lu - molecular cell - 2000

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Molecular Cell, Vol. 6, 233244, August, 2000, Copyright 2000 by Cell Press

Regulation of Skeletal Myogenesisby Association of the MEF2 Transcription Factorwith Class II Histone Deacetylases

differentiation in vitro (Ornatsky et al., 1997) demonstra-ting that MEF2 is essential for differentiation. In addition

to activating downstream muscle structuralgenes, myo-genic bHLH and MEF2 factors establish positive feed-back loops that amplifyand maintain their expressionas

Jianrong Lu, Timothy A. McKinsey,

Chun-Li Zhang, and Eric N. Olson*

Department of Molecular BiologyUniversity of Texas

Southwestern Medical Center at Dallasmyoblasts enter the differentiation pathway, providing aDallas, Texas 75235mechanism for stabilization of the myogenic phenotype(Edmondson et al., 1992; Cheng et al., 1993; Yee andRigby, 1993).

Summary The vertebrate MEF2 factors MEF2A, -B, -C, and -Dshare homology in the MADS domain, which mediates

Skeletal muscle differentiation is controlled by associ- dimerization and DNA binding, and an adjacent MEF2-ations between myogenic basic-helix-loop-helix and specific domain, which influences dimerization and co-MEF2 transcription factors. We show that chromatin factor interactions (reviewed in Black and Olson, 1998).associatedwith muscle genes regulatedby these tran- Recently, we and others discovered that the MADS/scription factors becomes acetylated during myogen- MEF2 domains of MEF2 interact with histone deacety-

lases (HDACs) 4 and 5 and the related protein MITR,esis and that class II histone deacetylases (HDACs),resulting in repressionof MEF2-dependent transcriptionwhich interact with MEF2, specifically suppress my-(Miska et al., 1999; Sparrow et al., 1999; Wang et al.,oblast differentiation. These HDACs do not interact1999a; Lu et al., 2000). Repression of MEF2 activitydirectly with MyoD, yet they suppress its myogenicon artificial reporters by HDACs 4 and 5 can be over-activity through association with MEF2. Elevating thecome by calcium calmodulindependent protein kinaselevel of M yoD can override the repression imposed by(CaMK)signaling through a mechanism involving disrup-HDACs on muscle genes. HDAC-mediated repressiontion of MEF2/HDAC complexes (Lu et al., 2000).of myogenesis also can be overcome by CaM kinase

HDACs participate in a dynamic process of chromatinand insulin-like growth factor (IGF) signaling. Theseremodeling, forming corepressor complexes that re-

findings reveal central roles for HDACs in chromatinpress transcription by deacetylating histones and tran-

remodeling during myogenesis and as intranuclearscription factors (reviewed in Workman and Kingston,

targets for signaling pathways controlled by IGF and1998). HDAC activity is antagonized by coactivator pro-

CaM kinase. teinssuch as CBPand p300that possess histoneacetyl-transferase (HAT) activity capable of acetylating core

Introduction histones of nucleosomes, resulting in relaxation of chro-matin. HDACs 4 and 5, classified as class II HDACs, are

Differentiation of skeletal muscle cells is coupled to expressed predominantly in heart, skeletal muscle, andwithdrawal from the cell cycle and is accompanied by brain (Fischle et al., 1999; Grozinger et al., 1999; Verdeltranscriptional activation of an array of muscle-specific and Khochbin, 1999), the same tissues that expressgenes. Membersof the MyoDfamily of basic-helix-loop- MEF2 at the highest levels (Edmondson et al., 1994;helix (bHLH) transcription factors, MyoD, myogenin, Lyons et al., 1995).Myf5, and MRF4, can activate the muscle differentiation In the present study, we investigated whether interac-program when introduced into nonmuscle cells and play tion of MEF2 with HDACs 4 and 5 influenced myoblastessential roles at multiple steps in the pathway for mus- differentiation. We show that these HDACs specificallycle development in vivo (reviewed in M olkentin and inhibit differentiation of skeletal myoblasts and blockOlson, 1996; Yun and Wold, 1996). Myogenic bHLH pro- the muscle-inducing function of MyoD by repressingteins activate muscle transcription as heterodimers with MEF2 activity. This form of repression can be overcomeubiquitousbHLH proteins known as E proteins and bind by elevating the levelof MyoDas normallyoccurs duringE boxes (CANNTG) in the regulatory regions of muscle myoblast differentiation.In addition, we show thatIGF-1,target genes (Lassar et al., 1991). a potent inducer of muscle differentiation and hypertro-

Activation of muscle gene expression by myogenic phy, actsthrough a CaMK signaling pathway to interfere

bHLH proteins is dependent on their association with with HDAC-mediated repression of MEF2. These find-members of the MEF2 family of MADS box transcription ingsdemonstratethat MEF2 activates orrepresses myo-factors (Molkentin et al., 1995), which bind a conserved genesis depending on its interactions with HDACs andA/T-rich sequence in muscle gene regulatory regions myogenic bHLH factors and reveal a key role for CaMK(Gossett et al., 1989). MEF2 factors lack myogenic activ- signaling in regulating chromatin remodeling events re-ity alone but potentiate the activity of myogenic bHLH quired for muscle gene activation.proteins (Molkentin et al., 1995). Loss-of-function muta-tions in the single Drosophila MEF2 gene prevent my- Resultsoblast differentiation(Bour et al., 1995; Lilly et al., 1995),and dominant-negative MEF2 mutants inhibit myoblast HDACs 4 and 5 Specifically Inhibit Myogenic

Activity of MyoDInlight of the ability of HDACs4 and5 to inhibit transcrip-* To whom correspondence should be ad dressed (e-mail: eolson@tional activity of MEF2 (Miska et al., 1999; Sparrow ethamon.swmed.edu).

These authors contributed equally to this work. al., 1999; Wang et al., 1999a; Lu et al., 2000) and the


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Molecular Cell234

Figure 1. HDACs 4 and 5 Inhibit Conversion

of 10T1/2 Cells to Skeletal Muscle by MyoD

(A) 10T1/2 cells were transiently transfected

with expression vectors for MyoD (0.5 g) or

the indicated HDACs (0.5 g) as described

in Experimental Procedures, and myogenic

conversion was scored by staining for MHC

expression. A value of 100 for cells trans-fected with MyoD expression plasmid corre-

sponds to 50 MHC-positive cells per 35 mm

dish. Assays were performed three indepen-

dent times with comparable results.

(B) Schematic diagram of HDACs and dele-

tion mutants used in transfection assays.

