RESEARCH ARTICLE

The Homeobox Gene Mohawk RepressesTranscription by Recruiting the Sin3A/HDACCo-repressor ComplexDouglas M. Anderson,1,2 Brian J. Beres,1,3 Jeanne Wilson-Rawls,1,4 and Alan Rawls1,4*

Mohawk is an atypical homeobox gene expressed in embryonic progenitor cells of skeletal muscle, tendon,and cartilage. We demonstrate that Mohawk functions as a transcriptional repressor capable of blockingthe myogenic conversion of 10T1/2 fibroblasts. The repressor activity is located in three small,evolutionarily conserved domains (MRD1–3) in the carboxy-terminal half of the protein. Point mutationanalysis revealed six residues in MRD1 are sufficient for repressor function. The carboxy-terminal half ofMohawk is able to recruit components of the Sin3A/HDAC co-repressor complex (Sin3A, Hdac1, and Sap18)and a subset of Polymerase II general transcription factors (Tbp, TFIIA1 and TFIIB). Furthermore, Sap18,a protein that bridges the Sin3A/HDAC complex to DNA-bound transcription factors, is co-immunoprecipitated by MRD1. These data predict that Mohawk can repress transcription throughrecruitment of the Sin3A/HDAC co-repressor complex, and as a result, repress target genes required for thedifferentiation of cells to the myogenic lineage. Developmental Dynamics 238:572–580, 2009.© 2009 Wiley-Liss, Inc.

Key words: Mohawk; Mkx; Iroquois; Irx; Sin3A; HDAC; Sap18; Tbp; repressor; MRD; myogenesis

Accepted 18 December 2008

INTRODUCTION

Mohawk (Mkx) is the sole member of anewly characterized class within theTALE superclass of atypical ho-meobox genes (PBC, MEIS, TGIF,IRO, and MKX)(Anderson et al., 2006;Murkerjee and Burglin, 2007). TALE(three amino acid loop extension) ho-meobox factors are defined by the in-clusion of three additional amino acidsin the loop between the first and sec-ond helices of their homeodomains.These proteins are essential for abroad set of developmental processes,

including cell proliferation, differenti-ation and positional specification(Selleri et al., 2001; Brendolan et al.,2005; Moens and Selleri, 2006; vanTuyl et al., 2006; diIorio et al., 2007).Initial characterization of mouse Mkxrevealed a dynamic transcription pat-tern restricted to progenitors of skele-tal muscle, tendon, and cartilage, aswell as the sex chords of the male go-nad and the ureteric bud tip of themetanephrogenic kidney (Anderson etal., 2006; Liu et al., 2006; Takeuchiand Bruneau, 2007). Because Mkx or-

thologs are highly conserved in bothprotostomes and deuterostomes, Mkxis believed to play an essential role inregulating decisions regarding differ-entiation and patterning of these tis-sues during development. To furtherour understanding of Mkx, we wereinterested in determining its functionas a transcriptional regulator and itsrole in tissue differentiation.

Among the TALE genes, Mkx ismost closely related to the Iroquois(Irx) class (Anderson et al., 2006; Liuet al., 2006; Takeuchi and Bruneau,

Additional Supporting Information may be found in the online version of this article.1School of Life Sciences, Center for Evolutionary Functional Genomics, Arizona State University, Tempe, Arizona2Molecular and Cellular Biology Graduate Program, Center for Evolutionary Functional Genomics, Arizona State University, Tempe,Arizona3Biology Graduate Program, Center for Evolutionary Functional Genomics, Arizona State University, Tempe, Arizona4Arizona Biodesign Institute, Arizona State University, Tempe, Arizona*Correspondence to: Alan Rawls, Box 874501, Life Sciences C Bldg., Room 544, Arizona State University, Tempe, Arizona,85287-4501. E-mail: alan.rawls@asu.edu

DOI 10.1002/dvdy.21873Published online 16 February 2009 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 238:572–580, 2009

© 2009 Wiley-Liss, Inc.

