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• W2024589812 abstract "Serum response factor (SRF) plays a pivotal role in cardiac myocyte development, muscle gene transcription, and hypertrophy. Previously, elevation of intracellular levels of Ca2+ was shown to activate SRF function without involving the Ets family of tertiary complex factors through an unknown regulatory mechanism. Here, we tested the hypothesis that the chromatin remodeling enzymes of class II histone deacetylases (HDAC4) regulate SRF activity in a Ca2+-sensitive manner. Expression of HDAC4 profoundly repressed SRF-mediated transcription in both muscle and nonmuscle cells. Protein interaction studies demonstrated physical association of HDAC4 with SRF in living cells. The SRF/HDAC4 co-association was disrupted by treatment of cells with hypertrophic agonists such as angiotensin-II and a Ca2+ ionophore, ionomycin. Furthermore, activation of Ca2+/calmodulin-dependent protein kinase (CaMK)-IV prevented SRF/HDAC4 interaction and derepressed SRF-dependent transcription activity. The SRF·HDAC4 complex was localized to the cell nucleus, and the activated CaMK-IV disrupted HDAC4/SRF association, leading to export of HDAC4 from the nucleus and stimulation of SRF transcription activity. Thus, these results identify SRF as a functional interacting target of HDAC4 and define a novel tertiary complex factor-independent mechanism for SRF activation by Ca2+/CaMK-mediated signaling. Serum response factor (SRF) plays a pivotal role in cardiac myocyte development, muscle gene transcription, and hypertrophy. Previously, elevation of intracellular levels of Ca2+ was shown to activate SRF function without involving the Ets family of tertiary complex factors through an unknown regulatory mechanism. Here, we tested the hypothesis that the chromatin remodeling enzymes of class II histone deacetylases (HDAC4) regulate SRF activity in a Ca2+-sensitive manner. Expression of HDAC4 profoundly repressed SRF-mediated transcription in both muscle and nonmuscle cells. Protein interaction studies demonstrated physical association of HDAC4 with SRF in living cells. The SRF/HDAC4 co-association was disrupted by treatment of cells with hypertrophic agonists such as angiotensin-II and a Ca2+ ionophore, ionomycin. Furthermore, activation of Ca2+/calmodulin-dependent protein kinase (CaMK)-IV prevented SRF/HDAC4 interaction and derepressed SRF-dependent transcription activity. The SRF·HDAC4 complex was localized to the cell nucleus, and the activated CaMK-IV disrupted HDAC4/SRF association, leading to export of HDAC4 from the nucleus and stimulation of SRF transcription activity. Thus, these results identify SRF as a functional interacting target of HDAC4 and define a novel tertiary complex factor-independent mechanism for SRF activation by Ca2+/CaMK-mediated signaling. Serum response factor (SRF) 1The abbreviations used are: SRF, serum response factor; SRE, serum response element; DAPI, 4′,6-diamidino-2-phenylindole; HAT, histone acetyltransferase(s); HDAC, histone deacetylase; CREB, cAMP-response element-binding protein; PBS, phosphate-buffered saline; CaMK, Ca2+/calmodulin-dependent protein kinase; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate; TSA, trichostatin A; FITC, fluorescein isothiocyanate; SRF-FL, full-length SRF; ANF, atrial naturatic factor; MHC, myosin heavy chain.1The abbreviations used are: SRF, serum response factor; SRE, serum response element; DAPI, 4′,6-diamidino-2-phenylindole; HAT, histone acetyltransferase(s); HDAC, histone deacetylase; CREB, cAMP-response element-binding protein; PBS, phosphate-buffered saline; CaMK, Ca2+/calmodulin-dependent protein kinase; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate; TSA, trichostatin