Amino acids are indicated.

obligate role of MEF2 in myogenesis, we tested whether or E12 in yeast two-hybrid or coimmunoprecipitationassays (data not shown).conversion of 10T1/2 fibroblasts to muscle by MyoD

was affected by these HDACs. In transientlytransfected To investigate theimportanceof HDAC catalytic activ-ity for repression of myogenesis, we mutated histidine-10T1/2 cells, bothHDACs completelyblocked myogenic

activity of MyoD, assayed by expression of myosin 803 in HDAC4 to leucine, shown previously to abolishenzymatic activity (Hassig et al., 1998). This mutantheavy chain (MHC) (Figure 1) or formation of multinucle-

ate myotubes (data not shown). In contrast, overexpres- (H803L) was severely impaired in its ability to repressmyogenesis, although it did retain residual repressivesion of HDACs 1 or 3, which do not interact with MEF2(Lu et al., 2000), did not affect the ability of MyoD to activity (Figure 1). As a further test of the possible

involvement of HDACs in repression of MyoD activity,initiate myogenesis (Figure 1). An amino-terminal dele-tion mutant of HDAC5 (HDAC5N) containing only the we attempted to convert HDACs 4 and 5 from inhibitors

to activators of myogenesis by fusion of their MEF2catalytic domain, or a mutant lacking only the MEF2-interaction domain (HDAC5175-192) also failed to in- binding regions to the VP16 coactivator. Chimeric pro-

teins containing the amino-terminal portions of HDACshibit MyoD activity (Figure 1), demonstrating a directcorrelation between MEF2 binding and repression of 4 and5, including the MEF2 binding motif, fusedto VP16

(see Figure 1B) enhanced the ability of MyoD to activatemyogenesis by HDAC5. We reasoned that inhibition ofMyoD activity by HDACs 4 and 5 could be mediated by myogenesis (Figure 1A) and the MCK enhancer (data

not shown) in transfected 10T1/2 cells. Together, therepression of MEF2 or by repression of MyoD or its Eprotein dimerization partners. However, we obtained no above results revealed a direct correlation between the

ability of HDACs to bind MEF2 and inhibit MyoD activity,evidence for interaction of HDACs 4 and 5 with MyoD


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Control of Myogenesis by HDAC-MEF2 Interactions235

implying that MEF2-dependent target genes were es-sential for conversion of 10T1/2 cells to skeletal muscleby MyoD.

Acetylated Histone H4 Associated withMEF2-Dependent Muscle Genes

Increases during MyogenesisTo determine whether the acetylation state of histonessurrounding functional MEF2 binding sites in skeletalmuscle genes was altered during myogenesis, we per-formed chromatin immunoprecipitation assays. Solublechromatin was prepared from cultures of proliferating,undifferentiated C2 myoblasts or multinucleated myo-tubesand immunoprecipitated withan antibodyspecificfor acetylated histone H4. Precipitated genomic DNAwas analyzed by PCR using primers designed to amplifysequences spanning the essential MEF2 binding sitesinthe MCKenhancer and myogeninpromoter. AsshowninFigure 2A,the amount of acetylated histone H4 associ-ated with these MEF2-responsive regulatory elementswas significantly higher in extracts derived from differ-

entiated myotubes compared to myoblasts. The pres-ence of equivalent amountsof chromatin ineach samplewas confirmed by PCR without prior immunoprecipita-tion. These data support the notion that transcriptionalrepression of skeletal muscle genes is coupled to his-tone deacetylation.

Overexpression of HDACs 4 and 5 PreventsDifferentiation of C2 CellsTo further investigate the potential of HDACs 4 and 5 toinhibit myogenesis, we stably transfected the C2 musclecellline withHDAC4and 5 expressionvectorsand exam-ined the consequences on differentiation. Analysis of sev-eral hundred independent C2 cell clones from each trans-fection showed a near complete block to differentiation

assayed by MHC staining and myotube formation. In con-trast, greater than 90% of clones stably transfected withan expressionvector encoding HDAC5N formed differ-entiated myotubes.

Several stable clones of HDAC-transfected C2 cellswere isolated and analyzed for expression of musclemRNAs by semiquantitative RT-PCR. Results obtainedwith representative clones are shown in Figure 2B. Con-sistent with the failure to form multinucleated myotubes

nonspecific precipitation of chromatin. Precipitated genomic DNA

was analyzed by PCR using primersdesigned to amplify sequences

spanning theM EF2binding sitesin the MCKenhancera nd myogenin

promoter. Positions of primers and MEF2 sites relative to the tran-

scription initiation sites of each gene are shown. The lower panelshows a DNA input control in which PCR amplification was per-

formed prior to immunoprecipitation to confirm that equivalent

amounts of DNA were present in each sample.

(B) C2 cells were stablytransfected withHDAC4 andHDAC5expres-

sion vectors, and stable transfectantswere isolated following selec-

tion in G-418. RNA was isolated from the parental C2 cell line andFigure 2. Induced Histone H4 Acetylationat MEF2 Target Sites dur- from representative clones in GM (day 0) or following t ransfer toing Skeletal Myogenesis and Inhibition of C2 Cell Differentiation by DM for the indicated number of days. Transcripts were detected byHDACs 4 and 5 semiquantitative RT-PCR as described inExperimentalProcedures.

(A) Soluble chromatin was prepared from cultures of proliferating (C) HDAC5 transcripts in wild-type C2 cells and in HDAC5-trans-

myoblasts in growthmedium(GM) or frommultinucleated myotubes fected cells (clone #6) were detected by RT-PCR using primers that

in differentiation medium (DM) and immunoprecipitated with an anti- distinguish the exogenous human HDAC5 and endogenous mouse

body specific for acetylated histone H4 (-AcH4). Parallel extracts HDAC5 transcripts. The exogenous transcript is expressed at a level

were exposed to normal rabbit serum (nonimmune) to control for approximately 4-fold higher than the endogenous.


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Molecular Cell236

Figure 3. HDAC5 Selectively Inhibits MyoD-Dependent Promoters that Contain M EF2 Sites

10T1/2 cellswere transiently transfected with expression vectors for MyoD, MEF2, or the indicated HDACs,and 4RE-luciferase, MCK-luciferase,

or 3 MEF2-luciferase expression plasmids, and luciferase activity was determined as described in Experimental Procedures.

(A) Activation of MCK-luciferase (0.3 g) by MyoD (0.3 g) is inhibited by HDAC5 (0.2 g). E stands for E box; M stands for MEF2 site.Sites for other enhancer binding factors are not shown.

(B) Activation of 4RE-luciferase (0.3 g) by MyoD (0.3 g) is unaffected by the presence of HDACs 4 and 5 (0.2 g).

(C) Increasing the amount of MyoD overcomes the inhibitory effect of HDAC5 on activation of MCK-luciferase, whereas an amount of MyoD

sufficient to fully activate the MCK enhancer in the presence of HDAC5 has no effect on activation of 3 MEF2-luciferase.

in differentiation medium (DM), transcripts for the mus- Gossett et al., 1989; Amacher et al., 1993; Cserjesi et al.,1994). HDAC5N had no effect on the MCK enhancer,cle differentiation markers MCK (Figure 2B) and MHC

(data notshown) were notexpressed above background consistent with the conclusion that repression requiredinteraction of MEF2 with the N terminus of HDAC5.levels in HDAC-transfected cells. Expressionof -skele-

tal actin and myogenin, early markers forthe differentia- HDACs 4 and 5 also blocked activation of the myogeninpromoter by MyoD (data not shown).tion pathway, was also diminished in HDAC-transfec-

tants, though not as dramatically as MCK and MHC. To determine whether inhibition of MyoD activity byHDACs required MEF2, we tested whether HDACs 4 andMEF2C, a late differentiation marker (Martinet al., 1993),

also was not expressed in HDAC-transfected cells, 5 influenced the ability of MyoD to activate the reporter

4RE-luciferase, which contains four copies of the rightwhereas MyoD was expressed at normallevelsin HDAC-transfectants that failed to differentiate. These findings E box from the MCK enhancer but lacks a MEF2 site.

