MOLECULAR AND CELLULAR BIOLOGY, Feb. 2007, p. 1280–1295 Vol. 27, No. 40270-7306/07/$08.00�0 doi:10.1128/MCB.00882-06Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Histone Deacetylase 3 Interacts with and DeacetylatesMyocyte Enhancer Factor 2�†

Serge Gregoire,1 Lin Xiao,1¶ Jianyun Nie,1¶ Xiaohong Zhang,2 Minghong Xu,1 Jiarong Li,1Jiemin Wong,3 Edward Seto,2 and Xiang-Jiao Yang1*

Molecular Oncology Group, Department of Medicine, McGill University Health Centre, Montreal, Quebec H3A 1A1, Canada1;H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, Florida 336122;

and Department of Molecular and Cellular Biology, Baylor College of Medicine,One Baylor Plaza, Houston, Texas 770303

Received 17 May 2006/Returned for modification 26 June 2006/Accepted 30 November 2006

The myocyte enhancer factor 2 (MEF2) family of transcription factors is not only important for controllinggene expression in normal cellular programs, like muscle differentiation, T-cell apoptosis, neuronal survival,and synaptic differentiation, but has also been linked to cardiac hypertrophy and other pathological conditions.Lysine acetylation has been shown to modulate MEF2 function, but it is not so clear which deacetylase(s) isinvolved. We report here that treatment of HEK293 cells with trichostatin A or nicotinamide upregulatedMEF2D acetylation, suggesting that different deacetylases catalyze the deacetylation. Related to the trichos-tatin A sensitivity, histone deacetylase 4 (HDAC4) and HDAC5, two known partners of MEF2, exhibited littledeacetylase activity towards MEF2D. In contrast, HDAC3 efficiently deacetylated MEF2D in vitro and in vivo.This was specific, since HDAC1, HDAC2, and HDAC8 failed to do so. While HDAC4, HDAC5, HDAC7, andHDAC9 are known to recognize primarily the MEF2-specific domain, we found that HDAC3 interacts directlywith the MADS box. In addition, HDAC3 associated with the acetyltransferases p300 and p300/CBP-associatedfactor (PCAF) to reverse autoacetylation. Furthermore, the nuclear receptor corepressor SMRT (silencingmediator of retinoid acid and thyroid hormone receptor) stimulated the deacetylase activity of HDAC3 towardsMEF2 and PCAF. Supporting the physical interaction and deacetylase activity, HDAC3 repressed MEF2-dependent transcription and inhibited myogenesis. These results reveal an unexpected role for HDAC3 andsuggest a novel pathway through which MEF2 activity is controlled in vivo.

Protein lysine acetylation refers to transfer of the acetylmoiety from acetyl coenzyme A (acetyl-CoA) to the ε-aminogroup of a lysine residue and is an important posttranslationalmodification that has recently emerged and rivals phosphory-lation (41, 61). Proteins known to be subject to lysine acetyla-tion include histones, over 50 transcription factors, and variousother proteins (10, 40, 41, 61, 77). This dynamic modification iscontrolled by the opposing actions of acetyltransferases anddeacetylases in vivo. Histones were the first substrates identi-fied, so these two families of enzymes are known as histoneacetyltransferases (HATs) and histone deacetylases (HDACs),although most of them also act on nonhistone proteins. In thepast decade, many proteins have been shown to possess HDACactivity (4, 22, 39, 66, 78). On the basis of homology to buddingyeast counterparts, human HDACs are grouped into fourclasses, with HDAC1, -2, -3, and -8, homologs of yeast Rpd3,forming class I. Class II comprises HDAC4, -5, -6, -7, -9, and-10, which possess deacetylase domains highly related to that ofyeast Hda1. HDAC4, -5, -7, and -9 have similar domain orga-nization and thus belong to a subgroup known as class IIa.

Class III consists of SIRT1 and other Sir2-related proteins. Arecent phylogenetic analysis revealed that HDAC11 representsclass IV (21). Members of classes I, II, and IV are zinc-depen-dent enzymes and display some sequence similarity to eachother but show no homology to Sir2-related proteins, whichrequire NAD� for deacetylation.

Human HDACs have both nuclear and cytoplasmic func-tions. Within the nucleus, these enzymes regulate gene expres-sion and other DNA-templated processes. According to ge-nome-wide analysis (56, 70), orthologs from budding andfission yeast not only display a clear “division of labor” but alsoact cooperatively. Consistent with this, systematic expressionand RNA interference knockdown experiments in Drosophilamelanogaster S2 cells have recently revealed distinct roles fordifferent deacetylases (7, 14), raising the possibility thatHDACs in Drosophila or higher organisms cooperate witheach other or have overlapping roles. Among others, the fol-lowing lines of evidence suggest that this is the case. First, bothHDAC1 and SIRT1 bind to and deacetylate p53, thereby reg-ulating its stability, DNA-binding ability, and transcriptionalactivity (46, 47, 63). Second, these two deacetylases interactwith and deacetylate MyoD and BCL6 (2, 15). Third, HDAC4has been shown to deacetylate Runx3 (33), whereas Runx1 andRunx2 are known to interact with members of classes I and II(59, 65, 69). Fourth, both HDAC1 and SIRT1 associate withp300/CBP-associated factor (PCAF) (15, 73). Fifth, as integralsubunits of HDAC3 complexes (24, 44, 68), the nuclear recep-tor corepressors SMRT and nuclear receptor corepressor (N-CoR) also interact with class II HDACs (31, 36). Finally, both

* Corresponding author. Mailing address: Molecular Oncology Group,Royal Victoria Hospital, Room H5.41, McGill University Health Center,687 Pine Avenue West, Montreal, Quebec H3A 1A1, Canada. Phone:(514) 934-1934, ext. 34490. Fax: (514) 843-1478. E-mail: xiang-jiao.yang@mcgill.ca.

† Supplemental material for this article may be found at http://mcb.asm.org/.

¶ These authors contributed equally to this work.� Published ahead of print on 11 December 2006.

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HDAC6 and SIRT2 efficiently deacetylate �-tubulin acetylatedon lysine 40 to regulate microtubule structure (32, 50). Thus, ageneral notion is that multiple HDACs are able to interactwith and/or deacetylate the same target protein in mammaliancells. This notion has led us to investigate whether myocyteenhancer factor 2 (MEF2), a well-known partner of HDAC4and homologs, is also targeted by other HDACs.

Mammalian MEF2 proteins, MEF2A, -B, -C, and -D, pos-sess a conserved N-terminal domain for specific binding toAT-rich sequences on promoters of target genes that are im-portant for biological processes, such as skeletal myogenesis,cardiac muscle growth and differentiation, T-cell apoptosis,neuronal survival, postsynaptic differentiation, growth factorresponse, and stress management (13, 49, 60). MEF2 proteinsare also of pathological importance by playing a role in cardiachypertrophy (27, 49), coronary artery disease (18), acute lym-phoblastic leukemia (82), virus propagation (45), and perhapsneurodegenerative disorders (13, 60). To repress transcription,MEF2 proteins recruit corepressors such as class IIa HDACs(39, 49, 66). Calcium/calmodulin-dependent kinases (CaMKs)and protein kinases D (PKDs) phosphorylate these deacety-lases, promote their nuclear export, and relieve repression (49,66, 78). Mitogen-activated protein kinases, such as p38 andextracellular signal-regulated kinase 5, directly phosphorylateMEF2 and activate transcription (26, 37, 38, 74, 84). Someother phosphorylation events stimulate sumoylation of MEF2on a conserved lysine residue and repress transcription (17, 19,20, 29, 35, 60, 85, 86; reviewed in reference 79). Calcineurinreverses such phosphorylation and derepresses transcription(13, 19, 60). Acetyltransferases like p300 acetylate MEF2 onspecific lysine residues to potentiate transcription (48, 60, 85).