A; FITC, fluorescein isothiocyanate; SRF-FL, full-length SRF; ANF, atrial naturatic factor; MHC, myosin heavy chain. is a key regulator of several extracellular stimuli-regulated genes important for cell growth, apoptosis, and differentiation. SRF was first identified by its ability to confer the serum-activated expression of the c-fos gene in replicating cells (1Greenberg M.E. Siegfried Z. Ziff E.B. Mol. Cell. Biol. 1987; 7: 1217-1225Crossref PubMed Scopus (154) Google Scholar). Paradoxically, SRF was also found to regulate expression of several muscle genes, which are expressed specifically in postmitotic myocytes (2Boxer L.M. Prywes R. Roeder R.G. Kedes L. Mol. Cell. Biol. 1989; 9: 515-522Crossref PubMed Scopus (116) Google Scholar). SRF acts through binding as a homodimer to the DNA consensus sequence CC(A/T)6GG, the serum response element (SRE), also referred as the CArG box, which is found essential for tissue-specific expression of numerous striated as well as smooth muscle-specific genes (2Boxer L.M. Prywes R. Roeder R.G. Kedes L. Mol. Cell. Biol. 1989; 9: 515-522Crossref PubMed Scopus (116) Google Scholar, 3Treisman R. Curr. Opin. Gen. Dev. 1994; 4: 96-101Crossref PubMed Scopus (618) Google Scholar). SRF plays a central role in the induction and maintenance of cardiac myogenic program, as exemplified by disruption of the SRF gene, which prevented mesoderm differentiation and cardiac development (4Arsenian S. Weinhold B. Oelgeschlager M. Ruther U. Nordheim A. EMBO J. 1998; 17: 6289-6299Crossref PubMed Scopus (305) Google Scholar). SRF is differentially expressed in embryonic and adult cardiac myocytes, being at least 2 orders of magnitude greater than those detected in cells of endodermal origin (5Belaguli N.S. Schildmeyer L.A. Schwartz R.J. J. Biol. Chem. 1997; 272: 18222-18231Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). SRF has the ability to physically interact and synergistically cooperate with many other known cardiac-myogenic factors, such as GATA-4, Nkx2.5, TEF-1, myocardin, and CRP1/2 (6Belaguli N.S. Sepulveda J.L. Nigam V. Charron F. Nemer M. Schwartz R.J. Mol. Cell. Biol. 2000; 20: 7550-7558Crossref PubMed Scopus (155) Google Scholar, 7Chen C.Y. Schwartz R.J. Mol. Cell. Biol. 1996; 16: 6372-6384Crossref PubMed Google Scholar, 8Gupta M. Kogut P. Davis F.J. Belaguli N.S. Schwartz R.J. Gupta M.P. J. Biol. Chem. 2001; 276: 10413-10422Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 9Wang D. Chang P.S. Wang Z. Sutherland L. Richardson J.A. Small E. Krieg P.A. Olson E.N. Cell. 2001; 105: 851-862Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar, 10Chang D.F. Belaguli N.S. Iyer D. Roberts W.B. Wu S.P. Dong X.R. Marx J.G. Moore M.S. Beckerle M.C. Majesky M.W. Schwartz R.J. Dev. Cell. 2003; 4: 107-118Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Functionally relevant CArG boxes in the promoter regions of several cardiac-restricted, contractile, Ca2+-transporting, and metabolic protein genes have been identified, indicating a direct role of SRF in their transcriptional regulation (6Belaguli N.S. Sepulveda J.L. Nigam V. Charron F. Nemer M. Schwartz R.J. Mol. Cell. Biol. 2000; 20: 7550-7558Crossref PubMed Scopus (155) Google Scholar, 11Cheng G. Hagen T.P. Dawson M.L. Barnes K.V. Menick D.R. J. Biol. Chem. 1999; 274: 12819-12826Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 12Moore M.L. Wang G.L. Belaguli N.S. Schwartz R.J. McMillin J.B. J. Biol. Chem. 2001; 276: 1026-1033Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). A large body of evidence also indicates that SRF-containing complexes are the end point targets in pathways associated with conversion of hypertrophic stimuli to cardiac cellular response (e.g. induction of “fetal gene program”) where re-expression of genes abundant in the embryonic heart occur (including activation of ANF, skeletal α-actin, and β-MHC genes, and repression of SRCa2+ATPase and α-MHC genes) (13Paradis P. MacLellan W.R. Belaguli N.S. Schwartz R.J. Schneider M.D. J. Biol. Chem. 1996; 271: 10827-10833Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 14Thuerauf D.J. Arnold N.D. Zechner D. Hanford D.S. DeMartin K.M McDonough P.M. Prywes R. Glembotski C.C. J. Biol. Chem. 1998; 273: 20636-20643Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 15Bristow M.R. Lancet. 1998; 352: 8-14Abstract Full Text Full Text PDF Google Scholar, 16Zhang X. Azhar G. Chai J. Sheridan P. Nagano K. Brown T. Yang J. Khrapko K. Borras A.M. Lawitts J. Misra R.P. Wei J.Y. Am. J. Physiol. 2001; 280: H1782-H1792Crossref PubMed Google Scholar, 17Hines W.A. Thorburn J. Thorburn A. Mol. Cell. Biol. 1992; 19: 1841-1852Crossref Scopus (25) Google Scholar). However, the underlying mechanism that enables SRF to transduce intracellular signals to the hypertrophic response of cardiac myocytes is not yet known. Several mechanisms have been shown to regulate the SRF transcription activity, including physical interaction of SRF with a number of positive and negative cofactors (6Belaguli N.S. Sepulveda J.L. Nigam V. Charron F. Nemer M. Schwartz R.J. Mol. Cell. Biol. 2000; 20: 7550-7558Crossref PubMed Scopus (155) Google Scholar, 7Chen C.Y. Schwartz R.J. Mol. Cell. Biol. 1996; 16: 6372-6384Crossref PubMed Google Scholar, 8Gupta M. Kogut P. Davis F.J. Belaguli N.S. Schwartz R.J. Gupta M.P. J. Biol. Chem. 2001; 276: 10413-10422Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 9Wang D. Chang P.S. Wang Z. Sutherland L. Richardson J.A. Small E. Krieg P.A. Olson E.N. Cell. 2001; 105: 851-862Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar, 10Chang D.F. Belaguli N.S. Iyer D. Roberts W.B. Wu S.P. Dong X.R. Marx J.G. Moore M.S. Beckerle M.C. Majesky M.W. Schwartz R.J. Dev. Cell. 2003; 4: 107-118Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), phosphorylation-dependent change in the DNA and/or protein binding ability of SRF (18Janknecht R. Hipskind R.A. Houthaeve T. Nordheim A. Stunnenberg H.G. EMBO J. 1992; 11: 1045-1054Crossref PubMed Scopus (106) Google Scholar), regulated nuclear translocation of SRF (19Camoretti-Mercado B. Liu H.W. Halayko A.J. Forsythe S.M. Kyle J.W. Li B. Fu Y. McConville J. Kogut P. Vieira J.E. Patel N.M. Hershenson M.B. Fuchs E. Sinha S. Miano J.M. Parmacek M.S. Burkhardt J.K. Solway J. J. Biol. Chem. 2000; 275: 30387-30393Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), and alternative splicing of SRF mRNA primary transcript (20Kemp P.R. Metcalfe J.C. Biochem. J. 2000; 345: 445-451Crossref PubMed Google Scholar, 21Belaguli N.S. Zhou W. Trinh T.T. Majesky M.W. Schwartz R.J. Mol. Cell. Biol. 1999; 19: 4582-4591Crossref PubMed Scopus (86) Google Scholar). Recently, we have shown that an alternative spliced isoform of SRF is highly expressed in the failing hearts of both humans and animals, which act as a dominant negative isoform to repress SRF-dependent cardiac muscle gene expression (22Davis F.J. Gupta M. Pogwizd S.M. Bacha E. Jeevanandam V. Gupta M.P. Am. J. Physiol. 