In contrast to the inhibition of MCKand myogeninregu-demonstrate that HDACs 4 and 5 block a step in thedifferentiation pathway downstream from MyoD. latory sequences, these HDACs had no effect on the

ability of MyoD to activate this reporter (Figure 3B).In wild-type C2 cells, HDACs 4 and 5 were expressedat constant levels in myoblasts and myotubes (Figure These results demonstrate the specificity of repression

by HDACs 4 and 5 and suggest that repression of MyoD-2B). Thus, the endogenous level of HDAC expression inC2 cells must be insufficient to prevent differentiation dependent target genes requires MEF2 sites that recruit

a MEF2-HDAC complex.when cells are cultured in differentiation medium andexpression of myogenic bHLH and MEF2 proteins is We next investigated whether MyoD could overcome

the inhibitory activity of HDACs on regulatory regionsupregulated. We were able to distinguish exogenous andendogenous HDAC5 using PCR primers that spanned a containing MyoD and MEF2 binding sites. In titration

experiments using the MCK enhancer as a target, we27 nucleotide insertion unique to the exogenous humanHDAC5 mRNA. Bysemiquantitative RT-PCR, the level of found that increasing the ratio of MyoD to HDAC5 re-

sulted in activation of the enhancer (Figure 3C). In con-exogenous HDAC expression in the HDAC5-transfected

cell line shownin Figure 2B was determined to be 4-fold trast, high levels of MyoD had no effect on repressionby HDAC5 of thereporter3MEF2-luc, containing threehigher than the level of the corresponding endogenousHDAC isoform (Figure 2C). tandem M EF2 sites (Figure 3 C) but no M yoD binding site.

Similar results were obtained with the c-jun promoter,which contains a single MEF2 site (data not shown).Inhibition of MyoD Activity by HDACs 4 and 5These findings suggest that M yoD can selectively over-Requires a MEF2 Sitecome the inhibitory effects of HDACs on promoters thatTo further understand the mechanism for HDAC-medi-containM yoD and MEF2 binding sites, whereas promot-ated inhibition of MyoDactivity, we examined theeffectsers lacking MyoD binding sites can be repressed byof HDACs on several MyoD- and MEF2-responsive re-HDACs irrespective of the presence of MyoD.porters. HDACs 4 and 5 were able to completely inhibit

activation of the MCKenhancer by MyoD in 10T1/2 cells(Figure 3A). This enhancer contains two E boxes, two Mapping the HDAC-Interacting Region of MEF2C

Because MyoD and HDACs 4 and 5 interact with theMEF2 sites, and sites for several other factors that coop-erate for full transcriptional activity (Lassar et al., 1989; MADS/MEF2 domains (residues 186) of MEF2 factors,


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it was of interest to determine whether they recognized was unable to activate the 3 MEF2-luciferase reporterbecause it lacks a TAD (Figure 5B). However, in thethe same amino acid determinants. We attempted to

more finely map the HDAC-interacting region of MEF2C presence of HDAC4-VP16 or HDAC5-VP16, MEF2 1117activated the reporter, reflecting recruitment of theseusing coimmunoprecipitation assays. Mutations in con-

served amino acids within the first 25 amino-terminal HDACs to the MEF2 binding site. In the absence ofMEF2 1117, HDAC4-VP16 and HDAC5-VP16 activatedresidues of M EF2C had no effect on HDAC4 binding

(Figures 4A and 4B), suggesting that the HDAC binding the reporter weakly, which we attribute to interactionwith endogenous MEF2D or other factors that bind themotif lay between residues 25 and 86. We were unable

to detect MEF2 mutants with amino acid substitutions promoter. Overall, these results support the conclusionthat interaction of MEF2 with HDACs 4 and 5 does notor deletions in this region by immunoprecipitations, so

we performed in vitro GST-HDAC4 binding assays with interfere with binding of MEF2 to DNA.[35S]-labeled MEF2 factors to further map the residuesin this region required for HDAC binding. CaMK Signaling Overcomes HDAC-Mediated

In vitro translation products of mouse MEF2C and Repression of Muscle Gene ExpressionDrosophila MEF2 interacted efficiently with GST-HDAC4, Recently, we found that CaMKs I and IV stimulate tran-whereas serum response factor (SRF), which shares ho- scriptional activity of MEF2 by preventing associationmology with the MADS domains of MEF2 factors, did with HDAC5 (Lu et al., 2000). CaMK activity has beennot interactwithGST-HDAC4(Figures4A and 4C). Muta- reported to increase during skeletal muscle differentia-tions of residues 39 through 67 of M EF2C prevented tion (Kim et al., 1992; Baek et al., 1994). Therefore, weinteractionwith GST-HDAC4, whereas mutations of resi- examined whether CaMK signaling could overcome thedues 68 through 80 did not (Figures 4A and 4C). Resi- inhibitory activity of HDACs 4 and 5 on MyoD. Indeed,dues 3967 are also required for dimerization of MEF2 asshownin Figure6A, MyoDwas able to activate muscle

factors (Molkentin et al., 1996). However, MEF2 dimer- gene expression in transiently transfected 10T1/2 cellsization is not sufficient for HDAC interaction because in the presence of activated CaMKI and HDAC5,mutant KYTEY68-72ECNDN homodimerizes (Molkentin whereas in the absence of CaMKI, HDAC5 inhibited allet al., 1996) but does not interact with HDAC. Thus, the myogenic activity of MyoD.HDAC binding region of MEF2 does not encompass The MAP kinase MKK6 hasalsobeen shownto stimu-the basic residues in the amino-terminal portion of the late M EF2 activity by activating the M AP kinase p38,MADS domain that mediates DNA binding but rather is which phosphorylates the MEF2 transcription activationlocalized to the carboxy-terminal subregion of the domain (Han et al., 1997; Yang et al., 1999; Zhao et al.,MADS/M EF2 domains. These residues are nearly identi- 1999). MKK6 weakly enhanced the myogenic activity ofcal in all known MEF2 proteins but are not conserved MyoD, but it could not overcome inhibition by HDACin SRF or other MADS box proteins (Figure 4D) (Shore (Figure 6A). In the presence of CaMKI and MKK6, weand Sharrocks, 1995). observed synergistic stimulation of MyoD activity and

an almost 10-fold increase in efficiency of myogenicconversion. Myotubes formed in the presence of acti-HDAC Associates with MEF2 on DNAvated CaMKI and MKK6 were also larger than with MyoDBecause the HDAC-interaction domain of MEF2 is lo-

alone (not shown). We conclude that CaM K signalingcated immediately adjacent to the DNA binding domain,acts as a stimulus for skeletal muscle differentiation andwe considered the possibility that inhibition of MEF2interferes with the inhibitory activity of HDACs towardtranscriptional activity by HDAC might occur through aMyoD.block to DNAbinding. To determine whether MEF2 could

bind DNA and interact with HDAC4 simultaneously, weperformed interaction assays with GST-HDAC4 and IGF-1 Signaling Stimulates Myogenesis by

Interfering with the Activity of HDACsMEF2C translated in vitro and a labeled MEF2 bindingsite. As shownin Figure 5A, GST-HDAC4 interacted with Insulin-like growth factors (IGFs)stimulate skeletalmus-

cle hypertrophy (Coleman et al., 1995; Musaro et al.,[35S]-labeled MEF2C bound to its [32P]-labeled targetsite. In contrast, there was no binding above back- 1999; Semsarian et al., 1999) and activate intracellular

calcium signaling (Kazaki et al., 1997; Wang et al.,ground of MEF2C or the labeled DNA probe to GSTalone. As a control, we performed this assay with the 1999b). To test whether CaMK activation is required for

IGF-1-mediated hypertrophy, we treated rat L6 muscleMEF2C mutant RKK3-5TNQ, which bound HDAC4 invivo (Figure 5B)but isdefective inDNA binding (Molken- cells with IGF-1 in the presence and absence of the