Although it is well-known that HDAC4 and other class IIaHDACs repress MEF2-dependent transcription, it remains un-clear whether they directly deacetylate MEF2. Here we reportthat HDAC3, but not HDAC4 and HDAC5, deacetylatesMEF2. This finding suggests a novel molecular mechanism bywhich MEF2 transcriptional activity is regulated in vivo. MEF2is of pathological importance, and HDAC inhibitors areemerging as promising therapeutic agents, so the HDAC3 linkalso sheds light on related therapeutic intervention.

MATERIALS AND METHODS

Cell culture. Human embryonic kidney HEK293 cells and mouse C3H10T1/2fibroblasts were maintained in Dulbecco’s modified Eagle’s medium (DMEM)(Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Sigma), peni-cillin, and streptomycin (Invitrogen). Mouse C2C12 cells were cultured in thesame medium containing 20% FBS.

Plasmid constructs. Mammalian expression plasmids for Flag- or hemagglu-tinin (HA)-tagged human MEF2D and MEF2C proteins, as well as baculovirusfor Flag-MEF2D, have been described elsewhere (20, 67). A full-length cDNAclone of mouse MEF2B (GenBank accession no. BC045147) was purchased fromOpen Biosystems, and the coding sequence was subcloned into a pcDNA3.1(�)derivative to express HA-tagged MEF2B (HA-MEF2B). For this, an oligonu-cleotide duplex consisting of MEF2BL1 (5�-AA TTC ATG GGG AGA AAGAAG ATC CA-3�) and MEF2BL2 (5�-GAT CTG GAT CTT CTT TCT CCCCAT G -3�) was linked to a BglII site overlapping with codons 7 and 8 of thecoding sequence. MEF2 mutants were generated by PCR with the Expandthermostable DNA polymerase (Roche) and subcloned into derivatives ofpcDNA3.1 (Invitrogen). Mutations were verified by sequencing. Expression vec-tors for short hairpin RNA (shRNA) specific to human and mouse HDAC3 werederived from pBS/U6 (83) and pSilencer3.0-H1 (Ambion), respectively. Anti-HDAC3 polyclonal rabbit antibody and expression plasmids for HDAC1, -2, -3,and -8 were previously described (43, 75). The HDAC3 mutant H134Q, which

contains the mutations H134Q and H135A, was engineered by PCR with primers(5�-CTG GTG GTC TGC AGG CTG CCA AGA AGT TTG-3� and 5�-CAAACT TCT TGG CAG CCT GCA GAC CAC CAGC-3�) cloned into a derivativeof pcDNA3.1 for expression of a Flag-tagged fusion protein. The mutations wereverified by DNA sequencing. Constructs for p300 (myc-tagged), SMRTs (shortform), and SMRTe (extended or long form) were provided by E. Chin (81), V.Giguere (6), and J. D. Chen (71), respectively. Baculovirus for Flag-tagged p300(Flag-p300) was a gift from Y. Nakatani (51), and anti-MEF2D polyclonal rabbitantibody was obtained from R. Prywes (28). Anti-PCAF polyclonal rabbit anti-body and baculovirus and the mammalian expression plasmid for Flag-PCAFwere described before (80).

Coimmunoprecipitation. To analyze the interactions of different HDACs withMEF2, expression plasmids for Flag-tagged HDACs were transfected intoHEK293 cells along with a construct expressing HA-MEF2. For analysis ofinteraction between HDAC3 and p300 (or PCAF), an expression plasmid forFlag- or HA-tagged HDAC3 was transfected into HEK293 cells along with aconstruct expressing Myc-p300 (or Flag-PCAF). About 48 h posttransfection,cells were washed twice with phosphate-buffered saline (PBS) and lysed in bufferK (20 mM sodium phosphate, pH 7.0, 150 mM KCl, 30 mM sodium pyrophos-phate, 0.1% NP-40, 5 mM EDTA, 10 mM NaF, 0.1 mM Na3VO4, and proteaseinhibitors) for extract preparation and affinity purification on M2 agarose(Sigma). Bound proteins were eluted with Flag peptide (Sigma), separated bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), anddetected by immunoblotting with anti-HA (Covance) and anti-Flag (Sigma)antibodies. PBS with 0.1% Tween 20 was used for membrane blocking andantibody incubation. Blots were developed with Supersignal chemiluminescentsubstrates (Pierce).

For analysis of interaction between endogenous proteins, subconfluentHEK293 and C2C12 cells were washed twice with PBS and lysed in buffer K.Soluble extracts were prepared for immunoprecipitation with anti-MEF2D oranti-PCAF polyclonal antibody, followed by immunoblotting with anti-HDAC3polyclonal antibody. To minimize detection interference from the immunoglob-ulin G (IgG) heavy chain (HDAC3 migrated as a 50- to 55-kDa band on anSDS-acrylamide gel), the light-chain-specific fraction of peroxidase-conjugatedmonoclonal mouse anti-rabbit IgG (catalog no. 211-032-171; Jackson Immunore-search Laboratories) was used as the secondary antibody.

Protein-protein interaction in vitro. Flag-tagged p300, PCAF, and MEF2Dwere expressed in Sf9 insect cells using recombinant baculoviruses. Extracts frominfected cells were used to immobilize these fusion proteins on M2 agarose.After rotation at 4°C for 10 min, agarose beads were incubated with putativeinteraction partners (i.e., MEF2D, HDAC3, HDAC4, SMRTs, and SMRTe),synthesized in vitro with TNT T7 RNA polymerase-coupled reticulocyte lysatesystem (Promega) in the presence of [35S]methionine (Amersham Biosciences).After rotation at 4°C for 1 h, agarose beads with bound proteins were washedfour times with buffer K. Bound proteins were eluted with Flag peptide forSDS-PAGE and autoradiography. Maltose-binding protein (MBP) pulldown as-says were performed as described previously (67).

Protein acetylation and deacetylation in vivo. Expression plasmids for Flag-tagged proteins were transfected into HEK293 cells as specified in the figurelegends. About 40 h posttransfection, cells were treated with 3 �M trichostatinA (TSA) for 1, 1.5, or 6 h. Extracts were prepared in buffer K containing 3 �MTSA for affinity purification on M2 agarose. Bound proteins were eluted withbuffer K containing Flag peptide for SDS-PAGE and immunoblotting with anti-Flag antibody (Sigma) and anti-acetyl lysine rabbit polyclonal antibody (CellSignaling Technology and ImmuneChem Pharmaceuticals Inc.). The latter anti-body needed overnight incubation and gentle washing. Specifically, membraneswere blocked in PBST (PBS plus 0.1 to 0.15% Tween 20) supplemented with20% horse serum (Invitrogen) at room temperature for 1 h, with gentle rocking.The membranes were then incubated with anti-acetyl lysine antibody (diluted1:300 in the blocking buffer) for 16 to 24 h at 4°C, with gentle agitation. After themembranes were washed four to six times with PBST (5 min each time at roomtemperature), they were incubated with peroxidase-conjugated secondary anti-body diluted in the blocking buffer and gently washed with PBST four to six times(5 min each time) prior to visualization with Supersignal chemiluminescentsubstrates.

Protein acetylation and deacetylation in vitro. Flag-tagged p300, PCAF, andMEF2D proteins were expressed in Sf9 cells, affinity purified on M2 agarose, andeluted with Flag peptide. Acetylation of the eluted proteins was carried out asdescribed previously (54).