2007). Mouse Mkx shares 56% homol-ogy with Irx2 over the entire home-odomain (35/63 residues) and 82%(14/17 residues) specifically in HelixIII, which physically interacts withDNA. This includes the conservationof an alanine in the DNA recognitiondomain of Helix III that is believed toparticipate in defining the target en-hancer sequence for Irx proteins. TheIrx genes can be distinguished fromMkx by the presence of a highly con-served IRO Box in the carboxy-termi-nal half of all Irx genes (Burglin,1997). Vertebrate members of the Irxfamily regulate a diverse set of devel-opmental events, including chamber-specific transcription in the heart,specification and positional identity ofthe vertebrate neuroectoderm,branching morphogenesis in thelungs, patterning of the nephron, andformation of the zebrafish organizer(Bao et al., 1999; Bruneau et al., 2001;Kudoh and Dawid, 2001; Gomez-Skarmeta and Modolell, 2002; Matsu-moto et al., 2004; Lecaudey et al.,2005; van Tuyl et al., 2006; Reggianiet al., 2007). Studies carried out withchick Irx2 and Drosophila Mirr pre-dict that the Irx family members func-tion to repress transcription (Matsu-moto et al., 2004; Bilioni et al., 2005).In addition, transcriptional repressionactivity by Irx4 has been shown to beessential for proper chamber specifi-cation in the chick heart. However,the mechanisms by which the Iroquoisgenes function are still unknown.

During embryonic development,Mkx transcription is first detected inthe dorsomedial and ventrolateral re-gions of the dermomyotome of matur-ing somites, which contain highly pro-liferative myogenic progenitor cells(Anderson et al., 2006). Tight regula-tion of the switch between prolifera-tion and differentiation of these cellsis essential for balancing the mainte-nance of the myogenic progenitor pooland the expansion of skeletal muscle(Amthor et al., 1999). Differentiationof myogenic progenitors can be mea-sured using the myogenic conversionassay, which measures the ability ofthe bHLH transcription factor MyoDto convert fibroblast cells to myo-blasts. In this assay, we found thatMkx is a strong inhibitor of myogenicdifferentiation, which was dependenton DNA-binding through the Mkx ho-

Fig. 1. Gal4-Mkx represses transcription in a dose-dependent manner. A: The DNA binding-dependent transcriptional properties of the Gal4-Mkx fusion protein were measured by co-expres-sion in NIH3T3 cells with the Gal4-responsive luciferase reporter, 5XUASpGL3Control. B: Gal4-Mkxis a potent transcriptional repressor at plasmid concentrations ranging from 1 ng to 100 ng.Repression is expressed as the percent luciferase activity in cells transfected with5XUASpGL3Control and Gal4DBD.

Fig. 2. Identification of the transcriptional repressor domains in Mkx. A: The Gal4-responsive lucif-erase reporter, 5XUASpGL3Control, was co-transfected into NIH3T3 cells with expression plasmids forGal4DBD fusions carrying either full-length Mkx (aa 1-354) or fragments of Mkx as indicated. Thehomeodomain (green) and Conserved Domains in the carboxy-terminal region (CD-A to E) are labeled.Data are presented for each Gal4DBD fusion as fold repression, which is the inverse of the foldluciferase activity relative to the luciferase activity determined for the Gal4 DNA-binding domain alone.

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meodomain. A biochemical analysisrevealed Mkx possesses transcrip-tional repressor activity, mediatedby three conserved domains in thecarboxy-terminal region of the pro-tein. This region forms a complexwith both the Sin3A/HDAC co-re-pressor complex and a subset ofPolymerase II general transcriptionfactors. Therefore, Mkx can functionas a repressor of genes required forthe differentiation of tissue progeni-tors.