CaMK inhibitor KN62. As shown in Figure 6B, IGF-1tin et al., 1996). This mutant interacted with GST-HDAC4

but did not bring the [32

P]-labeled DNA binding site into dramatically stimulated differentiation of L6 myoblastsand myotube hypertrophy in differentiation medium.the complex (Figure 5A). The DNA binding site also wasnot captured by GST-HDAC4 in the presence of the However, in the presence of KN62 the effects of IGF-1

were lost. KN62 had no discernable effect on cell viabil-MEF2C mutant LI45,46RN (Figure 5A), which was unableto interact with GST-HDAC4 (Figure 5B)and cannot bind ity, indicating that its effects likely reflect inhibition of

CaMK activity. The inactive analog KN92 had no effectDNA (Molkentin et al., 1996). Similarly, labeled SRF didnot associate significantly with GST-HDAC4 in this on the ability of IGF-1 to stimulate myogenesis (data not

shown).assay (Figure 5A).As an independent test of the potential effect of To test whether IGF-1 signaling was also directed at

the mechanism for HDAC inhibition of myogenesis, weHDACs 4 and 5 on MEF2 DNA binding, we performeda modified mammalian one-hybrid assay using the DNA treated the HDAC4-transfected C2 cell line with IGF-1.

Whereas these cells were completely blocked in theirbinding domain of MEF2C (amino acids 1117) andHDACs 4 and 5 mutants in which the carboxy-terminal ability to differentiate, when exposed to IGF-1 they

formed large multinucleate myotubes (Figure 6C). Thus,catalytic domainswere replaced withVP16. MEF2 1117


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Figure 4. Mapping Residues in the MADS/MEF2 Domains of MEF2C Required for Interaction with HDAC

(A) Amino acid sequence of the MADS and MEF2 domains of MEF2C. Mutants tested for interactions with HDACs 4 and 5 are shown below.

A dash indicates the wild-type residue at that position, and an X indicates residues that were deleted. The dimerization and DNA binding

activity of the mutants was reported previously (Molkentin, et al., 1996).

(B) Coimmunoprecipitations of HDAC4 and MEF2C mutants. 293 T cells were transiently transfected with expression vectors for Flag-tagged

HDAC4 and the indicated MEF2C mutants, and cell extracts were analyzed by immunoprecipitation and Western blot as described in

Experimental Procedures. The top panel shows the results of anti-HDAC4 (Flag) immunoprecipitation followed by anti-MEF2 Western blot,

and demonstrates interaction of all MEF2C mutants with HDAC4. The bottom panel shows the results of anti-MEF2 Western blot without anti-

HDAC immunoprecipitation, and demonstrates that comparable amounts of each MEF2 were expressed in transfected cells.

(C) Binding of MEF2C mutants to GST-HDAC4 in vitro. MEF2C and various deletion mutants, D-MEF2, and SRF were translated in vitro in the


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Figure 5. Simultaneous Binding of M EF2 to DNA and HDACs

(A) Binding of an MEF2-DNA complex to GST-HDAC4. GST-HDAC4 was incubated with [35S]-labeled in vitro translation products that were

premixed with a [32P]-labeled MEF2 site as described in Experimental Procedures. Beads were then washed and associated radioactivity was

determined.

(B) Modified one-hybrid assay. 10T1/2 cells were transiently transfected with 3 MEF2-luciferase reporter (0.3 g) and expression plasmids

for MEF2C 1-117 (0.3 g), HDAC4-VP16 (0.2 g), and HDAC5-VP16 (0.2 g) as indicated. HDAC4-VP16 and HDAC5-VP16 were created as

described in Experimental Procedures.

IGF-1 can rescue differentiation by interfering with the Selective Repression of MEF2 Activity by HDACs4 and 5inhibitory activity of HDAC4.

As a further test of the involvement of CaMK in the HDACs 4 and 5, classified as Class II HDAC enzymes,have been shown to deacetylate all four core histonesmechanism whereby IGF-1 suppressed the repressive

activity ofHDACs4 and 5, we examined whethera domi- invitro (Fischle et al., 1999;Grozinger el al., 1999; Verdeland Khochbin, 1999). These HDACs interact with aminonant-negative CaMK mutant could interfere with the

stimulatory effect ofIGF-1 onMyoD activityin transiently acids 3972 of MEF2 factors, spanning the junction ofthe MADS and M EF2 domains. This region of MEF2transfected 10T1/2 cells. As shown in Figure 6D, IGF-1

enhanced the ability of MyoD to activate myogenesis. encompasses the residues that mediate MEF2 homodi-merization, but interaction with HDACs does not affectRemarkably, MyoDlost allmyogenic activity inthe pres-

ence of dominant-negative CaMKIV. Moreover, whereas dimerization or DNA binding of MEF2.In contrast to the relatively confined region of MEF2IGF-1 could overcome the inhibition of MyoD activity

imposed by HDACs 4 and 5, it could not overcome the recognized by HDACs, MyoDinteracts with an extended

surface of MEF2 factors that includes residues through-effects of these HDACs in the presence of dominant-negative CaMKIV. These results suggest that CaMK ac- out the MADS and MEF2 domains (Molkentinet al., 1995,1996). Based on the strength of interactions in two-tivity is required for MyoD-mediated myogenesis and

for IGF-mediated stimulation of myogenesis. hybrid and coimmunoprecipitation assays, HDACs ap-pear to exhibit a much higher affinity than MyoD forMEF2. We have examined whether MyoD overcomesDiscussionHDAC-mediated repression by competing with HDACfor interaction with MEF2, but we have no evidence forThe results of this study demonstrate that HDACs 4such competition. Thus, we favor a model in which highand 5 inhibit myogenesis by repressing MyoD activityconcentrations of MyoD result in greater occupancy ofthrough association with MEF2 and support a modelE boxes, resulting in opposition to the inhibitory activityin which the decision of a myoblast to differentiate isof HDACs by the strong transcription activation domaindictated by a balance of positive and negativeinfluencesof MyoD or by recruitment of additional coactivators byon the transcriptional activity of MEF2. Consistent withMyoD.the conclusion that HDACs 4 and 5 repress muscle tran-

MyoD and MEF2 have been shown to interact withscription by deacetylating core histones associated withthep300/CBP coactivators(Eckner et al., 1996;Sartorellimuscle gene regulatory regions, the level of acetylatedet al., 1997, 1999) that possess HAT activity and wouldhistone H4 associated withthe MCKenhancer and myo-therefore be expected to antagonize the actions ofgeninpromoter, both of which are regulated directly byHDACs. It ispossible that HDACs 4 and 5 diminishM yoDMEF2 (Gossett et al., 1989; Edmondson et al., 1992),

increases during myogenesis. activity by direct deacetylation. However, this would

presence of [35S]methionine and incubated with GST-HDAC4 fusion protein containing residues 49233 of HDAC4 bound to glutathione-

agarose beads. Proteins bound to GST-HDAC4 were separated by SDSPAGE and analyzed by autoradiography (upper panel). As a control,

labeled proteins were also incubated with GST alone (middle panel). No labeled proteins were recovered under these conditions. Ten percent

of the labeled in vitro translation products used for GST-HDAC interaction assays was run on a separate gel (bottom panel). In the far right

lanes, [35S]methionine-labeled HDAC4 (residues 49233) was incubated with GST-M EF2C (residues 186), and bound protein was analyzed

as described above.

(D) Alignment of MADS/M EF2 domains of MEF2 factors and SRF. The region corresponding to the HDAC binding domain is conserved among

MEF2 factors but divergent in SRF.