For deacetylation, Flag-HDACs were expressed in and affinity purified fromHEK293 cells. Substrates were either acetylated in vitro or in vivo. For the latter,acetylated proteins were expressed in HEK293 cells with TSA treatment asspecified in figure legends and immobilized on M2 agarose. Beads were washed

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once with buffer H (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA, and0.1 mM phenylmethylsulfonyl fluoride [PMSF]) and mixed with affinity-purifiedFlag-HDAC proteins in 0.1 ml of buffer H to carry out deacetylation (67).Reaction tubes were rotated in a 37°C incubator for 2 h. The beads were washedonce with buffer K, and bound proteins were eluted in buffer K containing Flagpeptide. Acetylation levels of the eluted proteins were detected by immunoblot-ting with the anti-acetyl lysine antibody as described above.

For preparation of acetylated substrates in vitro, extracts from infected Sf9cells were used to immobilize Flag-tagged proteins on 40 �l of M2 agarose beads.After the agarose beads were washed four times with buffer K and once withbuffer A (50 mM Tris-HCl, pH 8.0, 10% glycerol, 1 mM dithiothreitol, 0.1 mMEDTA, 1 mM PMSF, and 10 mM sodium butyrate), they were mixed with bufferA (40 �l) and 2.5 nCi [14C]acetyl-CoA (51 mCi/mmol; Amersham Biosciences)for acetylation. Reaction tubes were rotated in a 30°C incubator for 1 h. After-wards, the beads were washed once with buffer H and mixed with purifiedFlag-HDAC proteins in buffer H for deacetylation. Reaction tubes were rotatedin a 37°C incubator for 2 h. The beads were washed once with 0.2 ml of buffer K,and bound proteins were eluted in 20 �l buffer K containing Flag peptide priorto SDS-PAGE and autoradiography.

Fluorescence microscopy and reporter gene and myogenic conversion assays.Fluorescence microscopy and the reporter gene and myogenic conversion assayswere performed as previously described (19, 20).

ChIP. C2C12 cells were grown in 10-cm dishes (five per time point) at 2 � 105

cells per dish and maintained in DMEM containing 20% FBS. Sixteen hourslater, the cells were transfected with the Flag-HDAC3 expression vector. Twen-ty-four hours posttransfection, cells were washed once with PBS and fed withDMEM containing 2% horse serum to induce differentiation. Chromatin immu-noprecipitation (ChIP) was carried out as described previously (42). On day 0 or3, cells were cross-linked in 1% formaldehyde for 10 min at room temperature.Briefly, cells were washed twice with cold PBS and harvested in PBS. The cellpellet was resuspended in 0.2 ml of ChIP lysis buffer (1% SDS, 10 mM EDTA,50 mM Tris-HCl, pH 8.1, and protease inhibitors) and incubated for 10 min onice. Cells were sonicated to obtain DNA fragments of about 300 to 2,000 bp(three pulses, 10 seconds each, at a power setting of 10 on a VirTis VirSonic 100sonicator linked to a microtip). After centrifugation at 4°C, the soluble chroma-tin was diluted in 1.2 ml of ChIP dilution buffer (1% Triton X-100, 2 mM EDTA,150 mM NaCl, and 20 mM Tris-HCl, pH 8.1) and then precleared with 45 �l ofprotein A/G-agarose beads. Ten percent of the precleared chromatin was kept asinput, and the rest was divided equally for immunoprecipitation with anti-Flag oranti-HA antibody (2 �l). After rotation overnight at 4°C, 45 �l of proteinA/G-agarose beads (preblocked by incubation with salmon sperm DNA) wasadded. After rotation for 2 h at 4°C, the beads were washed once by incubatingin 1 ml of wash buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mMTris-HCl, pH 8.1, and 150 mM NaCl) for 10 min at 4°C, with rotation. After abrief spin, the beads were similarly washed with 1 ml of buffer II (0.1% SDS, 1%Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, and 500 mM NaCl) andbuffer III (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, and 10mM Tris-HCl, pH 8.1). The beads were rinsed once with 1 ml Tris-EDTA andincubated in 0.15 ml of de-cross-linking buffer (1% SDS and 0.1 M NaHCO3)overnight at 65°C. The suspension was mixed with 1 ml of Wizard PCR prepsDNA purification resin (Promega). DNA fragments were eluted in 40 �l H2O,and 1 �l was used for 28 cycles of PCR with two primers (5�-TCT AGG CTGCCC ATG TAA GG-3� and 5�-CAT TCT TGG GAA AAC AAA CC-3�) span-ning the MEF2-binding site of the mouse muscle creatine kinase (MCK) pro-moter. The expected PCR fragment was 0.26 kb.

RESULTS

Multiple deacetylases target MEF2. To gain further insightsinto how lysine acetylation may coordinate with other modifi-cations such as phosphorylation and sumoylation in MEF2regulation, we sought to identify the responsible deacety-lase(s). As known partners of MEF2 (39, 49, 66), HDAC4 andhomologs were considered the first candidates. NeitherHDAC4 nor HDAC5 was found to deacetylate MEF2D (seebelow and data not shown), a ubiquitously expressed memberof the MEF2 family. Treatment of HEK293 cells with thedeacetylase inhibitor trichostatin A, however, promotedMEF2D acetylation (Fig. 1A, lanes 1 and 2), suggesting thatother zinc-dependent HDACs may be involved. Consistent

with the report that SIRT1 deacetylates MEF2 (85), nicotin-amide treatment upregulated MEF2 acetylation (lane 3).These findings suggest that different HDACs target MEF2acetylation in vivo.

Interaction of MEF2 with HDAC3. HDACs often directlyassociate with their substrates, so we next asked whether inaddition to HDAC4 and homologs, other HDACs directly bindto MEF2. Related to this, when HA-MEF2B was expressed inHEK293 cells, HA-MEF2B associated with Flag-HDAC3(data not shown). We thus investigated whether HDAC3 tar-gets MEF2D. For this, coimmunoprecipitation was first per-formed with epitope-tagged proteins. Expression plasmids forFlag-MEF2D and HA-HDAC3 were cotransfected intoHEK293 cells, and extracts were prepared for affinity purifica-tion on M2 agarose. Bound proteins were eluted by Flag peptidefor immunoblotting with anti-Flag and anti-HA antibodies. Asshown in Fig. 1B, HA-HDAC3 specifically coprecipitated withFlag-MEF2D, indicating that they are able to interact with eachother in vivo. We next determined whether MEF2D interactswith other members of class I deacetylases. For this, Flag-taggedHDAC1, -2, -3, and -8 were coexpressed individually with HA-MEF2D in HEK293 cells. Extracts were prepared for coimmu-noprecipitation and immunoblotting. Among these deacetylases,only HDAC3 coprecipitated with MEF2D (Fig. 1C). Moreover,the efficiency was comparable to that of HDAC4 or HDAC5 (Fig.1D). To analyze the interaction between endogenous HDAC3and MEF2D, anti-MEF2D antibody was used for coimmunopre-cipitation from extracts of normal HEK293 cells, which areknown to express MEF2D (20, 28). The precipitated proteinswere analyzed by immunoblotting with anti-MEF2D and anti-HDAC3 antibodies. This assay revealed that anti-MEF2D anti-body specifically coprecipitated HDAC3 (Fig. 1E), indicating thatendogenous HDAC3 and MEF2D proteins associate with eachother in HEK293 cells. Together, these results identify HDAC3as a class I deacetylase that interacts with MEF2D in vivo.