RESULTS

Mkx Is a TranscriptionalRepressor

The presence of a homeodomain inMkx predicts that it regulates tran-scription, however, neither its nativeDNA-binding site nor its transcrip-tional properties are known. There-fore, we used the well-characterizedGal4/UAS transcription assay to de-termine the DNA binding-dependenttranscription activity of Mkx. Full-length Mkx coding sequence wasfused to the amino-terminus of theGal4 DNA-Binding Domain (aa1-147; Gal4DBD). Because theclosely related Irx genes have beenreported to act as transcriptional re-pressors (Matusumoto et al., 2004; Bil-ioni et al., 2005), the Gal4-Mkx fusionprotein was co-transfected into NIH3T3cells with luciferase reporter5XUASpGL3Control. This reporter con-tains the SV40 promoter, the SV40 En-hancer and five copies of the Gal4 UAS,which provides a strong basal luciferaseactivity and is responsive to Gal4DBDfusion proteins (Fig. 1A). Gal4-Mkx wasa potent transcriptional repressor in adose-dependent manner over a 100-foldrange in concentration, as comparedwith transfection with Gal4DBD alone(Fig. 1B). A 2.6-fold reduction (38.5%) inluciferase activity of the reporter wasobserved when only 1 ng of Gal4-Mkxencoding plasmid DNA was co-trans-fected and an 18.2-fold reduction(95.5%) in luciferase activity was ob-served with 100 ng of Gal4-Mkx. Al-though the use of the Gal4 DNA-binding domain is artificial, our resultsdemonstrate that Mkx can function as atranscriptional repressor.

Identification of ThreeIndependent RepressorDomains Within Mkx

To identify domains of Mkx mediatingrepression activity, we again used theGal4/UAS assay system. For grossmapping, Gal4DBD fusion proteinswere generated containing the Mkxhomeodomain [Gal4-Mkx(70-132)],the amino-terminal region [Gal4-Mkx(1-69)] or carboxy-terminal region[Gal4-Mkx(133-354)] to the homeodo-main (Fig. 2). Co-transfection of thereporter with the carboxy-terminal fu-sion protein resulted in an 18.3-foldrepression of luciferase activity, rela-tive to the Gal4DBD alone (Fig. 2).Neither the amino-terminal domainnor the homeodomain demonstratedany repressor activity. Therefore, thecarboxy-terminal region contains allof the repressor activity.

Potential repression domainswithin the carboxy-terminal regionwere identified based on amino acidconservation among vertebrate or-thologs of Mkx. A multiple sequencealignment of Mkx orthologs from Musmusculus (GenBank Accession #NM_177595), Homo sapiens (Gen-Bank accession no. NM_173576), andthe two orthologs from Danio rerio,(MkxA, GenBank accession no.EU280785; MkxB, GenBank accession

no. EU280786), revealed five highlyconserved domains (CD-A throughCD-E) in the carboxy-terminal region(sequence alignment and CDs areidentified in Supp. Fig. S1, which isavailable online). Recently, CD-A, -B,and -D were also reported to be con-served among protostomes (Murkerjeeand Burglin, 2007). Of interest, CD-Eis found within 25 residues at the car-boxy-terminus that are not present inprotostomes.

Fragments containing individualCDs (CD-A [aa 133-174], CD-B [aa207-241], CD-C [aa 247-283], CD-D[aa 301-322] and CD-E [aa 327-354])were subcloned in-frame with theGal4DBD and tested for their abilityto repress the 5XUASpGL3Control re-porter. CD-A, CD-A with the adjacenthomeodomain, and CD-C were unableto repress luciferase transcription(Fig. 2). In contrast, CD-B was a po-tent repressor and resulted in a 21.5-fold repression in luciferase activity,relative to the Gal4DBD alone. Amongprotostomes and deuterostomes, onlythe central 13 aa of CD-B (M. muscu-lus KYKSSLLNRYLND; aa 281-230)are highly conserved (Murkerjee andBurglin, 2007). When fused to theGal4DBD these residues were able torepress transcription of the reporter30-fold. We renamed this sequenceMohawk Repressor Domain 1

Fig. 3. Residues required for MRD1 repressor function. A: The Gal4-responsive luciferase reporter,5XUASpGL3Control, was co-transfected into NIH3T3 cells with expression plasmids for Gal4DBDfusions carrying mouse MRD1, Drosophila MRD1, or mouse MRD1 alanine substitution mutants.Data are presented for each Gal4DBD fusion as fold repression, which is the inverse of the foldluciferase activity relative to the luciferase activity determined for the Gal4 DNA-binding domainalone. B,C: I-TASSER program predicted models of MRD1 (B) and the artificial MRD1 (C) secondarystructures with the side chains.

