar). Different Mediator subunits are targets for interactions with various DNA-binding transcriptional activators. Mediator complexes with the same subunit composition are proposed to have distinct activities through interactions with different transcriptional activation domains (10Kornberg R.D. Trends Biochem. Sci. 2005; 30: 235-239Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 12Taatjes D.J. Kim M.T. Tjian R. Nat. Rev. Mol. Cell Biol. 2004; 5: 403-410Crossref PubMed Scopus (127) Google Scholar). In addition, studies addressing the functions of the intact mammalian Mediator complex, as well as individual subunits, are beginning to emerge (13Stumpf M. Kim C. Krotschel M. van Essen D. Rodriguez P. Zhang X. Guyot B. Roeder R.G. Borggrefe T. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 18504-18509Crossref PubMed Scopus (82) Google Scholar, 14Yang F. Kim B.W. Satterlee J.S. Walker A.K. Jim Sun Z.Y. Watts J.L. DeBeaumont R. Saito R.M. Hyberts S.G. Yang S. Macol C. Iyer L. Tjian R. van den Heuvel S. Hart A.C. Wagner G. Naar A.M. Nature. 2006; 442: 700-704Crossref PubMed Scopus (297) Google Scholar). To evaluate whether MED28 plays a role in controlling specific transcriptional programs and to determine putative target genes regulated by MED28, we performed whole genome expression profiling of Med28 RNAi knockdown in NIH3T3 cells and observed that Med28 suppression results in a significant induction of many genes involved in smooth muscle cell (SMC) differentiation. Overexpression of MED28 represses the expression of SMC genes consistent with our RNAi knockdown data. Intriguingly, our results also show that Med28 suppression in the multipotent mesenchymal-derived murine precursor cell line C2C12 results in transdifferentiation into SMCs, a differentiation pathway not previously associated with this cell line. Furthermore, induction of SMC differentiation by Med28 knockdown involves destabilization of several other subunits in the mammalian Mediator head module. The results presented here demonstrate for the first time that mammalian Mediator subunit MED28 functions as a repressor of SMC differentiation, and this observation could have wide medical implications since abnormalities in SMC growth and differentiation are linked to many disorders, including atherosclerosis, asthma, hypertension, and smooth muscle tumors. Reagents and Antibodies—Human full-length MED28 (hMED28) was cloned into the pEGFP-N1 (Clontech) plasmid, as well as the CSCW2 lentiviral plasmid. The CSCW2 vector, packaging, and viral supernatants were provided through the Neuroscience NINDS, National Institutes of Health, Vector Core at Massachusetts General Hospital. For RNAi, SMARTpool® reagents were obtained from Dharmacon RNA Technologies and included murine Med28 (NM_ 025895; catalog number M-063448-00) and Med6 (NM_027213; catalog number M-055846-00), individual siRNA reagent for murine Med28 (accession number D-063448-02), as well as non-targeting siControl (catalog number D-001206-13-05). Primary antibodies used included the anti-MED28 antibodies Tim3 (rabbit polyclonal) and 7E1 (mouse monoclonal), which have been previously described (1Wiederhold T. Kim M.F. James M. Neujahr R. Smith N. Murthy A. Hartwig J. Gusella J.F. Ramesh V. Oncogene. 2004; 23: 8815-8825Crossref PubMed Scopus (58) Google Scholar); M2 (anti-FLAG monoclonal; Sigma), MED6, CDK8, and SM22α/transgelin (Santa Cruz Biotechnology); SM-γActin (MP Biomedicals); GAPDH (Chemicon); and SM-αActin, α-tubulin, and normal rabbit serum (Sigma). Rabbit polyclonal antibodies for MED8, MED17, and MED18 were a kind gift from Drs. R. C. Conaway and J. W. Conaway, and a second MED17 rabbit polyclonal antibody was a kind gift from Dr. R. G. Roeder. Secondary antibodies included horseradish peroxidase-conjugated anti-mouse and anti-rabbit (Amersham Biosciences) and horseradish peroxidase-conjugated anti-goat (Santa Cruz Biotechnology) for Western analysis and Alex Fluor 594-phalloidin (Molecular Probes) and Cy3-donkey anti-mouse for immunofluorescence. Cell Culture—The mouse fibroblast line NIH3T3 was maintained in Iscove’s Dulbecco’s modified Eagle’s medium (Cellgro) containing 10% fetal bovine serum (Sigma). The multipotent, mesenchymal-derived C2C12 line was