HDAC3 binds to the MADS box of MEF2. To determinewhich region of MEF2D mediates the interaction, we com-pared the full-length protein with a truncation mutant, 1-86,which contains the N-terminal 86 residues corresponding tothe MADS box and the adjacent MEF2-specific domain. Thesetwo regions mediate DNA binding and are highly conservedamong MEF2 proteins (49). As shown in Fig. 2A, HDAC3interacted with the mutant, suggesting that the DNA-bindingdomain mediates HDAC3 binding. Consistent with this find-ing, HDAC3 also coprecipitated with two MEF2C mutants,1-178 and 1-116, which contain the N-terminal 178 and 116residues, respectively (Fig. 2B). Like mutant 1-86, the DNA-binding domain is intact in these two mutants. These resultsare consistent with the fact that the DNA-binding domain ofMEF2D is highly conserved in MEF2C (49). Thus, the DNA-binding domain of MEF2 mediates HDAC3 interaction.

To test whether the interaction is direct, we carried out invitro pulldown assays. For this, three MEF2C mutants, 1-116,1-86, and 1-64, were expressed in Escherichia coli as MBPfusion proteins to pull down HDAC3 translated in vitro. Mu-tant 1-64 contains the N-terminal 64 residues and encompassesonly the MADS box. As shown in Fig. 2C, all three mutantswere able to interact with HDAC3, indicating that the MADSbox is sufficient for the interaction (Fig. 2C). Related to this,the C-terminal part of MEF2D failed to associate with

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HDAC3 (Fig. 2D). Since HDAC4, as well as HDAC5/7/9, iswell-known to interact with the DNA-binding domain ofMEF2, we tested whether coexpression of HDAC4 interfereswith HDAC3 binding. As shown in Fig. 2E, coexpression ofHDAC4 had no detectable effects on association of HDAC3with MEF2D. This is also consistent with the results presentedin Fig. 1D. Therefore, HDAC3 binds directly to the MADSbox, a domain that is located away from the MEF2-specificregion important for interaction with HDAC4/5/7/9.

Subcellular colocalization of MEF2 with HDAC3. We nextassessed association of HDAC3 with MEF2 by immunofluo-rescence microscopy. For this, HA-MEF2D and green fluores-cent protein-tagged HDAC3 (GFP-HDAC3) were expressedindividually or together in HEK293 cells. As expected, MEF2D

signals were found exclusively in the nucleus (Fig. 3A),whereas GFP-HDAC3 was enriched in the nucleus (Fig. 3B).When GFP-HDAC3 and MEF2D were coexpressed, GFP-HDAC3 remained predominantly nuclear, but some MEF2Dsignals were detectable in the cytoplasm (Fig. 3C). In thenucleus, MEF2D and HDAC3 colocalized to distinct dots in�50% transfected cells. These dots were observed with theHDAC3 mutant 1-313 but not with 122-428 (Fig. 3D), suggest-ing that the N-terminal region of HDAC3 is required for tar-geting MEF2 and HDAC3 to the nuclear dots. Consistent withthis, mutant 1-313, but not 122-428, coprecipitated withMEF2D (see Fig. S1 in the supplemental material). In �50%of the cells with coexpression of GFP-HDAC3 and HA-MEF2D, some HA signals were also detected in the cytoplasm

FIG. 1. Interaction of MEF2D with HDAC3. (A) HEK293 cells were transfected with an expression plasmid for Flag-MEF2D. Beforeharvesting, cells were treated with 3 �M trichostatin A for 6 h or 50 mM nicotinamide for 24 h. Extracts were prepared in buffer K containing therespective inhibitor for affinity purification on M2 agarose, and bound proteins were eluted with Flag peptide for Western blotting (WB) withanti-acetyl lysine (�Acetyl-K) or anti-Flag (�Flag) antibody. (B to D) An expression construct for Flag- or HA-tagged MEF2D was transfected intoHEK293 cells along with expression constructs for Flag- or HA-tagged HDACs as indicated. The exception is HDAC1, whose expression vectorwas for an HA- and Flag-tagged form, leading to a very faint band on lane 5 in panel C (marked with an asterisk). Extracts were prepared in bufferK for coimmunoprecipitation (IP) on M2 agarose, and bound proteins were eluted with Flag peptide for immunoblotting with anti-Flag or anti-HAantibody. The positions of molecular mass markers (in kilodaltons) are shown to the left of the gel. (E) HEK293 extracts were prepared in bufferK for immunoprecipitation with anti-GFP (�GFP) or anti-MEF2D antibody (�MEF2D) and immunoblotting with anti-HDAC3 or anti-MEF2Dantibody. For immunoblotting with the former, an IgG light chain (IgG L)-specific secondary antibody was used to avoid detection interferencefrom the IgG heavy chain (IgG H). The asterisk denotes a nonspecific signal in lane 1.

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FIG. 2. Mapping the HDAC3-binding site on MEF2. (A) Flag-tagged MEF2D and deletion mutant 1-86 were expressed with HA-HDAC3 inHEK293 cells as specified. Extracts were prepared in buffer K for purification on M2 agarose, and bound proteins were eluted with Flag peptidefor Western blotting (WB) with anti-HA (�HA) or anti-Flag (�Flag) antibody. (B) HA-tagged MEF2C mutants 1-116 (HA-1-116) and 1-178(HA-1-178) were expressed with Flag-HDAC3 in HEK293 cells as indicated. Extracts were prepared and analyzed as described above for panelA. IP, immunoprecipitation. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gel. (C) Extracts from bacteriaexpressing MEF2C fragments fused to MBP were incubated with amylose resin to immobilize the fusion proteins and pull down HA-HDAC3,synthesized in vitro in the presence of [35S]methionine. The bound proteins were resolved by SDS-PAGE for Coomassie blue staining (bottom)and autoradiography (top). Extracts with MBP fused to a small portion of �-galactosidase were used as a negative control (lane 2). About 20%of the total HA-HDAC3 protein used per binding reaction was analyzed on the input lane. (D) Extracts from Sf9 insect cells expressingFlag-HDAC3 were incubated with M2 agarose to immobilize this fusion protein to pull down MEF2D or its deletion mutant 148-522, synthesizedin vitro in the presence of [35S]methionine. Plain Sf9 extracts were used as a negative control (lanes 2 and 5). About 20% of each protein usedper binding reaction was analyzed on the input lanes. (E) HEK293 cells were transfected with expression constructs for Flag-MEF2D (2 �g) andHA-HDAC3 (6 �g) along with increasing amounts of the expression vector for HA-HDAC4 (0, 1, and 2 �g). Extracts were prepared and analyzedas described above for panel A.

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FIG. 3. Subcellular localization of MEF2D and HDAC3. (A to C) HA-MEF2D and GFP-HDAC3 were expressed individually (A and B) ortogether (C) in HEK293 cells for green fluorescence microscopy to detect GFP signals. Expression of HA-MEF2D was determined by immuno-staining with the anti-HA antibody and a Cy3-labeled secondary antibody. Hoechst 33258 was used to stain nuclei. (D) Same as panel C exceptthat GFP-tagged HDAC3 mutants 1-313 and 122-428 were expressed with HA-MEF2D as indicated.