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(Zetser et al., 1999). However, theability of CaMK signal-ing to activate myogenesis and to synergize with MAPkinases to superinduce muscle-specific genes has notbeen described. Our results show that IGF-1, a potentinducer of skeletal muscle hypertrophy (Florini et al.,1991; Coleman et al., 1995; M usaro and Rosenthal,1999), can overcome the inhibitory effects of HDAC4 ondifferentiation of C2 cells and that inhibition of CaMKactivity abolishes the hypertrophic activity of IGF-1. Inthis regard, IGFs have been shown to activate L-typecalcium channels(Kazaki et al., 1997; Bruton et al., 1999;Semsarian et al., 1999; Wang et al., 1999b). IGF-1 alsoinduces hypertrophy through a pathway involving cal-cineurin (Musaro et al. 1999; Semsarian et al., 1999).How the calcineurin pathway may cross-talk with theCaMK signaling pathway in skeletal muscle cells re-mains to be established, but in cardiac myocytes thesesame pathways have beenshown to synergize to inducehypertrophy.

Remarkably, a CaMK dominant-negative mutant com-pletely blocked the ability of MyoD to activate myogen-esis, revealing an essential role for CaMK signaling in

Figure 7. A Model for the Role of HDACs in Skeletal Myogenesis the transcriptional pathway for muscle gene activation.(A) Three typesof muscle targetgenes distinguished by theirrespon- What might be the target for CaMK? We favor the possi-siveness to MyoD, MEF2, and HDAC. bility that HDACs are targets, either directlyor indirectly,(B) Schematic diagram showing the potential roles of MyoD, MEF2,

for CaMK signaling and that in the absence of a CaMKand HDACs in muscle gene expression. In myoblasts, association

signal, MEF2 activity is repressed by association withof HDACwith MEF2resultsin repressionof muscle genes controlledHDACs. Myoblast differentiation has been shown to beby MyoD. When myoblasts are triggered to differentiate, myogenicaccompanied by an increase in CaMK activity (Kim etbHLH and MEF2 levels rise, which overcomes repression by HDAC

of genes that contain E boxes and MEF2 sites. CaMK signaling, al., 1992; Baek et al., 1994) that would be predicted towhich dissociates M EF2-HDAC complexes, and MAPK signaling, stimulate MEF2 activity.which phosphorylates the MEF2 transcription activation domain, A simple model for the role of HDAC and MEF2 infurther stimulate muscle gene expression by enhancing MEF2 ac- myogenesis is shown in Figure 7B. According to thistivity. model, HDACs 4 and 5 associate with MEF2 in my-

oblasts and repress muscle-specific genes. When my-oblasts are triggered to differentiate, MyoD upregulates

7A. Muscle genes that contain E boxes but not MEF2 expression of MEF2, and together MEF2 and MyoD acti-sites would be activated by myogenic bHLH factorsand

vate myogenin transcription and establish a positivewould be insulated from the inhibitory effects of HDACs. feedback loop that amplifies expression of both factorsThis type of MyoD target gene might be expressed in as well as other myogenic bHLH factors (Edmondsonproliferating myoblasts. Othergenes containM EF2 sites et al., 1992). Thus, although HDAC expression remainsbut noMyoD sites. Thisclass of gene would be activated constant in myoblasts and myotubes, increasing levelsby MEF2 and repressed by HDACs and would be unaf- of myogenic bHLH and MEF2 factors in differentiatingfected by the presence of MyoD. Finally, many muscle muscle cells would exceed the capacity of HDAC togenes such as MCKarecontrolledby E boxes andM EF2 repress MEF2-dependent genes, resulting in musclesites. Expression of these genes would be dependent gene activation. CaMK signaling stimulates myogenesison the balance between MyoD and HDAC activity. by dissociating HDACs from MEF2, and MAPKs further

enhance MEF2 activityby phosphorylation of the activa-tion domain. Dissociation of HDACs from MEF2 in re-Stimulation of Myogenesis by Signal-Dependentsponse to CaMK signaling (Lu et al., 2000) may resultDissociation of HDACs from MEF2inactivation of MEF2 not onlythrough relieffrom HDAC-Our results show that MEF2 is a signal-dependent acti-mediated repression but may also facilitate associationvator of skeletal myogenesis that responds to CaMKwith CBP and p300 coactivators that also interact with

and MAP kinase pathways. CaMK signaling overcomes the DNA binding domain of MEF2C (Eckner et al., 1996;the inhibitoryactivityof HDAC by preventing associationSartorelli et al., 1997).of HDAC with MEF2 (Lu et al., 2000), whereas MKK6,

which activates p38 (Enslen et al., 1998), stimulatesMEF2 activity by phosphorylation of the carboxy-termi- MEF2: A Transcriptional Target for Growth

and Differentiation Signalsnal transcription activation domain (Han et al., 1997;Yang et al., 1999; Zhao et al., 1999). Previously, we In addition to regulating skeletal muscle differentiation,

MEF2 factors have been implicated in cardiac morpho-showed that MKK6 can only activate MEF2 in cardiacmyocytes if HDAC is dissociated from the DNA binding genesis, vascular development, and neuronal differenti-

ation, as well as in the controlof growth factorinducibledomain (Lu et al., 2000). Thus, the CaMKand MAPkinasepathways synergize to activate MEF2-dependent tran- genes (reviewed in Black and Olson, 1998). Calcium-

dependent signals have also been shown to connectscription by targeting different domains of MEF2.Previous studies have demonstrated that MAP kinase MEF2 to cell survival (Mao et al., 1999) and apoptotic

(Youn et al., 1999) pathways. How MEF2 discriminatessignaling can stimulate myoblast differentiation via MEF2


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Molecular Cell242

were resolved by SDSPAGE, transferred to PVDF membranes, andbetween the different sets of target genes involved insequentially immunoblotted with polyclonal antisera raised againstthese processes and whether the type of signal-depen-the indicated MEF2 protein (Han and Prywes, 1995) and an anti-dent derepression of HDACs described here for IGF-1Flag monoclonal antibody (Sigma). Proteins were visualized usingand CaMK participate in these gene regulatory pro-a chemiluminescence system (Santa Cruz).

grams remains to be determined. Given the selectiveexpression of ClassII HDACs and MEF2 inskeletalmus-

GST Capture Experimentscle, heart, and brain, and the importance of calcium

Ten microliters of in vitro translated [35

S]-labeled MEF2 and HDACsignaling in these tissues, it seems likely that the type of proteins was incubated with bead-bound GST-HDAC and GST-regulatory circuitry through which these transcriptional MEF2 fusion proteins, respectively, in 200 l of GST binding bufferregulators connect extracellular signaling with chroma- (20 mM Tris [pH 7.3], 150 mM NaCl, 0.5% NP-40, and protease

inhibitors [Boehringer Mannheim]) for 1 hr at 4C. Beads were col-tin remodeling in skeletal muscle cells will have rele-lected by centrifugation at 2500 rpm for 5 min and washed threevance to multiple aspects of gene expression in thesetimes with 500 l GST binding buffer followed by SDSPAGE.tissues.