Fig. 4. The carboxy-terminal region of Mkx forms a stable complex with the Sin3A/HDAC co-repressor complex. The ability of Mkx to form a stable complex with Sin3A, Hdac1, and Sap18 wasexamined by co-immunoprecipitation/western blot. Myc-tag Mkx-specific fusion proteins wereco-expressed in NIH3T3 cells with HA-tag Hdac1, Sin3A or Sap18. Whole cell lysates wereimmunoprecipitated with an anti-myc antibody and western blots were probed with an anti-HAantibody. A: HA-tag Hdac1 was co-immunoprecipitated by myc-tag Mkx and not by the myc-tagonly control. B: HA-tag Sin3A was co-immunoprecipitated by full-length Mkx and the carboxy-terminal domain (aa 133-354), but not by the amino-terminal region (aa 1-69), the homeodomain (aa70-132) or by MRD1. HA-tag Sap18 bound to Mkx in a similar manner as Sin3A, but additionally tothe homeodomain and MRD1.

Fig. 5. Mkx interacts with Tbp, TFIIA1, and TFIIB. The ability of Mkx to form a stable complex withmembers of the RNA Polymerase II general transcription factors was examined by co-immuno-precipitation/Western blot. A: The myc-tag Mkx fusion proteins were co-expressed in NIH3T3 cellswith HA-tag versions of Tbp, Tbpl1, TFIIA1 and TFIIB. Whole cell lysates were immunoprecipitatedwith an anti-myc antibody and subsequently probed with an anti-HA antibody. Tbp, TFIIA1 andTFIIB all interacted with full-length myc-tag Mkx. B: HA-tag Tbp was co-immunoprecipitated byfull-length Mkx and the carboxy-terminal domain (aa 133-354), but not by the amino-terminal region(aa 1-69) or the homeodomain (aa 70-132).

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Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6. Mkx inhibits the differentiation of MyoD-converted myoblasts. A: Mouse 10T1/2 fibroblast cells were transfected with expression vectors carryingMyoD or MyoD and Mkx and challenged to differentiate into myotubes by culturing in low mitogen containing conditions. B: The degree of differentiation wasassessed by counting the number of myotubes, as identified by immunostaining for an antibody against skeletal muscle-specific myosin heavy chain (MHC).The myogenic activity of Mkx or Mkx(�H3), a mutation that deletes the DNA-recognition helix of the homeodomain, is expressed as the percentage ofMHC-positive myotubes, compared with the total number of myotubes formed as a result of MyoD transfection alone, which was given the arbitrary valueof 100%. The average of three independent experiments reveal that Mkx is able to inhibit muscle differentiation, while Mkx(�H3) relieved most, but not allof the inhibition. As well, Mkx, Mkx(�H3) and the myc-tag empty vector were alone not sufficient to induce muscle differentiation.

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(MRD1). A carboxy-terminal fragmentcontaining both CD-D and CD-E[Gal4-Mkx(284-354)] led to an 11.9-fold reduction in luciferase activity.Individual analysis of fragments con-taining either CD-D or CD-E revealeda 6.0- and 5.0-fold reduction, respec-tively (Fig. 2). We renamed these re-gions MRD2 and MRD3, respectively.