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FIG. 4. Deacetylation of MEF2D by HDAC3. (A) The expression construct for hHDAC3i (shRNA specific to human HDAC3) (lane 1) or thecorresponding empty vector (lane 2) was transfected into HEK293 cells. Extracts were prepared for immunoblotting with anti-HDAC3 (�HDAC3)and anti-MEF2D (�MEF2D) antibodies. (B) The Flag-MEF2D expression plasmid was transfected into HEK293 cells along with the hHDAC3iexpression construct or the corresponding empty vector as indicated. Before harvesting, cells were treated with 3 �M trichostatin A for 1.5 h (�)

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(Fig. 3C and data not shown). In the cytoplasm, colocalizationbetween MEF2 and HDAC3 could be detected (Fig. 3C).Thus, consistent with protein-binding assays, the results offluorescence microscopic analysis support the notion thatMEF2D can colocalize with HDAC3 in vivo.

Deacetylation of MEF2 by HDAC3. Having established thatHDAC3 physically associated with MEF2 (Fig. 1 to 3) and thatTSA treatment elevated MEF2D acetylation in HEK293 cells(Fig. 1A), we then assessed the ability of HDAC3 to deacety-late MEF2. For this, a previously described shRNA expressionvector (83) was employed to deplete endogenous HDAC3 inHEK293 cells. As expected, the shRNA vector was effective inknocking down HDAC3 expression (Fig. 4A). As shown in Fig.4B, HDAC3 knockdown upregulated MEF2D acetylation, in-dicating that HDAC3 negatively regulates the acetylation invivo. Consistent with this, expression of wild-type HDAC3reduced MEF2D acetylation in HEK293 cells (Fig. 4C, lanes 1and 2). In contrast, expression of the deacetylase-deficientmutant H134Q slightly increased the acetylation (lane 3), per-haps due to a dominant-negative effect.

We next examined direct MEF2D deacetylation in vitro.Flag-tagged HDAC3 was expressed in HEK293 cells and af-finity purified on M2 agarose. For comparison, HDAC1, -2, -4,-5, and -8 were similarly prepared. Eluted proteins were incu-bated with acetylated Flag-MEF2D, which was separately ex-pressed in and purified from HEK293 cells. As shown in Fig.4D, HDAC3, but not the other three class I HDACs, deacety-lated MEF2D (lanes 1 to 5). Despite their well-characterizedinteraction of MEF2 (49, 66), neither HDAC4 nor HDAC5displayed detectable deacetylase activity towards MEF2D(lanes 6 and 7). Consistent with this, expression of CaMKIV-ca, a constitutively active form known to promote nuclearexport of HDAC4/5/7/9 (72, 78), had minimal effects onMEF2D acetylation (Fig. 4E).

We subsequently determined the ability of HDAC3 to re-verse MEF2 acetylation catalyzed by known acetyltransferases.As reported for MEF2C (48), PCAF and p300 efficiently acety-lated recombinant MEF2D protein (Fig. 4F, lanes 1 and 2, andFig. 4G, lane 1). Consistent with this, both acetyltransferasesphysically associated with MEF2D (Fig. 4H and data notshown) (58). In agreement with what was observed with acetyl-MEF2D purified from HEK293 cells (Fig. 4D), HDAC3 cat-alyzed the removal of [14C]acetyl groups from MEF2D acety-lated by PCAF and p300 in vitro (Fig. 4F, lane 3, and Fig. 4G,

lanes 2 and 3). Similarly, coexpression of HDAC3 inhibitedacetylation of MEF2D by PCAF and p300 in HEK293 cells(Fig. 4I). Taken together, the above results indicate thatHDAC3 deacetylates MEF2D in vitro and in vivo.

HDAC3 directly targets PCAF and p300. Reminiscent ofautophosphorylation of protein kinases, many HATs are auto-acetylated. Moreover, autoacetylation is important for activat-ing p300 (3, 62) and promoting nuclear localization of PCAF(57). During analysis of in vitro deacetylation of MEF2, wenoticed that HDAC3 inhibited autoacetylation of p300 andPCAF (Fig. 4F and G), suggesting that HDAC3 not only tar-gets MEF2 but also its acetyltransferases. To substantiate this,we first investigated whether HDAC3 could directly bind toPCAF and p300. Expression plasmids for Flag-PCAF and HA-HDAC3 were cotransfected into HEK293 cells. Proteins wereaffinity purified on M2 agarose for immunoblotting with anti-Flag and anti-HA antibodies. As shown in Fig. 5A, interactionbetween HDAC3 and PCAF could be readily detected. Inter-action between the endogenous proteins was then analyzed bycoimmunoprecipitation with anti-PCAF and anti-HDAC3polyclonal antibodies. As shown in Fig. 5B, anti-PCAF anti-body specifically coprecipitated endogenous HDAC3. Interac-tion of HDAC3 with p300 could also be detected in HEK293cells and in vitro (Fig. 5C and D). Functionally, coexpression ofHDAC3 promoted cytoplasmic localization of PCAF (Fig. 5Eand F), which is consistent with an earlier report that auto-acetylation is required for nuclear localization of PCAF (57).Therefore, HDAC3 not only deacetylates MEF2 but also actson its acetyltransferases.

SMRT promotes deacetylation of MEF2 and PCAF byHDAC3. As integral subunits of HDAC3 complexes (24, 44,68), the SMRT and N-CoR corepressors activate HDAC3 toefficiently deacetylate histone substrates (9, 23). However, itremains to be determined whether this is the case with otherprotein substrates. The ability of HDAC3 to deacetylate MEF2,PCAF, and p300 prompted us to ask whether the SMRT andN-CoR corepressors could activate HDAC3 to deacetylate thesenonhistone substrates. To address this, we performed in vitrodeacetylation assays in the presence of SMRTs and SMRTe,the short and extended isoforms of the SMRT corepressor,respectively (6, 71). SMRTe, but not SMRTs, contains theSANT domain required for activating the histone-deacetylat-ing activity of HDAC3 (9, 23). Coexpression of SMRTe but notSMRTs stimulated the deacetylase activity of HDAC3 towards

or not treated with 3 �M TSA (�). Extracts were prepared in buffer K supplemented with the inhibitor for affinity purification on M2 agarose,and bound proteins were eluted for Western blotting (WB) with anti-acetyl lysine (�Ac-K) or anti-Flag (�Flag) antibody. (C) The Flag-MEF2Dexpression plasmid was transfected into HEK293 cells with or without plasmids expressing Flag-tagged HDAC3 and mutant H134Q. Beforeharvesting, cells were treated with 3 �M TSA for 1 h. Extracts were prepared in buffer K with the inhibitor for immunoprecipitation on M2 agarose,and bound proteins were eluted for immunoblotting with anti-acetyl lysine (�Ac-K) or anti-Flag (�Flag) antibody. (D) Flag-HDAC proteins,expressed in and affinity purified from HEK293 cells, were used for in vitro deacetylation of Flag-MEF2D, which was expressed separately in andaffinity purified from HEK293 cells. Before harvesting, the cells expressing Flag-MEF2D were treated with 3 �M TSA for 6 h. Flag-MEF2D wasthen affinity purified on M2 agarose using buffer K containing 3 �M TSA. (E) Same as panel C except that the effects of HDAC4 and CaMKIV-ca(a constitutively active form) were analyzed. (F and G) Flag-tagged HDAC1 and HDAC3 proteins, affinity purified from HEK293 cells, were usedfor deacetylation of Flag-MEF2D, acetylated in vitro with [14C]acetyl-CoA by Flag-PCAF (F) or Flag-p300 (G). Flag-tagged MEF2D, PCAF, andp300 proteins were affinity purified from Sf9 cells. (H) Expression plasmids for Flag-PCAF and HA-MEF2D were transfected into HEK293 cellsindividually or in combination. Extracts were prepared for immunoprecipitation (IP) and immunoblotting or Western blotting (WB) with anti-Flagor anti-HA antibody as in Fig. 2A. (I) HEK293 cells were transfected with plasmids expressing the indicated proteins. Before harvesting, cells weretreated with 3 �M TSA for 1.5 h. Extracts were prepared in buffer K with the inhibitor for immunoprecipitation on M2 agarose, and bound proteinswere eluted for immunoblotting with anti-acetyl lysine or anti-Flag antibody.