2001; 282: H1521-H1533Google Scholar). While studying the mechanism of SRF in the hypertrophic growth of cardiac myocytes, several recent reports have evoked our interest to examine the role of chromatin-remodeling enzymes, histone acetyltransferases (HAT) and deacetylases (HDAC), in SRF-mediated cardiac muscle gene activation. By changing the acetylation state of histones, histone acetylases/ deacetylases modify the chromatin structure that alters the accessibility of DNA to transcription factors. These enzymes are often recruited as cofactor complexes on the promoter of different genes, where they associate with transcription factors involved in expression of the target gene (23Imhof A. Yang X.J. Ogryzko V.V. Nakatani Y. Wolffe A.P. Ge H. Curr. Opin. Biol. 1997; 7: 689-692Abstract Full Text Full Text PDF Scopus (531) Google Scholar). During differentiation of P19 cells to smooth muscle cells, SRF was shown to be recruited to target genes along with hyperacetylation of histones at smooth muscle-specific regulatory regions of chromatin (24Manabe I. Owens G.K. Circ. Res. 2001; 88: 1127-1134Crossref PubMed Scopus (141) Google Scholar). Similarly, SRF and the HAT-containing co-activator, cAMP-response element binding protein (CREB)-binding protein, was shown recruited to the CArG box of the SM22 promoter during gene activation (25Qiu P. Li L. Circ. Res. 2002; 90: 858-865Crossref PubMed Scopus (81) Google Scholar). SRF also physically interacts with the HAT-containing activators, CREB-binding protein/p300, during CArG box-mediated activation of the c-fos gene expression (26Ramirez S. Ait-Si-Ali S. Robin P. Trouche D. Harel-Bellan A. J. Biol. Chem. 1997; 272: 31016-31021Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Moreover, Rho-A signaling mediated activation of SRF function on the c-fos promoter was observed only when local chromatin was hyperacetylated, indicating that histone modulation is required for the conversion of intracellular signals to cellular response via a CArG box-dependent mechanism (27Alberts A.S. Geneste O. Treisman R. Cell. 1998; 92: 475-487Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Recently, several myogenic factors, including MyoD, twist and another MADS-containing factor, MEF2, have been shown to associate directly with cofactors having HAT (CREB-binding protein, p300, p300/CBP-associated factor) or HDAC (HDAC4/5) activities, leading to a change in their muscle gene activation potential (28Hamamori Y. Sartorelli V. Ogryzko V. Puri P.L. Wu H.Y. Wang J.Y. Nakatani Y. Kedes L. Cell. 1999; 96: 405-413Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 29Sartorelli V. Huang J. Hamamori Y. Kedes L. Mol. Cell. Biol. 1997; 17: 1010-1026Crossref PubMed Scopus (318) Google Scholar, 30Lu J. McKinsey T.A. Nicol R.L. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4070-4075Crossref PubMed Scopus (411) Google Scholar). Based on these reports, a pertinent question may be raised of whether SRF-mediated cardiac muscle gene transcription could also be regulated by histone acetylases/deactetylases and whether this regulation is altered by hypertrophic signals that up-regulate cardiac muscle gene expression? We were particularly interested in class II HDACs, because, as opposed to class I HDACs, the members of class II, which include HDAC4, -5, -7, and -9, have been shown to exhibit tissue-specific expression. HDAC4 and HDAC5 are expressed at high levels in the heart, skeletal muscle, and brain, where they have been suggested to be involved in cell differentiation and development (31Miska E.A. Langley E. Wolf D. Karlsson C. Pines J. Kouzarides T. Nucleic Acids Res. 2001; 29: 3439-3447Crossref PubMed Scopus (111) Google Scholar, 32Grozinger C.M. Hassig C.A. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4868-4873Crossref PubMed Scopus (642) Google Scholar, 33Wang A.H. Bertos N.R. Vezmar M. Pelletier N. Crosato M. Heng H.H. Th'ng J. Han J. Yang X.J. Mol. Cell. Biol. 1999; 19: 7816-7827Crossref PubMed Scopus (253) Google Scholar). In this study, we demonstrate that SRF directly associates with HDAC4 in cardiac myocytes via its MADS domain, resulting in repression of SRF gene activation potential. Both proteins, upon interaction, co-localize to the nucleus of cardiac myocytes. We also show that the SRF/HDAC4 association is a target for upstream signaling events that increase intracellular Ca2+ levels and activate CaMK signaling during hypertrophic growth of myocytes. The increased activity of Ca2+/CaMK signaling results in dissociation of SRF from HDAC4, leading to export of HDAC4 from the nucleus and restoration of the SRF transcriptional activity. These results demonstrate that under basal conditions the transcription activity of SRF is negatively controlled by HDAC4 and that this is reversed upon activation of Ca2+/CaMK signaling. The unmasking of SRF transcription activity in this manner through dissociation from HDACs could be a key regulatory mechanism involved in transducing multiple and diverse intracellular signals that activate SRF-dependent cardiac muscle gene transcription. Primary cultures of cardiac myocytes were prepared from 2-day-old neonatal rats as previously described (34Nemoto, S., Sheng, Z., and Lin, A. Mol. Cell. Biol., 18, 3518–3526Google Scholar). After differential plating to eliminate fibroblasts, myocytes were further purified using a Percoll density gradient (Amersham Biosciences) and plated at a density of 4 × 106 in 100-mm plates, precoated with 2% gelatin. Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 units/ml penicillin and 100 mg/ml streptomycin. COS and C2C12 cells were typically plated in Falcon six-well tissue culture dishes at a density of ∼3–5 × 105 cells/well and maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and penicillin/streptomycin combination. Cells were transfected 48 h after plating, using LipofectAMINE reagent (Invitrogen) according to the manufacturer's protocol. The pCMV-β gal was used as a reference plasmid in all transfections. After 48 h, transfected cells were harvested, and cell lysates were prepared and assayed for luciferase (Luciferase Assay System, Promega, Madison, WI), β-galactosidase activities, and protein content (Bio-Rad protein reagent). pcDNA3Gal4-HDAC4 (designated as Gal-HDAC4), pcDNA3Gal4-HDAC4-D840N (designated as Gal-HDACD840N), pcDNA3Myc-HDAC4 (having a Myc tag at the COOH terminus of HDAC4), and pcDNA3Myc-HDAC4-D840N were kindly provided by Dr. T. Kouzarides (Wellcome/CRC Institute, University of Cambridge, Cambridge, UK) and have been described (35Miska E.A. Karlsson C. Langley E. Nielsen S.J. Pines J. Kouzarides T. EMBO J. 1999; 18: 5099-5107Crossref PubMed Scopus (455) Google Scholar). Luciferase reporter constructs for skeletal α-actin and expression vectors pCGN and pCGNSRF-FL have been previously described (7Chen C.Y. Schwartz R.J. Mol. Cell. Biol. 1996; 16: 6372-6384Crossref PubMed Google Scholar). pBS-SRF constructs having deletions of different regions of SRF were kindly provided by Dr. R. Prywes (Columbia University, New York, NY). The reporter construct with five SREs was purchased from Stratagene. pcDNA-SRF-M (lacking exon 5) was obtained from Dr. P. Kemp (Cambridge University, Cambridge, UK) (20Kemp P.R. Metcalfe J.C. Biochem. J. 2000; 345: 445-451Crossref PubMed Google Scholar). pcDNA-SRF-S (lacking exons 4 and 5) has been described elsewhere (22Davis F.J. Gupta M. Pogwizd S.M. Bacha E. Jeevanandam V. Gupta M.P. Am. J. Physiol. 2001; 282: H1521-H1533Google Scholar). pcDNA3-CaMK-IVdCT (active) and pcDNA3-CaMK-IVdCTK75E (kinase-dead) plasmids were provided by Dr. D. L. Black (Howard Hughes Medical Institute, UCLA, Los Angeles, CA) (36Jiuyong X. Black D.L. Nature. 2001; 410: 936-939Crossref PubMed Scopus (204) Google Scholar). The plasmids SR-α-CaMK-II active (α-CAMK-T286D) and SR-α-CaMK-II inactive (α-CaMK-K42M) were provided by Dr. M. R. Rosner (The Ben May Institute, University of Chicago) (37Waldmann R. Hanson P.I. Schulman H. Biochemistry. 