Assays for DNA Binding and HDAC Interaction of MEF2Experimental ProceduresTo test whether MEF2 could bind DNA and HDAC simultaneously,

5 l of an in vitro translation reaction of MEF2C was incubated withCell Culture and Transfections1 105 cpm of [32P]-labeled MEF2 site for 30 min at 25C. The10T1/2 and C2 cells were maintained in Dulbeccos modified Eaglesequence of the M EF2 oligomer was 5-GGCTCTAAAAATAACCmedium (DMEM) with 10% fetal bovine serum (FBS) (GIBCO/BRL),CCC-3. Two hundred microliters of GST binding buffer (20 mMand L6 cells were grown in DMEM with 20% FBS. One day prior toTris [pH 7.3], 150 mM NaCl, 0.5% NP-40, and protease inhibitorstransfection, 101/2 cells were plated on 35 mm dishes at a density[Boehringer Mannheim]) was then added to the reaction with 20 lof 1 105 cells/dish. Lipid-mediated transfections were performedglutathione-agarose beads-bound GST-HDAC (amino acids49233)using FuGENE6 Reagent (Boehringer Mannheim). Forty-eight hours

and incubation was continued for 1 hr at 4C. Beads were thenlater, cells were harvested for luciferase activity or were switchedcollected by centrifugation at 2500 rpm for 5 min, washed twice into differentiation medium (DMEM with 2% horse serum) for 5 daysGST binding buffer, and associated radioactivity was determined.for MHC staining in myogenic conversion assays. For stable trans-

fections, C2 cells growing at a density of 2 106 cells/100 mmRNA Isolation and RT-PCR Analysisdish were transfected by the calcium-phosphate method with theRT-PCR was performed essentially as described (Rawls et al.,1998).indicated expression constructs (10 g each), and stable clonesTotal cellular RNA was prepared from C2C12 cells using Trizol Re-were isolated followingselection inG- 418 (400g/ml)fortwo weeks.agent (GIBCO), and 1.5 g aliquots were converted to cDNA usingThefollowing plasmids were used: EMSV-MyoD (Lassaret al., 1989),random hexamers and MMLV reverse transcriptase (GIBCO). ForpCDNA1-MEF2 (Molkentin et al., 1995), pBJ5-HDAC1,-4,-5, andeach PCR reaction, 3% of the cDNA pool was amplified using TaqpCDNA6-HDAC3 (Grozinger et al., 1999). HDAC4-VP16 and HDAC5-polymerase (Perkin-Elmer) in the presence of [32P]-dCTP. In allVP16 were generated by subcloning cDNAs encoding HDAC4cases,PCR wasperformed for2025 cycles, and10% ofthe reaction(amino acids 1233) and HDAC5 (amino acids 9214) into pVP16was resolved through a 5% polyacrylamide gel. The amplified prod-(Clontech). The MKK6 expression plasmid encoded an active formuctswere visualized byautoradiography. Sequencesof PCR primersof the enzyme with serine 207 and threonine 211 to glutamate muta-for myogenin, MyoD, MCK, -skeletal actin, MEF2C, and L7 havetions (Jiang et al., 1996). The activated CaMKI plasmid encoded abeen described (Valdez et al., 2000). Other primers are as follows:constitutively activated form of the enzyme in which isoleucine-294HDAC4 (), 5-GAGAGAATTCTGCTAGCAATGAGCTCCCAA-3; (),was replaced with a stop codon.

5-GAGACTCGAGCTATGCAGGTTCCAAGGGCAGTGA-3; HDAC5Full length rat CaMKIV was amplified, and the K71E mutation was(), 5-GAAGCACCTCAAGCAGCAGCTGG-3; (), 5-CACTCTCTTinserted using PCR (primer sequences available upon request). TheTGCTCTTCTCCTTGTT-3.PCR fragment was cloned as an NheI to XbaI fragment into Blue-

script-HA3 (triple HA epitope tag), and the insert of the resulting

ImmunostainingpBSHA3-CaMKIVK71Eplasmidwas sequenced.A KpnIto XbaI frag-

Myosin expression in L6 cells was assessed by immunoperoxidasement containing HA3-CaM KIVK71E was subsequently transferred

staining withthe VectastainElite ABCkit andan anti-skeletal myosininto like digested pCMV5 expression vector. An equivalent lysine in

monoclonal antibody (Sigma). Indirect immunofluorescence analy-human CaMKIV (lysine 75) has been shown to be required for ATP

sis ofC2 cellswas performedusing theM F-20 anti-myosinantibody,binding by the enzyme. Mutation of this lysine to glutamate results

a fluorescein-conjugated anti-mouse secondary antibody (Vectorina catalyticallyinactive enzyme that stillbindscalciumand calmod-

Laboratories), and Hoechst 33342 dye (Molecular Probes).ulin (Chatila et al., 1996; Anderson et al., 1997).

Reporter plasmidswere used as follows:M CK-luciferasecontains

the 301 bp mouse MCK enhancer upstream of the 246 bp basal Chromatin Immunoprecipitation Assaypromoter. This enhancer fragment was previously referred to as Chromatinimmunoprecipitation experimentswere performed as de-fragment e4 (Sternberg et al., 1988). 4RE-luciferase contains four scribed (Braunstein et al., 1993). Briefly, soluble chromatinwas pre-tandem copies of the right E box from the MCK enhancer upstream pared from proliferating myoblasts at 60% confluence on a 15 cmof thymidine kinase promoter and luciferase as described (Wein- dish or confluent multinucleated myotubes on a 10 cm dish. Equal

traubet al.,1990). 3MEF2-luciferasecontains threetandem copies amounts of chromatin from each sample (normalized by ethidiumof the mouse MCK enhancer MEF2 site upstream of the thymidine bromide staining of DNA) were immunoprecipitated with either ankinase promoter in luciferase as described (Molkentin et al., 1996). anti-acetylated histone H4 antibody (Upstate Biotechnology)or nor-

mal rabbit serum. Ten percent of each immune complex was sub-

jected to 25 cycles of PCR with primers specific for either the MCKCoimmunoprecipitation Assays

For coimmunoprecipitation experiments, 5 105 293T cells were enhancer ([], 5-GACACCCGAGATGCCTGGTT-3; [], 5-GATC

CACCAGGGACAGGGTT-3) or the myogenin promoter ([], 5-GAAtransfected using FuGENE 6 reagent with expression vectors (1 g

each)encoding the indicated Flag epitope-tagged HDAC and MEF2 TCACATGTAATCCACTGGA-3; [], 5-ACGCCAACTGCTGGGTG

CCA-3). As a control for DNA content, PCR reactions were alsoproteins. Forty-eight hours later, cellswere harvestedin phosphate-

buffered saline containing 0.5% Triton X-100, 1 mM EDTA, 1 mM performed on chromatin samples prior to immunoprecipitationwith

anti-acetylated histone H4 antibody. To facilitate visualization andPMSF, and protease inhibitors (Complete, Boehringer Mannheim).

Cells were subjected to brief sonication, and cellular debris was quantitation of the products, [32P]-dCTP was included in the reac-

tions. Ten percent of each reaction mixture was resolved throughremoved by centrifugation. Flag-tagged HDAC proteinswere immu-

noprecipitated from cell lysates using anti-Flag M2 affinity resin a 5% native acrylamide gel, visualized by autoradiography, and

quantified using a phosphorimager (Molecular Dynamics).(Sigma)and washed fivetimes withlysisbuffer.Precipitatedproteins


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Acknowledgments Edmondson, D.G., Lyons, G.E., Martin, J.F., and Olson, E.N. (1994).