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MEF2D in vitro (Fig. 6A). To test whether this is the case invivo, HEK293 cells were transfected with expression plasmidsfor Flag-MEF2D, HA-HDAC3, SMRTs, and/or SMRTe. Ex-pression of SMRTe, but not SMRTs, potentiated MEF2Ddeacetylation by HDAC3 (Fig. 6B). HDAC3 knockdown coun-teracted the effect of SMRTe expression (Fig. 6C), suggestingthat SMRTe acts through HDAC3. SMRTe also promoted

deacetylation of PCAF by HDAC3 (Fig. 6D). Therefore, asreported for histone substrates (9, 23), SMRTe stimulates thedeacetylase activity of HDAC3 towards both MEF2 andPCAF.

SMRTe could promote MEF2 deacetylation through thefollowing mechanisms: (i) directly affecting the structure ofHDAC3, (ii) enhancing the interaction between HDAC3

FIG. 5. Interaction of HDAC3 with PCAF and p300. (A) Expression plasmids for Flag-PCAF and HA-HDAC3 were cotransfected intoHEK293 cells as indicated. Extracts were prepared in buffer K for affinity purification on M2 agarose, and bound proteins were eluted for Westernblotting (WB) with anti-HA (�HA) or anti-Flag (�Flag) antibody. (B) HEK293 cells were washed and lysed in buffer K in situ to prepare extractsfor immunoprecipitation (IP) with anti-PCAF antibody (�PCAF) and immunoblotting with anti-HDAC3 or anti-PCAF antibody. Immunoblottingwith anti-HDAC3 antibody was carried out as described in the legend to Fig. 1E. (C) Flag-HDAC3 and Myc-tagged p300 were expressed andpurified as in described above for panel A by immunoblotting anti-Flag and anti-Myc monoclonal antibodies, respectively. (D) Extracts from Sf9cells expressing Flag-p300 (lane 3) or Flag-PCAF (lane 4) were used to immobilize these fusion proteins on M2 agarose for incubation withHA-HDAC3, synthesized in vitro in the presence of [35S]methionine. Plain Sf9 extracts were used as a negative control (lane 2). About 20% ofthe total HA-HDAC3 protein used per binding reaction was analyzed on lane 1. (E and F) Flag-PCAF was expressed alone (E) or withGFP-HDAC3 (F) in HEK293 cells. Green fluorescence microscopy was used to detect GFP signals. Expression of Flag-PCAF was visualized byimmunostaining with anti-Flag antibody and a Cy3-labeled secondary antibody. Hoechst 33258 was used to stain nuclei.

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and MEF2, and (iii) forming a trimeric enzyme-substratecomplex. To gain further mechanistic insights, we examinedthe interaction between MEF2D and HDAC3 in the pres-ence and absence of SMRT isoforms. For this, Flag-MEF2Dwas expressed in Sf9 cells and immobilized on M2 agarose topull down HDAC3, SMRT, and SMRTe translated in vitro.As shown in Fig. 2E, the presence of HDAC4 had minimaleffects on HDAC3 binding to MEF2D (Fig. 6E, lanes 1, 4 to6, and 10). Neither SMRT isoform affected the interaction

between HDAC3 and MEF2D (Fig. 6E, lanes 6 to 9). In-terestingly, SMRTe interacted with MEF2D (lane 9). Theseresults indicate that MEF2D, HDAC3, and SMRTe couldform a trimeric complex. Within this complex, HDAC3 maybe more active for MEF2D deacetylation. The above find-ings suggest that SMRT is a cofactor required for efficientdeacetylation of MEF2 and PCAF by HDAC3, although theprecise mechanism through which this occurs awaits furtherinvestigation.

FIG. 6. Effects of SMRT on deacetylation of MEF2D and PCAF by HDAC3. (A) Flag-HDAC3 was coexpressed with SMRTs or SMRTe inHEK293 cells to affinity purify deacetylase complexes for in vitro deacetylation of Flag-MEF2D, which was expressed separately in and affinitypurified from HEK293 cells. Before harvesting, cells expressing Flag-MEF2D were treated with 3 �M TSA for 6 h. After deacetylation, Flag-taggedproteins and the acetylation level of Flag-MEF2D were analyzed by Western blotting (WB) with anti-Flag (�Flag) and anti-acetyl lysine(�Acetyl-K) antibodies, respectively. (B) HEK293 cells expressing the indicated proteins were treated with 3 �M TSA for 1.5 h. Extracts wereprepared in buffer K in the presence of the inhibitor for immunoprecipitation on M2 agarose and immunoblotting with anti-Flag and anti-acetyllysine antibodies. (C) HEK293 cells were transfected with plasmids expressing Flag-MEF2D, SMRTe, and hHDAC3i as indicated. Beforeharvesting, cells were treated with 3 �M TSA for 1 h. Extracts were prepared in buffer K with the inhibitor for immunoprecipitation on M2 agarose,and bound proteins were eluted for immunoblotting with anti-acetyl lysine or anti-Flag antibody. (D) Flag-PCAF autoacetylated with [14C]acetyl-CoA was used for in vitro deacetylation. Flag-PCAF was expressed in and affinity purified from Sf9 cells. Flag-HDAC3 was coexpressed withSMRTe in HEK293 cells for affinity purification of the deacetylase complex. (E) Extracts from Sf9 cells expressing Flag-MEF2D were used toimmobilize this fusion protein on M2 agarose. Agarose beads were incubated, as indicated, with HA-HDAC3, SMRT isoforms, and/or HA-HDAC4, synthesized in vitro in the presence of [35S]methionine. Plain Sf9 extracts were used as a negative control (lane 5). For the labeledproteins, 20% of the total amount used per binding reaction was analyzed separately on lanes 1 to 4. The positions of molecular mass markers (inkilodaltons) are indicated to the left of the gel.

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FIG. 7. Repression of MEF2D-dependent transcription by HDAC3. (A) The luciferase reporter 3xMEF2-luc (0.2 �g) and a �-galactosidaseexpression plasmid (0.05 �g) were cotransfected into HEK293 cells with or without the expression vector for Flag-MEF2D (0.1 �g). Wherespecified, the expression vector for hHDAC3i (0.1 �g) or the empty shRNA vector (0.1 �g) was cotransfected. About 48 h posttransfection, cells