1990; 29: 1679-1684Crossref PubMed Scopus (102) Google Scholar). The plasmid pCMV-p300 was obtained from Dr. D. M. Livingston (Dana-Faber Institute, Harvard University, Boston, MA) (38Eckner R. Ewen M.E. Newsome D. Gerdes M. DeCaprio J.A. Lawrence J.B. Livingston D.M. Genes Dev. 1994; 8: 869-884Crossref PubMed Scopus (920) Google Scholar). Unless otherwise specified, all common salts and reagents were obtained from Sigma. Whole cell lysates from frozen tissues or from cultured cells were prepared according to the method described before (22Davis F.J. Gupta M. Pogwizd S.M. Bacha E. Jeevanandam V. Gupta M.P. Am. J. Physiol. 2001; 282: H1521-H1533Google Scholar). For co-immunoprecipitation studies, whole cell lysate (∼500–700 μg of protein) was first incubated with 0.5 μg of normal rabbit IgG and 20 μl of protein A/G-agarose beads at 4 °C for 30 min. The precleared lysate was obtained by separation of beads by centrifugation at 1,000 × g for 5 min at 4 °C and incubated with 20 μg of anti-SRF antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4 °C for 60 min. The absence of primary antibody in parallel reaction mixes served as negative control. Protein A/G-agarose (20 μl) was then added to each sample and incubated at 4 °C overnight on a rotating device. Pellets were obtained by centrifugation, washed four times in phosphate-buffered saline (PBS; 9.1 mm dibasic sodium phosphate, 1.7 mm monobasic sodium phosphate, and 150 mm sodium chloride, pH 7.4) and reconstituted in 50 μl of PBS. The SRF immunoprecipitate (25 μl) or the negative control was subjected to SDS-PAGE on a 10% gel. Proteins were transferred to polyvinylidene difluoride membrane in a tank transfer system with a buffer (25 mm Tris-HCl, pH 8.3, 0.192 m glycine, 20% methanol), at 4 °C overnight. Membranes were blocked with 10% nonfat milk in PBST (PBS, Tween 20, 0.5%). Anti-HDAC4 (H-92) or anti-Gal4 (RK5C1) polyclonal antibody (1:500) (Santa Cruz Biotechnology) was used as the primary antibody. The immunoblot analysis was carried out with the appropriate secondary antibody (1:2000) coupled to horseradish peroxidase. An enhanced chemiluminescence kit (Amersham Biosciences) was used for the signal detection. The blots were routinely stripped and reprobed with the SRF antibody to ensure expression of the SRF protein. GST Pull-down Assay—GST fusion proteins were expressed in bacteria and purified as described previously (9Wang D. Chang P.S. Wang Z. Sutherland L. Richardson J.A. Small E. Krieg P.A. Olson E.N. Cell. 2001; 105: 851-862Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar). Five micrograms of GST or GST-SRF-FL bound to glutathione-agarose beads was incubated with [35S]Myc-HDAC4 in a protein interaction buffer of the following composition: 20 mm HEPES, pH 7.5, 75 mm KCl, 1 mm EDTA, 2 mm MgCl2, 2 mm dithiothreitol, and 0.1% Nonidet P-40. (Note that to assay the role of Ca2+ on the binding of SRF and HDAC4, the basal protein interaction buffer was altered by the addition of increasing concentrations of Ca2+ (0–4 mm) or EGTA (10 mm).) The binding reaction was conducted for2hat room temperature on a rocking platform. The beads were then pelleted at 7500 rpm for 3 min, washed three times with 1× protein interaction buffer, and suspended in Laemmli buffer (Bio-Rad), and proteins bound to beads were resolved on SDS-PAGE and detected by autoradiography. In Vitro Co-immunoprecipitation (Myc Pull-down Assay)—Equal amounts (10-μl aliquots) of [35S]SRF-FL and [35S]Myc-HDAC4 were incubated in a co-immunoprecipitation buffer (50 mm Tris, pH 7.4, 75 mm NaCl, 5 mm EDTA, and 0.1% Nonidet P-40) for 1 h at room temperature. A negative control included a binding reaction with [35S]Myc-HDAC4 and either unprogrammed TNT lysate or 35S-labeled luciferase. One