MEF2 gene expression marks the cardiac and skeletal muscle lin-

eages during mouse embryogenesis. Development 120, 12511263.We thank S. Schreiber, R. Prywes, A. R. Means, and R. Bassel-Duby

for reagents and R. Valdez for advice. We are also grateful to J. Enslen,H., Raingeaud, J., andDavis, R.J. (1998). Selective activationPage and A. Tizenor for assistance with the manuscript and R. Nicol of p38 mitogen-activated protein (MAP)kinase isoformsby the MAPfor dominant-negative CaMKIV. This work was supported by grants kinase kinases MKK3 and MKK6. J. Biol. Chem. 273, 17411748.to E. N. O. from NIH, the Robert A. Welch Foundation, and the

Fischle, W., Emiliani, S., Hendzel, M .J., Nagase, T., Nomura, N.,

Muscular Dystrophy Association. T. A. M. is a Pfizer fellow of the Voelter, W., and Verdin, E. (1999). A new family of human histoneLife Sciences Research Foundation.deacetylases related to Saccharomyces cere visiae HDA1p. J. Biol.

Chem. 274, 1171311720.Received December 6, 1999; revised June 5, 2000.

Florini, J., Ewten, D., and Magri, K. (1991). Hormone growth factors

and myogenin differentiation. Annu. Rev. Physiol. 53, 201216.References

Gossett, L.A., Kelvin, D.J., Sternberg, E.A., Olson, E.N. (1989). A

new myocyte-specific enhancer-binding factor that recognizes aAmacher, S.L., Buskin, J.N., and Hauschka, S.D. (1993). Multipleconserved element associated with multiple muscle-specific genes.regulatory elements contribute differentially to muscle creatine ki-Mol. Cell. Biol.. 9, 50225033.nase enhancer activity in skeletal and cardiac muscle. Mol. Cell.Grozinger, C.M., Hassig, C.A., and Schreiber, S.L. (1999). Three pro-Biol. 13, 27532764.teins define a class of human histone deacetylases related to yeastAnderson, K.A., Ribar,T.J., Illario, M., andMeans,A.R.(1997). Defec-Hda1p. Proc. Natl. Acad. Sci. USA 96, 48684873.tive survival and activaiton of thymocytes in transgenic mice ex-Han, T.H., and Prywes, R. (1995). Regulatoryrole of MEF2D inserumpressing a catalytically inactive form of Ca/calmodulin-dependentinduction of the c-junpromoter. Mol. Cell. Biol. 15, 29072915.protein kinase IV. Mol. Endocrinol. 11, 725737.

Han, J., Jiang, Y., Li, Z., Kravchenko, V.V., and Ulevitch, R.J. (1997).Baek, H.Y., Jeon, Y.J., Kim, H.S., Kang, M-S., Chung, C.H., and Ha,Activation of the transcription factor MEF2C by the MAP kinase p38D.B. (1994). Cyclic AMP negatively modulates both Ca 2/calmodu-

in inflammation. Nature 386, 296299.lin-dependent phosphorylation of the 100-kDa protein and mem-

brane fusion of chick embryonicmyoblasts. Dev. Biol. 165, 178184. Hassig, C.A., Tong, J.K., Fleischer, T.C., Owa, T., Grable, P.G., Ayer,

D.E., andSchreiber, S.L. (1998). A role forhistone deacetylase activ-Black,B.L., and Olson, E.N. (1998). Transcriptional control of muscleity in HDAC1-mediated transcriptional repression. Proc. Natl. Acad.development by myocyte enhancer factor-2 (MEF2) proteins. Annu.Sci. USA 95, 35193524.Rev. Cell Dev. Biol. 14, 167196.

Hasty, P., Bradley, A., Morris, J.H., Edmondson, D.G., Venuti, J.M.,Bour, B.A., OBien, M.A., Lockwood, W.L., Goldstein, E.S., Bodmer,Olson, E.N., and Klein, W.H. (1993). M uscle deficiency and neonatalR., Taghert, P.H., Abmayr, S.M., andNguyen,H.T. (1995). Drosophiladeathin mice witha targetedmutationin the myogeningene. NatureMEF2, a transcription factor that is essential for myogenesis. Genes364, 501506.Dev. 9, 730741.

Jiang Y., Chen C., Li Z., Guo W., Gegner J.A., Lin S., and HanBraunstein, M., Rose, A.B., Holmes, S.G., Allis, C.D., and Broach,J. (1996). Characterization of the structure and function of a newJ.R. (1993). Transcriptional silencing in yeast is associated withmitogen-activated protein kinase (p38beta). J.Biol. Chem. 271,reduced nucleosome acetylation. Genes Dev. 7, 592604.1792017926.Breitbart, R.E., Liang, C.S., Smoot, L.B., Laheru, D.A., Mahdavi, V.,Kazaki, M., Nie, L., Shibata, H., and Kojima, I. (1997). Activation ofand Nadal-Ginard, B. (1993). A fourth human MEF2 transcriptiona calcium-permeable cation channel CD20 expressed in Balb/C 3T3factor, hMEF2D, is an early marker of the myogenic lineage. Devel-cells by insulin-like growth factor-1. J. Biol. Chem. 272, 49644969.opment 118, 10951106.

Kim, H.S., Lee, I.H., Chung, C-H., Kang, M-S., and Ha, D.B. (1992).Bruton, J.D., Katz, A., and Westerblad, H. (1999). Insulin increasesCa2/calmodulin-dependent phosphorylationof the 100-kDa proteinnear-membrane but not global Ca2 in a isolated skeletal muscle.in chick embryonic muscle cells in culture. Dev. Biol. 150, 223230.Proc. Natl. Acad. Sci. USA 96, 32813286.

Lassar, A.B., Buskin, J.N., Lockshon, D., Davis, R.L., Apone, S.,Buchberger, A., Ragge, K., and Arnold H.H. (1994). The myogeninHauschka, S.D., and Weintraub, H. (1989). MyoD is a sequence-gene is activated during myocyte differentiation by pre-existing, notspecific DNA binding protein requiring a region of myc homologynewly synthesized transcription factor MEF-2. J. Biol. Chem. 269,to bind to the muscle creatine kinase enhancer. Cell 58, 823831.1728917296.

Lassar, A.B., Davis, R.L., Wright, W.E., Kadesch, T., Murre, C., Voro-Chatila, T., Anderson, K.A., Ho, N., andM eans, A.R. (1996). A uniquenova, A., Baltimore, D., and Weintraub, H. (1991). Functional activityphosphorylation-dependent mechanism for the activation of Ca2/of myogenicbHLH proteinsrequiresheterooligomerizationwith E12/calmodulindependent protein kinase type IV/GR. J. Biol. Chem.E47-like proteins in vivo. Cell 66, 305315.271, 2154221548.

Lilly, B., Zhao, B., Ranganayakulu, G., Paterson, B.M., Schultz, R.A.,Cheng, T.-C., Wallace, M., Merlie, J.P., andOlson, E.N. (1993). Sepa-and Olson, E.N. (1995). Requirement of MADS domain transcriptionrable regulatory elements govern myogenin transcription in embry-factor D-MEF2 for muscle formation in Drosophila. Science 267,onic somites and limb buds. Science 261, 215218.688693.

Coleman, M.E., DeMayo, F., Yin, K.C., Lee, H.M., Geske, R., Mont-Lu, J., McKinsey, T.A., Nicol, R.L., and Olson, E.N. (2000). Signal-gomery, C., and Schwartz, R.J. (1995). Myogenic vector expressiondependent activation of the MEF2 transcription factor by dissocia-of insulin-like growth factor I stimulates muscle cell differentiationtion from histone deacetylases.Proc. Natl. Acad. Sci. USA97, 4070and myofiber hypertrophy in transgenic mice. J. Biol. Chem. 19,4075.1210912116.