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Repression of MEF2-dependent transcription by HDAC3. Wenext performed reporter gene assays to assess whetherHDAC3 regulates the transcriptional activity of MEF2D. Forthis, 3xMEF2-Luc, a reporter with luciferase expression con-trolled by three copies of a MEF2-binding site (48), was used.As shown in Fig. 7A, TSA treatment of HEK293 cells stimu-lated the transcriptional activity of MEF2D, suggesting the in-volvement of zinc-dependent HDACs. To determine whetherHDAC3 plays a role, we expressed shRNA to knock downHDAC3 expression in HEK293 cells. As shown in Fig. 7A,expression of shRNA specific to human HDAC3 increasedMEF2D-dependent reporter activity. Expression of HDAC3,but not its point mutant H134Q, reduced the reporter activity(Fig. 7B). Similar to what was observed in HEK293 cells, ex-pression of shRNA specific to mouse HDAC3 in murineC3H10T1/2 fibroblasts potentiated MEF2-dependent tran-scription and expression of HDAC3 reduced the reporter ac-tivity (Fig. 7C). Moreover, expression of SMRTe repressed thereporter activity in a dose-dependent manner (Fig. 7C), sug-gesting that HDAC3 may cooperate with SMRT to repressMEF2-dependent transcription. We also analyzed how HDAC3affects the ability of PCAF to coactivate MEF2-dependenttranscription. As shown in Fig. 7C, PCAF potentiatedMEF2D-dependent transcription, but expression of HDAC3or SMRTe diminished this potentiation. Supporting the syn-ergy between SMRTe and HDAC3, knockdown of thisdeacetylase reduced the repressive effect of SMRTe (Fig. 7D).To assess the relative contribution of HDAC3 and HDAC4/5/7/9, we expressed CaMKIV-ca. As reported previously (49, 66),this kinase significantly relieved the repression of HDAC4expression (Fig. 7E). However, it had much smaller effectswhen no exogenous HDAC4 was present (Fig. 7F), suggestingthe existence of HDAC4/5/7/9-independent repression. Moreinterestingly, this kinase slightly stimulated the inhibitory ac-tivity of HDAC3 (Fig. 7E) but acted synergistically withHDAC3 knockdown to upregulate MEF2-dependent reporteractivity (Fig. 7F). Together, these results indicate that HDAC3represses MEF2-dependent transcription in a manner that isindependent of and different from HDAC4/5/7/9.

We also utilized MyoD-dependent myogenic conversion as-says to analyze the repressive effect of HDAC3 (55). For this,pluripotent C3H10T1/2 cells were cotransfected with a MyoDexpression construct along with the expression plasmid forMEF2D. As expected, wild-type MEF2D stimulated the myo-genic potential of MyoD (Fig. 8A and B). Importantly, thisactivity was reduced in the presence of HDAC3 (Fig. 8A andB). Consistent with results from reporter gene assays (Fig. 7),SMRTe cooperated with HDAC3 to downregulate the myo-genic activity of MEF2D (Fig. 8A and B), whereas HDAC3

knockdown increased the myogenic potential in a manner syn-ergistic with CaMKIV-ca expression (Fig. 8C). As shown inFig. 8D, ChIP assays revealed association of HDAC3 with amuscle-specific promoter with a known MEF2-binding site invivo. Together, these results suggest that interaction withHDAC3 and SMRTe keeps MEF2 in a repressed state in vivo.

Effect of myogenesis on the interaction of HDAC3 withMEF2. Results shown in Fig. 7 and 8A to D raise an intriguingissue as to whether the repressive role of HDAC3 is regulatedin vivo. We thus investigated how the interaction betweenHDAC3 and MEF2D may be modulated during myogenesis.For this, C2C12 cells were induced to differentiate in 2% horseserum. On days 0 and 3, cells were harvested in buffer K toprepare soluble extracts for immunoprecipitation with anti-MEF2D antibody. Precipitated proteins were analyzed by im-munoblotting with anti-MEF2D and anti-HDAC3 antibodies.As shown in Fig. 8E, interaction of HDAC3 with MEF2D wasstrong on day 0 but diminished on day 3 of differentiation (Fig.8E), suggesting that the interaction is signal responsive. There-fore, HDAC3 may inhibit MEF2-dependent transcription in asignal-dependent manner (see Discussion below).

DISCUSSION

HDAC3 interacts with and deacetylates MEF2. Numerousstudies have established HDAC4/5/7/9 as signal-responsivebinding partners of MEF2 transcription factors (39, 49, 66).Unexpectedly, these deacetylases were unable to deacetylateMEF2 (Fig. 4D and data not shown). Instead, HDAC3deacetylated MEF2D in vitro and in vivo (Fig. 4). Under sim-ilar conditions, other class I HDACs exhibited no obviousdeacetylase activity towards MEF2D (Fig. 4D). In agreementwith this, HDAC3, but not HDAC1, -2, or -8, physically asso-ciated with MEF2D (Fig. 1). Interaction of HDAC3 withMEF2D is also supported by their subcellular colocalization(Fig. 3). Like MEF2D, both MEF2B and MEF2C appeared tobind HDAC3 (Fig. 2 and data not shown). Consistent with this,the HDAC3-binding site was mapped to the DNA-bindingdomain (Fig. 2), a region that is highly conserved among meta-zoan MEF2 proteins (49). Functionally, HDAC3 kept MEF2in a transcriptionally inactive state to prevent myogenesis (Fig.7 and 8). These results suggest that HDAC3 association may becommon to metazoan MEF2 proteins. Therefore, repressionby HDAC3 represents an important mechanism for MEF2regulation in vivo.

HDAC3 may target MEF2 through multiple mechanisms,including direct deacetylation and physical association. Re-lated to the latter, MEF2 may recruit HDAC3 to promoters oftarget genes for histone deacetylation (Fig. 8D and 9). In

were harvested for determination of luciferase and �-galactosidase activities. Treatment with TSA (0.3 �M) was carried out overnight beforeharvesting. Normalized luciferase activities from transfection without any effector plasmids were arbitrarily set at 1.0, and the values are shown asaverage values plus standard deviations (error bars) of three representative experiments. (B) Same as panel A except that expression plasmids forthe wild-type and H134Q mutant of HDAC3 were compared. (C) Same as panel A except that C3H10T1/2 cells were used. Expression vectors forshRNA specific to mouse HDAC3 (mHDAC3i) (0.15 or 0.4 �g), HA-HDAC3 (0.15 or 0.4 �g), SMRTe (0.15 or 0.4 �g), and Flag-PCAF (0.05�g) were cotransfected as indicated. (D) Same as panel A except that the effect of HDAC3 knockdown on SMRTe-mediated repression wasdetermined. (E) Same as panel A except that the effect of CaMKIV-ca expression (0.2 �g) on repression by HDAC3 and HDAC4 was examined.(F) Same as panel A except that the synergy between HDAC3 knockdown and CaMKIV-ca expression was analyzed. In panels D and E, 0.2 �gof the Flag-MEF2D expression plasmid was used.

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FIG. 8. Inhibition of myogenic conversion by HDAC3. (A to C) An MyoD expression plasmid (0.4 �g) was transfected into murine C3H10T1/2fibroblasts along with constructs expressing Flag-MEF2D (0.6 �g), HA-HDAC3 (0.3 �g), SMRTe (0.3 �g), mHDAC3i (0.3 �g), and CaMKIV-ca(0.3 �g). On day 7, myosin heavy chain (MHC) expression was detected by indirect immunofluorescence microscopy with anti-MHC monoclonalantibody and a Cy3-labeled secondary antibody. Average values of three independent experiments are illustrated in panels A and C, with somerepresentative images shown in panel B. (D) C2C12 cells were transfected with the Flag-HDAC3 expression vector. Sixteen hours later, cells werewashed once with PBS and fed with DMEM containing 2% horse serum to induce differentiation. Cells were harvested in ChIP lysis buffer on day0 for chromatin immunoprecipitation with anti-Flag antibody to determine association of Flag-HDAC3 with the MCK promoter. An anti-HAantibody (aHA) was used as a negative control (lane 2). (E) C2C12 cells, maintained in DMEM with 20% FBS, were washed once with PBS andfed with DMEM containing 2% horse serum to induce differentiation. Cells were harvested in buffer K on days 0 and 3 to prepare extracts forimmunoprecipitation with anti-MEF2D antibody and immunoblotting with anti-HDAC3 and anti-MEF2D antibodies. An anti-GFP antibody wasused as a negative control for immunoprecipitation (IP) (lane 3). Western blotting (WB) with anti-HDAC3 antibody was carried out as describedin the legend to Fig. 1E.