cultured at ≤60% confluency in growth medium containing Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 20% fetal bovine serum (Sigma) to prevent myoblast differentiation. Transfection and Infection—All transfections were performed using Lipofectamine 2000 reagent (Invitrogen), according to the manufacturer’s protocol, utilizing reverse transfection methods to increase transfection efficiency. For rescue experiments, C2C12 cells were transfected with either small interfering control or siRNA-mMed28 plus GFP-hMED28 plasmid. Transfections were harvested at 48 (for NIH3T3) and 72 h (for C2C12) after transfection. Lentiviral infections were carried out in NIH3T3 cells using low titer viral supernatants of either CSCW2 vector alone or hMED28-CSCW2 at a multiplicity of infection of 100 transforming units/cell. At 72 h after infection, cells were analyzed for enhanced GFP expression and fluorescence-activated cell sorter-sorted by the Analytical and Quantitative Cytology Core Facility at Massachusetts General Hospital (MGH). Nuclear Fractionation, Co-immunoprecipitation, and Western Blotting—Isolation of nuclear fractions was carried out using a modification of the Dignam et al. method (28Dignam J.D. Kim R.M. Roeder R.G. Nucleic Acids Res. 1983; PubMed Scopus Google Scholar). was carried out as described previously (1Wiederhold T. Kim M.F. James M. Neujahr R. Smith N. Murthy A. Hartwig J. Gusella J.F. Ramesh V. Oncogene. 2004; 23: 8815-8825Crossref PubMed Scopus (58) Google Scholar). for of Med28 and Med6, NIH3T3 were with anti-MED28 or normal rabbit serum with protein and normal rabbit or protein For from NIH3T3 nuclear were with an of Dignam and to using and normal rabbit serum as a negative Western was carried out as described previously (1Wiederhold T. Kim M.F. James M. Neujahr R. Smith N. Murthy A. Hartwig J. Gusella J.F. Ramesh V. Oncogene. 2004; 23: 8815-8825Crossref PubMed Scopus (58) Google Scholar). were and were by to medium and with antibodies by horseradish peroxidase-conjugated were using the (Amersham RNA was harvested using the according to the manufacturer’s control of the RNA and on mouse from were performed by the Cancer Core The results were analyzed using the and the from and was from RNA using the synthesis was carried out to Med28 as well as the differentiation protein and differentiation including and SM-γActin To was with and mouse for CRP1, SM-αActin, and SM-γActin, as well as the control gene were carried out using a and analyzed with and in gene expression were using the using was also carried out for to in expression for SM-γActin, SM-αActin, CRP1, and GAPDH, as well as the smooth muscle heavy are provided in for have been previously described A. Kim L. A. A. Y. Y. L. M. S. Sun Y. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). of Med28 in NIH3T3 of a of SMC an earlier we isolated MED28 as a novel protein, we observed Med28 in with the actin cytoskeleton of the mouse cell line as determined by cofractionation, immunofluorescence, and electron (1Wiederhold T. Kim M.F. James M. Neujahr R. Smith N. Murthy A. Hartwig J. Gusella J.F. Ramesh V. Oncogene. 2004; 23: 8815-8825Crossref PubMed Scopus (58) Google Scholar). MED28 also been identified as a subunit of the mammalian Mediator complex in the nucleus as a transcriptional (6Brower C.S. Kim S. Tomomori-Sato C. Kamura T. Pause A. Stearman R. Klausner R.D. Malik S. Lane W.S. Sorokina I. Roeder R.G. Conaway J.W. Conaway R.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10353-10358Crossref PubMed Scopus (73) Google Scholar, 7Sato S. Kim C. Parmely T.J. Florens L. Zybailov B. Swanson S.K. Banks C.A. Jin J. Cai Y. Washburn M.P. Conaway J.W. Conaway R.C. Mol. Cell. 2004; 14: 685-691Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Thus MED28 is a Mediator subunit with possible functions in the cytoskeleton and in the nucleus. In an to the genes that are regulated by MED28, we the gene expression in NIH3T3 cells Med28 expression was by RNAi, a non-targeting siRNA by knockdown of Med28 was carried out in and of RNAi was by Western analysis profiling the mouse expression genes that at least a in expression upon knockdown of Med28 and data not the significant we observed a of genes, including and after Med28 suppression