To determine the necessity ofMRD1, 2, and 3 for the repression ac-tivity of Mkx, individual and combina-torial deletions of these domains weremade in the context of the full-lengthprotein. Deletion of MRD1 led to areduction in repressor activity, (6.3-fold vs. 30.0-fold), while deletion of allthree domains [Gal4-Mkx(�MRD1-3)]resulted in a complete loss of repres-sor function (Fig. 2). MRDs do notshare any significant sequence homol-ogy with known functional domains,based on a TBLASTN search of theavailable sequenced genomes (datanot shown). Therefore, MRDs 1–3 rep-resent novel repression domains thatcan function independently to represstranscription and are necessary forthe repressor function of Mkx.

Determination of ResiduesRequired for MRD1Function

A comparison of MRD1 sequence frommouse to the distantly related Drosoph-ila melanogaster revealed that only 5 of13 amino acids are identical, while 5 arehighly similar (Fig. 3A). As a fusion tothe Gal4DBD domain, Drosphila MRD1was able to repress transcription of the5XUASpgl3Control reporter in mouseNIH3T3 cells, albeit less strongly thanmouse MRD1 (Fig. 3A). This suggeststhat distantly related protostome or-thologs may share a conserved mecha-nism of MRD1-mediated repression.

To determine the specific amino acidsrequired for MRD1 function, alaninesubstitution mutants were created asfusions to the Gal4DBD and tested fortheir ability to repress the Gal4 re-sponsive 5XUASpgl3Control reporter.Alanine substitution of residues K218,Y219, K220, L223, R226, and Y227greatly reduced MRD1 repressor func-tion, whereas substitution of L224 orD230 had little effect (Fig. 3A). Wenext tested the highly substituted arti-ficial MRD1 (aMRD1 sequence: KY-KAALAARYAAA), which only con-

tained those residues found to becritical for repressor function. aMRD1was able to repress transcription at alevel equivalent to MRD1.

The three-dimensional structure ofMRD1 and aMRD1 were predicted us-ing the web-based I-TASSER pro-gram, which is a hierarchical proteinstructure modeling approach based onsecondary-structure enhanced pro-file–profile threading and the interac-tive implementation of the threadingassembly refinement program (Wu etal., 2007; Zhang, 2007, 2008). Basedon this program, both MRD1 andaMRD1 are predicted to form shortalpha helices (Fig. 3B,C). From themodels, it is clear that the predictedcritical residues for MRD1 functionare present on the same side of thealpha helix (Fig. 3B). To further dem-onstrate the importance of secondarystructure for MRD1 function, we sub-stituted a proline residue in place ofasparagine at position 225. The pro-line substitution mutant, which is pre-dicted to break the alpha helix second-ary structure, resulted in a completeloss of repressor function (Fig. 3A).Therefore, secondary structure ap-pears to be critical for MRD1 function.

Mkx Associates WithComponents of the Sin3A/HDAC Co-repressor Complex

Chromatin remodeling by the recruit-ment of Histone Deacetylases (HDACs)to a gene locus is a common mechanismfor transcriptional repression (Burkeand Baniahmad, 2000). HDACs are tar-geted to specific gene loci complexedwith other proteins, notably as part ofthe Sin3A/HDAC or NuRD co-repressorcomplexes (Ayer, 1999). We tested theability of components of the Sin3A/HDAC complex to form stable interac-tions with Mkx using co-immunopre-cipitation. Myc-epitope tagged Mkx(MT-Mkx) and HA-epitope taggedmembers of the Sin3A/HDAC complex(Hdac1, Sin3A, and Sap18) were co-ex-pressed in NIH3T3 cells. MT-Mkx wasimmunoprecipitated from cell lysatesusing an anti-myc antibody and co-pre-cipitation of members of the Sin3A/HDAC co-repressor complex was deter-mined by Western Blot using ananti-HA antibody. MT-Mkx, but not themyc-tag only control, was able to co-precipitate both Hdac1 and Sin3A (Fig.