Lyons, G.E., M icales, B.K., Schwarz, J., M artin, J.F., Olson, E.N.Cserjesi, P., Lilly, B., Hinkley, C., Perry, M., and Olson, E.N. (1994).(1995). Expression of MEF2 genes in the mouse central nervousHomeodomainprotein MHox and MADS protein myocyte enhancer-system suggests a role in neuronal maturation. J. Neurosci. 15,binding factor-2 converge on a common element in the muscle57275738.creatine kinase enhancer. J. Biol. Chem. 269, 1674016745.

Mao, Z., Bonni, A., Xia, F., Nadal-Vicens, M., Greenberg, M.E. (1999).Eckner, R., Yao, T.P., Oldread, E., and Livingston, D.M. (1996). Inter-Neuronal activity-dependent cell survival mediated by transcriptionactionand functional collaboration of p300/CBP and bHLH proteinsfactor MEF2. Science 286, 785790.in muscle and B-cell differentiation. Genes Dev. 10, 24782490.

Martin,J.F., Schwarz, J.J., andOlson,E.N. (1993). MyocyteenhancerEdmondson, D.G., Cheng, T.-C., Cserjesi, P., Chakraborty, T., andfactor (MEF) 2C: A tissue-restricted member of the M EF-2 family ofOlson, E.N. (1992). Analysis of the myogenin promoter reveals antranscription factors. Proc. Natl. Acad. Sci. USA 90, 52825286.indirect pathway for positive autoregulation mediated by the mus-

cle-specific enhancer factor MEF-2. Mol. Cell. Biol. 12, 36653677. Miska, E.A., Karlsson, C., Langley, E., Nielsen, S.J., Pines, J., and


12/12

Molecular Cell244

Kouzarides, T. (1999). HDAC4 deacetylase associates with and re- mitogen-activated protein kinases to MEF2 transcription factors.

Mol. Cell. Biol. 19, 40284038.presses the MEF2 transcription factor. EMBO J. 18, 50995107.

Yee, S.P., and Rigby, P.W. (1993). The regulation of myogenin geneMolkentin, J., and Olson, E.N. (1996). Combinatorial control of mus-

expression during the embryonic development of the mouse. Genescle development by bHLH and M ADS box transcription factors.

Dev. 7, 12771289.Proc. Natl. Acad. Sci. USA 93, 93669373.

Youn, H-D., Sun, L., Prywes, R., and Liu, J.O. (1999). Apoptosis ofMolkentin, J.D., Black, B.L., M artin, J.F., and Olson, E.N. (1995).

T cells mediated by Ca2-induced release of the transcription factorCooperative activation of muscle gene expression by MEF2 and

MEF2. Science 286, 790793.myogenic bHLH proteins. Cell 83, 11251136.

Yun, K., and Wold, B. (1996). Skeletal muscle determination andMolkentin, J.D., Black, B.L., M artin, J.F., and Olson, E.N. (1996).

differentiation: a story of a core regulatory network and its context.Mutational analysis of the DNA binding, dimerization, and transcrip-

Curr. Opin. Cell Biol. 8, 877899.tional activation domains of MEF2C. Mol. Cell. Biol. 16, 26272636.

Zetser, A., Gredinger, E., and Bengal, E. (1999). p38 mitogen-acti-Musaro, A., and Rosenthal, N. (1999). Maturation of the myogenic

vated protein kinase pathway promotes skeletal muscle differentia-program is induced by post-mitotic expression of IGF-1. Mol. Cell.

tion. J. Biol. Chem. 274, 51935200.Biol. 19, 31153124.

Zhao, M., New, L., Kravchenko, V.V., Kat, Y., Gram, H., Di Padova,Musaro, A., Karl, M., Naya, F.J., Olson, E.N., and Rosenthal, N.F., Olson, E.N., Ulevitch, R.J., and Han, J. (1999). Regulation of the(1999). IGF-1 induces skeletal myocyte hypertrophy through cal-MEF2 family of transcription factors by p38. M ol. Cell. Biol. 19,cineurin in association with GATA-2 and NF- ATc1. Nature 400,

2130.581585.

Ornatsky, O.I., Andreucci, J.J., and McDermott, J.C. (1997). A domi-

nant-negative form of transcription factor M EF2 inhibits myogen-

esis. J. Biol. Chem. 272, 3327133278.

Rawls, A., Valdez, M.R., Zhang, W., Richardson, J., Klein, W.H., and

Olson, E.N. (1998). Overlapping functions of the myogenic bHLH

genes MRF4 and MyoD revealed in double mutant mice. Develop-

ment 125, 23492358.

Sartorelli, V.,Huang, J., Hamamori, Y., andKedes, L. (1997). Molecu-

lar mechanisms of myogenic coactivationby p300:direct interaction

with the activation domain of MyoD and with the M ADS box of

MEF2C. Mol. Cell. Biol. 17, 10101026.

Sartorelli, V.,Puri, P.L., Hamamori, Y., Ogryzko, V., Chung, G., Naka-

tani, Y., Wang, J.Y., and Kedes, L. (1999). Acetylation of MyoD di-

rected by PCAF is necessary for the execution of the muscle pro-

gram. Mol. Cell 4, 725734.

Semsarian, C., Wu, M-J., Marcintec, T., Harvey, R.P., and Graham,

R.M. (1999). Skeletal muscle hypertrophy is mediated by a cal-

cineurin-dependent signaling pathway. Nature 400, 576581.

Shore, P., and Sharrocks, A.D. (1995). The MADS box family of

transcription factors. Eur. J. Biochem. 229, 113.

Sparrow, D.B., Miska, E.A., Langley, E., Reynaud-Deonauth, S., Ko-

techa, S., Towers, N., Spohr, G., Kouzarides, T., and Mohun, T.J.

(1999). MEF-2 function is modified by a novel co-repressor MITR.

EMBO J. 18, 50855098.

Sternberg, E.A., Spizz, G., Perry, W.M., Vizard, D., Weil, T., and

Olson, E.N. (1988). Identification of upstream and intragenic regula-

tory elements that confer cell-type restricted and differentiation-

specific expression on the muscle creatine kinase gene. Mol. Cell.

Biol. 8, 28962909.

Valdez, M .R., Richardson, J.A., Klein, W.H., and Olson, E.N. (2000).

Failure of Myf5 to support myogenic differentiation without myo-

genin, MyoD, and MRF4. Dev. Biol. 219, 287298.

Verdel, A., and Khochbin, S. (1999). Identification of a new family of

higher eukaryotic histone deacetylases. J. Biol. Chem. 274, 2440

2445.

Wang, A.H., Bertos, N.R., Vezmar, M., Pelletier, N., Crosato, M.,

Heng, H.H., Thng, J., Han, J., and Yang, X-J. (1999a). HDAC4, a

human histone deacetylase related to yeast HDA1, is a transcrip-

tional corepressor. Mol. Cell. Biol. 19, 78167827.

Wang, Z-M., Messi, M.L.,Renganathan, M., andBelbono, O. (1999b).

Insulin-like growth factor-I enhances rat skeletal muscle charge

movement and L-type Ca2 channel gene expression. J. Physiol.

(Lond.) 516, 331341.

Weintraub, H., Davis, R., Lockshon, D., and Lassar, A. (1990). MyoD

binds cooperatively to two sites in a target enhancer sequence:

occupancy of two sites is required for activation. Proc. Natl. Acad.

Sci. USA 87, 56235627.

Workman, J.L., and Kingston, R.E. (1998). Alteration of nucleosome

structure as a mechanism of transcriptional regulation. Annu. Rev.

Biochem. 67, 545579.

Yang, S., Galanis, A., and Sharrocks, A.D. (1999). Targeting of p38

lu - molecular cell - 2000

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