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addition, HDAC3 is the catalytic subunit of deacetylase com-plexes containing the nuclear receptor corepressors SMRT andN-CoR (24, 44, 68). The extended form of SMRT formed atrimeric complex with HDAC3 and MEF2D (Fig. 6E). Inagreement with this, SMRT recruits MEF2 to nuclear dots(71). SMRT and N-CoR activate HDAC3 to deacetylate his-tones (9, 23). Similarly, SMRT acted synergistically withHDAC3 to deacetylate MEF2D (Fig. 6A and B). Moreover,SMRT repressed MEF2-dependent transcription (Fig. 7 and8). Therefore, in cooperation with SMRT and perhaps alsoN-CoR, HDAC3 associates with and deacetylates MEF2 fortranscriptional repression (Fig. 9).

HDAC3 targets autoacetylation of PCAF and p300/CBP. Inaddition to MEF2, HDAC3 associated with PCAF and p300(Fig. 5) and inhibited autoacetylation (Fig. 4F and G). More-over, the SMRT corepressor promoted PCAF deacetylation byHDAC3 (Fig. 6D). Autoacetylation is known to promote nu-clear localization of PCAF (57). Consistent with this, expres-sion of HDAC3 led to translocation of PCAF to the cytoplasm

(Fig. 5E and F). Similar to HDAC3, HDAC1 and SIRT1directly associate with PCAF (15, 73). Thus, multiple HDACsseem to target PCAF. Autoacetylation has been linked to stim-ulation of acetyltransferase activity and other functions of p300(3, 62). Its two neighboring acetylatable lysine residues aresubject to sumoylation and transcriptional repression (16). Re-lated to this, SIRT1 inhibits acetylation of p300 to upregulatesumoylation (5). By analogy, HDAC3 may act through theacetyltransferase activity and sumoylation of p300 to control itsability to potentiate MEF2-dependent transcription. Thesefindings suggest that deacetylation of acetyltransferases, suchas PCAF and p300, is a common mechanism by which differentHDACs repress transcription. Consistent with this, a recentreport showed that HDAC3 interacts with and deacetylatesCBP (8). Therefore, similar to PCAF, different HDACs act onp300 and CBP. These findings also suggest that in addition topromoter-associated histones, HDAC3 targets MEF2 and itsacetyltransferase coactivators (Fig. 9).

Cell signaling utilizes distinct pathways for MEF2 regula-tion. Mapping the HDAC3-binding site to the MADS box ofMEF2 (Fig. 2) raises two intriguing issues. First, this box isconserved in the MADS superfamily of transcription factors,so an interesting question is whether other MADS boxes alsoassociate with HDAC3. Of relevance, the serum response fac-tor SRF interacted with HDAC3 (see Fig. S2 in the supple-mental material). Second, dependent on its association withcoactivators or corepressors, MEF2 can activate or represstranscription (39, 49, 66). Reporter gene and myogenic con-version assays revealed that HDAC3 is a MEF2 corepressor(Fig. 7 and 8). Similar corepressors include Cabin1 (52) andHDAC4/5/7/9 (39, 49, 66), all of which bind to the DNA-binding domain of MEF2 (25). More interestingly, p300 bindsto a similar region (25, 58), suggesting that MEF2 may switchcoactivator and corepressor partners by direct competition. Inaddition, CaMKs phosphorylate HDAC4/5/7/9 (39, 49, 66, 78),as well as Cabin1 (52), to promote nuclear export of thesecorepressors and derepress MEF2-dependent transcription.Other kinases such as PKDs also negatively regulate nuclearlocalization of HDAC4 and homologs (11, 53, 64). HDAC3was found to bind the DNA-binding domain of MEF2 (Fig. 2).By analogy to Cabin1 and class IIa HDACs, the corepressorrole of HDAC3 needs to be regulated. In support of this,HDAC3 association with MEF2 diminished after C2C12 myo-blasts differentiated (Fig. 8E), and MEF2 acetylation was re-ported to increase during myogenesis (48), raising an impor-tant question as to how cell signaling regulates HDAC3association with MEF2 (Fig. 9). Potential candidates includeIB kinase � and other kinases known to regulate SMRT andN-CoR (1, 30, 34).

As a MEF2 corepressor, HDAC3 displays several differ-ences from HDAC4/5/7/9. (i) HDAC3 is widely expressed (12,76), whereas HDAC4/5/7/9 exhibit tissue-specific distribution(66). (ii) SMRT and N-CoR form stable deacetylase complexeswith HDAC3 and are required for the deacetylase activity (9,23, 24, 44, 68), so cell signaling pathways acting on SMRT andN-CoR may also regulate the activity of HDAC3. (iii) Whilethe nuclear localization of HDAC4/5/7/9 is negatively regu-lated by CaMK- or PKD-mediated phosphorylation and 14-3-3binding (49, 66), HDAC3 possesses no obvious sites for 14-3-3binding (12, 76), and at least CaMKIV did not block the re-

FIG. 9. Cartoon illustrating distinct pathways by which HDAC3and HDAC4/5/7/9 repress MEF2-dependent transcription. Associationwith p300, CBP, and PCAF acetylates MEF2 and proximal nucleo-somes (gray ovals) to activate transcription. HDAC3 cooperates withSMRT and perhaps also N-CoR to maintain MEF2 and the proximalnucleosomes in a hypoacetylated state for transcriptional repression.In addition to MEF2 and histones, HDAC3 deacetylates coactivatorslike PCAF, p300, and CBP. HDAC4/5/7/9 repress MEF2-dependenttranscription through multiple repression domains, with the deacety-lase domain perhaps deacetylating proximal nucleosomes. While it iswell established that CaMKs and PKDs act through HDAC4/5/7/9 toactivate MEF2 (pathway a), investigation is needed to identify kinasesthat may act through HDAC3 to regulate MEF2-dependent transcrip-tion (pathway b). HDAC3 is widely expressed, and HDAC4/5/7/9 dis-play tissue-specific expression, so pathway b may be the default in mosttissues. HDAC4/5/7/9 directly target MEF2, and there is no evidenceindicating direct interaction of these HDACs with the coactivatorsPCAF and p300/CBP. The broken arrow indicates potential cross talkbetween the two pathways. IKK, IB kinase; Ac, acetyl group.

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pression activity of HDAC3 (Fig. 7E). (iv) HDAC3 interactedwith the MADS box of MEF2 (Fig. 2), whereas HDAC4/5/7/9preferentially bind to the MEF2-specific region (49). Of note,related to the ubiquitous expression of HDAC3 (12, 76),MEF2D is widely expressed in different tissues (49). Thus, theHDAC3 link suggests a novel, and perhaps default, pathwayfor MEF2 regulation (Fig. 9). It will be interesting to elucidatethe upstream regulatory events of this pathway and to deter-mine how it may cross talk with the well-established HDAC4/5/7/9 pathways (Fig. 9) to gain effective spatiotemporal controlof MEF2 activity in vivo.

ACKNOWLEDGMENTS

We thank Ron Prywes, Zhenguo Wu, Minoru Yoshida, Jiahuai Han,Eric Olson, J. Don Chen, and Vincent Giguere for plasmids andantibodies.

This work was supported by NIH grants (to J.W. & E.S.) and fundsfrom Canadian Institutes for Health Research and National CancerInstitute of Canada (to X-.J.Y.).

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