microgram of c-Myc antibody (mouse monoclonal; Santa Cruz Biotechnology) was added to each binding reaction, and the reaction was further allowed to incubate for 1 h at room temperature. Protein-agarose beads (10 μl) were then added, and reactions were continued at room temperature with continuous rocking for an additional 1 h. The beads were pelleted and washed three times with 1× co-immunoprecipitation buffer by repeated centrifugation at 7,500 rpm for 3 min. Beads were then resuspended in 20 μl of Laemmli sample buffer, denatured by boiling for 3 min, and run on a 10% SDS-PAGE gel. Full-length protein synthesis was confirmed on a separate SDS-PAGE gel. For mapping the domain of SRF required for its interaction with HDAC4, reactions with the various 35S-labeled fragments of SRF and [35S]Myc-HDAC4 were subjected to Myc pull-down assays as described above and visualized by autoradiography. In Vitro CaMK Phosphorylation of GST-SRF or Myc-HDAC4 —GST-SRF, Myc-HDAC4, or GST alone (1 μg) was incubated in a kinase reaction buffer (50 mm HEPES, pH 7.4, 10 mm MgCl2, 100 μm ATP, 5 mm CaCl2, 30 μg/ml calmodulin, and 0.6 ng/μl CaMK-II) in a 50-μl reaction volume. [γ-32P]ATP was added to specified reaction tubes at 0.1 μCi/reaction. Reactions were allowed to proceed at room temperature for 1 h. A 10-μl aliquot of the kinase reaction was subjected to SDS-PAGE to ensure phosphorylation of the proteins. A positive control included a known peptide substrate, autocamtide-2 (Biomol, Plymoth Meeting, PA). Subsequent GST pull-down assays were conducted with stated combinations of phosphorylated/unphosphorylated GST-SRF and phosphorylated/unphosphorylated Myc-HDAC4 as described above. Beads were pelleted, washed thoroughly, and resuspended in 50 μl of Laemmli's sample buffer. Protein bound to beads were resolved on 10% SDS-PAGE and subsequently analyzed by autoradiography. Double-stranded oligonucleotides were 5′-end-labeled with T4 polynucleotide kinase (Invitrogen) and [γ-32P]ATP. The binding reaction was carried out in a total volume of 25 μl containing ∼10,000 cpm (0.1–0.5 ng) of the labeled DNA, 2–5 μg of the specified nuclear extract, and 1 μg of poly(dI-dC). The binding buffer consisted of 10 mm Tris-HCl (pH 7.4), 100 mm NaCl, 0.1 mm EGTA, 0.5 mm dithiothreitol, 0.3 mm MgCl2, 8% glycerol, and 0.5 mm phenylmethylsulfonyl fluoride. After incubation at room temperature for 20 min, the reaction mixtures were loaded on 5% native polyacrylamide gels (44:1, acrylamide/bisacrylamide), and electrophoresis was carried out at 150 V in a 0.5× TBE buffer in a cold room. For competition and antibody experiments, unlabeled competitor DNA or the antibody was preincubated with nuclear extracts at room temperature for 15–20 min in the reaction buffers prior to the addition of the labeled DNA probe. Probe was human α-cardiac actin CArG (sense): 5′-AAG GGG ACC AAA TAA GGC AAG GTG G-3′. Myocytes grown on 2% gelatin-coated coverslips and COS cells grown on two-well chamber slides (Nalge Nunc International) were transfected with specified DNA and subjected to the following treatments. Cells were washed with PBS, fixed with ice-cold methanol for 5 min, rehydrated with 5% Triton-X, and permeabilized with 0.02% Nonidet P-40. Cells, shielded from light, were blocked with a 0.3% bovine serum albumin solution and incubated with specified primary antibody at room temperature for 2 h. For localization of overexpressed Myc-HDAC4, we utilized the fluorescein-conjugated c-Myc (9E10) FITC antibody (Santa Cruz Biotechnology). For localization of overexpressed full-length HA-SRF, we utilized the rhodamine-conjugated HA (F-7) antibody (HA (F-7) TRITC; Santa Cruz Biotechnology). After three rapid washes, cells were mounted using the