4A,B). The fragment of Mkx carboxy-terminal to the homeodomain [MT-Mkx(133-354)] also bound Sin3A. Incontrast, neither the homeodomain[MT-Mkx(70-132)] nor the region ami-no-terminal to the homeodomain [MT-Mkx(1-69)] was able to bind HA-Sin3A.This is consistent with our previousfinding that the repressor function ofMkx is in the carboxy-terminal domainof the protein. However, the 13 aaMRD1 was not sufficient to co-precipi-tate Sin3A (Fig. 4B).

Sin3A-associated protein, 18 kDa(Sap18), has been shown to mediatethe interaction between Sin3A andDNA-bound transcription factors(Zhang et al., 1997; Sheeba et al.,2007). Using the same co-immunopre-cipitation assay, Sap18 was found tobind to full-length Mkx, the Mkx ho-meodomain, the region carboxy-termi-nal to the homeodomain, but not theregion amino-terminal to the home-odomain (Fig. 4B). Of interest, MRD1was sufficient to co-immunoprecipi-tate Sap18 as well. These results indi-cate that Mkx recruits the Sin3A/HDAC co-repressor complex.

Mkx Interacts With a Subsetof Polymerase II GeneralTranscription Factors

Components of the RNA PolymeraseII general transcription factors(GTFs) are common targets of tran-scriptional repressors, and representan HDAC-independent mechanism ofrepression. This occurs through directbinding or interaction with co-repres-sor complexes, which has been notedfor the Sin3A/HDAC complex (Heinzelet al., 1997; Laherty et al., 1997; Nagyet al., 1997). To determine whetherMkx interacts with GTFs, co-immuno-precipitations were performed withMT-Mkx and HA-epitope tagged Tbp,Tbp-like 1 (Tbpl1) and GTFs that hadbeen previously shown to interactwith co-repressors, including TFIIA1and TFIIB, (Burke and Baniahmad,2000). Full-length Mkx was able to co-immunoprecipitate TFIIA1, TFIIB,and Tbp (Fig. 5A). Of interest, Mkxdid not co-precipitate Tbpl1, a genethat is 73% similar to Tbp and thatcan also bind TATA box elements andinitiate transcription (Fig. 5A). Wefurther show that Tbp is specificallyco-immunoprecipitated by the region

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carboxy-terminal to the Mkx homeo-domain, but by neither the region ami-no-terminal to the homeodomain northe homeodomain itself (Fig. 5B). Thisdemonstrates that Mkx can interactwith a specific subset of RNA Poly-merase II GTFs, which potentially cancontribute to a second mechanism oftranscriptional repression.

Mkx Inhibits theDifferentiation of MyoD-Initiated Myoblasts

It has been previously reported thatMkx is expressed in the myogenic pro-genitor cells in the dermomyotomecompartment of mouse somites(Anderson et al., 2006). Initiation ofMkx transcription appears in progres-sively posterior somites at a rate sim-ilar to the appearance of myoblasts inthe myotome of these somites. Thissuggests that Mkx may play a role inregulating the onset of muscle differ-entiation. To test this, we examinedthe impact of Mkx on the conversion of10T1/2 mouse fibroblasts to myoblastsby MyoD. Ectopic expression of MyoDin 10T1/2 cells induces the completemyogenic program, characterized byan exit from the cell cycle and theformation of multinucleate myotubesthat express skeletal muscle-specificstructural proteins (Tapscott et al.,1988).

MyoD, driven by the EMSV pro-moter (Davis et al., 1987), was ex-pressed in 10T1/2 cells for 36 hoursand then the cells were allowed to dif-ferentiate for 5 days under low mito-gen conditions. Differentiation to themuscle lineage was assessed by im-munolabeling for the skeletal muscle-specific protein myosin heavy chain(MHC; Fig. 6A,B). Co-expression ofMyoD with full-length Mkx reducedthe number of MHC-positive cells andappeared to reduce the number ofmultinucleated myotubes comparedwith MyoD alone. Quantifying thenumber of MHC-positive cells wasused to assess the impact of Mkx onmyoblast differentiation. MyoD ex-pression alone was used as a referenceof 100% differentiation (Fig. 6B). Co-expression of Mkx with MyoD resultedin a 48.2% reduction in the number ofMHC-positive myotubes (Fig. 6B). Theinhibition of myogenesis by Mkx wasdisrupted (14.0% vs. 48.2%), when He-

lix III, the DNA-binding recognitionhelix of the homeodomain (aa114-130), was deleted (Fig. 6B). Thisdemonstrates that Mkx inhibits myo-genesis in a DNA-dependent manner.Given that Mkx is expressed in myo-genic progenitors in the mouse em-bryo, this argues that Mkx plays a rolein restricting the differentiation ofprogenitors to the myogenic pathway.