and the expression of several SMC genes in Med28 knockdown cells In addition, we NIH3T3 cells MED28 was and observed down-regulation of the SMC genes, that regulation of SMC genes by Med28 is and specific Western of Med28 knockdown and cells were consistent with results results suggest that MED28 functions as a negative regulator of smooth muscle differentiation. Med28 in C2C12 into the of Med28 suppression in a more cell we the expression of Med28 in a smooth muscle cell line as well as multipotent mesenchymal-derived murine myoblast precursors the expression of Med28 was low in smooth muscle we C2C12 cells for with results obtained with NIH3T3 suppression of Med28 in C2C12 cells also in the of SM-αActin, CRP1, and SM-γActin, as by Western and and The also an by that the expression of Med28 genes and associated with muscle differentiation, not increase upon Med28 knockdown not More C2C12 cells Med28 was by RNAi a distinct and cell smooth muscle cells when with cells transfected with non-targeting control siRNA analysis of the presence of smooth muscle cells in Med28 knockdown C2C12 cells when with control cells Our data demonstrate for the first time that Med28 suppression can in transdifferentiation of C2C12 into smooth muscle In addition, of SM-MHC, a of smooth muscle that the observed Med28 suppression is that of smooth muscle cells and not of MED28 the by Med28 in C2C12 determine whether the observed were in to down-regulation of we carried out rescue by siRNA of Med28 with Overexpression of GFP-hMED28 the smooth muscle by Med28 knockdown in C2C12 cells a and cells the smooth muscle cell a and These results that MED28 functions as a repressor of smooth muscle differentiation in MED28 within a of the Mediator to Cell MED28 is in the nucleus and in the associated with the we the whether the negative regulation of SMC differentiation by Med28 is or of its function as a mammalian Mediator an anti-MED28 antibody first we the of Med28 with mammalian Mediator subunits Med6, and in NIH3T3 cells and that Med28 can at the with Mediator subunits of head module and a kinase module component We whether Med28 suppression the cellular of Mediator complex subunits in C2C12 we observed a consistent down-regulation of head module including Med6, Med8, and in Med28 knockdown cells of head module not In addition, a subunit of the kinase a down-regulation We the whether suppression of Mediator upon Med28 also results in of SMC differentiation We Med6 as a to out suppression in C2C12 cells and observed that Med6 to Med28 in of and expression with other SMC including Similar to Med28 Med6 down-regulation in transdifferentiation of C2C12 into smooth muscle cells a and results suggest that Med28 functions as a negative regulator of SMC differentiation in concert with Med6 and other subunits within the head module. Our results the that Med28 may function as a scaffolding protein by maintaining the stability of a submodule within the head module. The Mediator complex functions as a transcription at and RNA polymerase at the (8Conaway R.C. Kim S. Tomomori-Sato C. Yao T. Conaway J.W. Trends Biochem. Sci. 2005; 30: 250-255Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 9Malik S. Kim R.G. Trends Biochem. Sci. 2005; 30: 256-263Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 10Kornberg R.D. Trends Biochem. Sci. 2005; 30: 235-239Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). Mediator is of at least 30 subunits to is into and (9Malik S. Kim R.G. Trends Biochem. Sci. 2005; 30: 256-263Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, L. Kim S. S. S. P. Nat. Mol. Biol. 2006; PubMed Scopus Google Scholar). MED28, which we originally identified as a interactor is as of the head module subunits Kim F.J. Trends Biochem. Sci. 2005; 30: Full Text Full Text PDF PubMed Scopus Google Scholar, A.C. Kim T.J. Tomomori-Sato C. S. D. Conaway R.C. Conaway J.W. Florens L. Washburn M.P. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: PubMed Scopus Google Scholar). Our results presented here show that knockdown of Med28 expression of several SMC including SM-MHC, and overexpression of MED28 represses SMC gene More importantly, multipotent myoblast cells can transdifferentiate into smooth muscle cells when Med28 is down-regulated. Furthermore, Med28 knockdown results in down-regulation of at least other subunits in the head module as well as a subunit in the kinase module. significant in Med6 and in Med28 knockdown cells is of a possible interaction subunits. been previously in cell for two other subunits, and were a subunits J.Y. Kim G. J. Kim J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Our results for the first time demonstrate that a submodule within the mammalian Mediator complex could function in a specific to smooth muscle differentiation. Our data also suggest that MED28 may function as a scaffolding protein by maintaining the stability of this and the of MED28 may for this submodule to smooth muscle differentiation. Mediator complex is to for transcription of genes the RNA polymerase II the presence of specific subunits to determine the regulation of distinct expression programs interactions with transcriptional activators. The of subunit composition of the Mediator complex as a for specificity of gene expression also been (9Malik S. Kim R.G. Trends Biochem. Sci. 2005; 30: 256-263Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, S. Kim Roeder R.G. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Mediator subunits a crucial role in the activation of eukaryotic mRNA synthesis through interactions with transcriptional RNA polymerase transcription and other Mediator complex also the to function in transcriptional repression when associated with kinase module subunits such as MED12, and (9Malik S. Kim R.G. Trends Biochem. Sci. 2005; 30: 256-263Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 10Kornberg R.D. Trends Biochem. Sci. 2005; 30: 235-239Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). The of different kinase with Mediator is thought to regulate a distinct of genes S. Kim C. Parmely T.J. Florens L. Zybailov B. Swanson S.K. Banks C.A. Jin J. Cai Y. Washburn M.P. Conaway J.W. Conaway R.C. Mol. Cell. 2004; 14: 685-691Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, A.M. Kim D.J. W. E. Tjian R. 2002; PubMed Scopus Google Scholar). recent report that the of subunits in Mediator that Mediator through MED28 of kinase module components and RNA polymerase II A.C. Kim T.J. Tomomori-Sato C. S. D. Conaway R.C. Conaway J.W. Florens L. Washburn M.P. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: PubMed Scopus Google Scholar). Our for the first time that MED28 functions as a repressor of SMC differentiation, are in concordance with this report and the that the presence of the kinase module could a role in this negative regulation. we out the that MED28 can an and a by or cellular specificity as in the of a transcription which differentially to or Mediator complexes as determined by a X. Kim E. A. Mol. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). that out is that MED28 may in function in an the expression of an SMC repressor and as a repressor of SMC SMCs are and from of are and can and in to in SMC growth and differentiation are in a of disorders such as atherosclerosis, hypertension, and smooth muscle Kim Rev. 2004; PubMed Scopus Google Scholar). muscle genes are by a transcription that within SMC gene and transcription to transcription of SMC genes Kim 2004; 14: PubMed Scopus Google Scholar). transcription are in the and to the nucleus in to signaling Res. PubMed Scopus Google Scholar). The presence of MED28 in the and in the nucleus the whether of MED28 could also a role in controlling its function in SMC In to repression of SMC genes is for the of SMCs, and recent studies have several repressor Kim J. PubMed Scopus Google Scholar). growth is a of SMC differentiation, and several transcription and phosphorylated a crucial role in growth SMC suppression Kim J. PubMed Scopus Google Scholar, Y. Kim S. Y. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). recent identified (for in smooth a novel protein, as a transcriptional repressor of smooth muscle cells C.A. Kim M. G. S. Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar). Our results presented here MED28 and associated Mediator subunits to the of SMC and the whether MED28 can either suppress the expression of with binding to studies are to the by which MED28 functions as an SMC with