Slow-Fade Antifade kit with DAPI (Molecular Probes, Inc., Eugene, OR). Cells were observed using a ×63 Planapo objective and photographed using a Zeiss Axioplan microscope equipped with a PXL cooled charged coupled device camera (Roper, Tucson, AZ). Cellular detail in each field was obtained by Nomarski (differential interference contrast) imaging. For color florescence in each field, nuclear DAPI staining was visualized using 330–380-nm excitation and 460–470-nm emission filters; FITC staining was visualized by 493–509-nm excitation and 510–550-nm emission filters; and rhodamine staining was visualized using a 556–580-nm excitation filter and 600–660-nm emission filters. Single images were processed by no neighbor deconvolution, using Openlab 3 (Improvision, Coventry, UK) with digital confocal processing. All imaging was carried out in the Cancer Center Digital Light Microscopy Laboratory at the University of Chicago. HDAC4 Repressed the Transcription Activity of SRF—To test the hypothesis that a functional relationship exists between SRF and HDAC4, cells were co-transfected with various combinations of expression plasmids encoding either full-length SRF or HDAC4 and the skeletal-α-actin reporter plasmid, containing –394/+24 bp promoter fragment linked to the luciferase gene. The promoter region of skeletal α-actin gene used here contained two CArG boxes and several other adjacent cis-regulatory elements (Fig. 1). Overexpression of HDAC4 reduced (>80%) the basal activity of the skeletal α-actin gene in COS cells as well as in differentiated C2C12 cells, whereas SRF overexpression activated (4-fold) expression of the reporter gene in both cell types. When SRF was co-expressed with HDAC4, the trans-activation effect of SRF was markedly suppressed (>75%) (Fig. 1). We then used an artificial promoter/reporter construct, which has five repeated and adjacent sequences of the CArG box motifs to determine whether this HDAC4-dependent repression was mediated through SRF binding to the CArG box sequence. The basal activity of this construct was also repressed (>80%) by overexpression of HDAC4 (Fig. 1), whereas HDAC4 abrogated SRF-mediated activation of the reporter gene activity. However, HDAC4 did not inhibit reporter activity of the plasmid in which CArG sequences were either mutated or deleted (not shown), thus indicating that HDAC4 repressed SRF-dependent transcription mediated through intact CArG box sequences. All members of the histone deacetylase superfamily contain a conserved Asp840 residue. Mutation of this residue (D840N) and of the analogous residue in members of class I HDACs (e.g. Asp176 in HDAC1) has been shown to eliminate the catalytic activity of the enzyme (35Miska E.A. Karlsson C. Langley E. Nielsen S.J. Pines J. Kouzarides T. EMBO J. 1999; 18: 5099-5107Crossref PubMed Scopus (455) Google Scholar, 39Hassig C.A. Tong J.K. Fleischer T.C. Owa T. Grable P.G. Ayer D.E. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3519-3524Crossref PubMed Scopus (327) Google Scholar). To determine whether the deacetylase activity of HDAC4 was responsible for the repression of SRF function, we examined the effect of a catalytic defective mutant of HDAC4 (D840N) on SRF-induced gene expression. As shown in Fig. 2A, HDAC4 with mutation of the Asp840 residue (D840N) also reduced (50%) SRF-mediated gene transcription, albeit to an extent lesser than what resulted from the wild-type HDAC4. This finding in" @default.
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• W2024589812 title "Calcium/Calmodulin-dependent Protein Kinase Activates Serum Response Factor Transcription Activity by Its Dissociation from Histone Deacetylase, HDAC4" @default.
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