DISCUSSION

Mkx is a recently identified homeoboxgene transcribed in the discrete em-bryonic progenitor cells of the muscu-loskeletal system, including skeletalmuscle, tendon, and cartilage (Ander-son et al., 2006). Mkx has the poten-tial to play a critical role in the geneticpathways that lead to the develop-ment of these tissues. In this regard,the expression pattern of Mkx over-laps with Pax3 and paraxis in skeletalmuscle progenitors, scleraxis in ten-don progenitors and Sox9 in cartilageprogenitors of the axial skeleton(Anderson et al., 2006). Here, we de-scribe the results of our analysis of thebiochemical function of Mkx in cellculture. Mkx can function as a tran-scriptional repressor and containsthree small repressor domains. Co-IPexperiments predict that Mkx re-presses transcription through eitherHDAC-dependent chromatin remodel-ing or association with the generaltranscription machinery. Finally, amyogenic conversion of 10T1/2 cellsdemonstrated that expression of Mkxwas able to block MyoD-directed mus-cle differentiation. Overall, this studyidentifies Mkx as potent regulator ofskeletal muscle differentiation thatfunctions through three domains to di-rect transcriptional repression.

The TALE superclass of homeodo-main-containing proteins have beenshown to be important regulators ofcell proliferation, differentiation, andpatterning of a diverse set of tissuesduring the development of both proto-stomes and deuterostomes. Severalmembers of the TALE superclass havebeen reported to act as transcriptionalrepressors, including genes in theclosely related Irx, Pbc, and TGIFclasses (Asahara et al., 1999; Sharmaand Sun, 2001; Wotton et al., 2001;Matsumoto et al., 2004). We foundthat Mkx can also act as a transcrip-

tional repressor. This activity wasmapped to three short domains thatwere able to function independentlywhen fused to a heterologous DNA-binding domain. The short MRD’slikely function as protein–protein in-teraction domains with co-repressors.This is highlighted by the fact thatresidues critical for MRD1 function alloccur on the same side of an alpha-helix, creating a potential protein in-teraction surface. Short alpha-heliceshave been shown to participate in pro-tein–protein interactions for severaltranscription factors, including BCL2and MDM2 (Arkin and Wells, 2004).In the case of MRD1, we were able toidentify Sap18 as a binding partner.Short modular repression motifs havebeen described for other transcrip-tional repressors. Most notable arethe 55 aa en D domain of the engrailedprotein, the 57 aa C2D2 domain of eve,and the 13 aa Sin3A Interacting Do-main (SID) of the Mad family (Jaynesand O’Farrell, 1991; Han and Manley,1993a,b; Eilers et al., 1999). The MRDsequences of Mkx do not share signif-icant homology with these motifs andtherefore represent novel repressiondomains. It is interesting to note thatMRD3 only appears in vertebrate or-thologs of this gene. Considering theability of MRD3 to independently re-press transcription, this gives verte-brate Mkx orthologs an additionalregulatory function.

The Sin3A/HDAC core complex hasbeen well studied for its ability to di-rect gene-specific transcriptional re-pression. In addition to the acetyl-transferase activity of HDACs, thecore complex serves as a scaffold forbringing other enzymes that modifythe nucleosome and general transcrip-tion machinery (Yang et al., 2002,2003). Neither Sin3A nor HDACs pos-sess intrinsic DNA-binding activity,indicating that interaction with DNA-binding transcription factors is neces-sary to localize the repressor complex.To date, several transcription factors,including TALE superclass homeodo-main proteins Pbx1 and TGIF, havebeen shown to bind Sin3A (Sharmaand Sun, 2001; Wotton et al., 2001;Silverstein and Ekwall, 2005). Here,we demonstrate that Mkx also bindsthe Sin3A/HDAC complex. Sin3A,Hdac1 and Sap18 all interacted withthe carboxy-terminal region of Mkx,

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which acts as a functional repressor invivo. Interestingly, we were able toshow that Sap18 can interact withMRD1 alone. Sap18 has been reportedto play a role in stabilizing the Sin3A/HDAC complex and interacts withSin3A (Zhang et al., 1997). Localiza-tion of Sap18 through a heterologousDNA-binding domain to a reportergene results in transcriptional repres-sion (Zhang et al., 1997). Thus, Sap18recruitment can explain the repres-sion observed by MRD1 and may alsobe important for bridging Mkx to thelarger Sin3A/HDAC complex. TheMkx homeodomain was also able toco-immunoprecipitate Sap18, whichwas surprising, considering that thisdomain has no repressor activity. Forfuture work, it will be interesting todetermine the targets of MRD2 andMRD3 and determine whether theyhelp stabilize the Sin3A/HDAC com-plex interaction of Mkx or functionthrough novel mechanisms.

The ability to co-immunoprecipitateTFIIA1, TFIIB, and Tbp raises thepossibility that Mkx is able to represstranscription through an HDAC-inde-pendent mechanism. General tran-scription factors are essential for theformation of the RNA Polymerase IIinitiation complex at the transcrip-tional start site. Binding to these pro-teins can lead to a disruption of theinitiation complex, resulting in genesilencing (Heinzel et al., 1997; La-herty et al., 1997; Nagy et al., 1997).Our analysis has demonstrated thatthe large carboxy-terminal region ofMkx can bind Tbp, which is the sameregion that also bound the Sin3A/HDAC complex. Since Tbp and TFIIBbinding to Sin3A has been reported(Burke and Baniahmad, 2000), it re-mains a possibility that Mkx mediat-ed-repression occurs through onelarge complex.

Development of skeletal muscle isdependent on the balance between theself-renewal of myogenic progenitorcells associated with the periphery ofthe muscle and their differentiationinto myofibers. Cells at the dorsome-dial and ventrolateral lips (DML andVLL, respectively) of the dermomyo-tome are the source of the self-renew-ing premyogenic cells that contributeto the expanding myotome, the anlagefor skeletal muscle (Christ and Or-dahl, 1995; Cossu et al., 1996; Denet-

claw et al., 1997; Venters and Ordahl,2002). Specification of these cells bythe bHLH muscle regulator factorsMyoD, Myf5, Mrf4, and myogenin, isbalanced by muscle antagonists suchas Notch, Pax3, Pax7, and follistatin,which promote proliferation and blockthe transcription of muscle-specificgenes and muscle agonists such asmyostatin. We became interested inthe role Mkx plays in skeletal muscledifferentiation based on its dynamicexpression in the myogenic progeni-tors during development. Mkx is tran-scribed specifically in the regions ofthe DML and VLL, indicating a role incontrolling differentiation of the pre-myogenic cells (Anderson et al., 2006).To test this hypothesis, we used theclassic myogenic conversion cell-cul-ture assay. We found that Mkx inhib-ited the differentiation of MyoD-in-duced myoblasts in a DNA-bindingdependent manner. While the geneticpathway through which this occurredis unclear at this time, Mkx may func-tion upstream of the myogenic factorsand repress their transcription. Thisis also supported by the observationthat Mkx is not transcribed in themyotome compartment of the somite,thus indicating that down-regulationof Mkx transcription may be a neces-sary step in differentiation of the myo-genic lineage.

EXPERIMENTALPROCEDURES

Plasmids

Full-length gene-coding sequences andsubfragments were cloned into theCS2MT (6 � amino-terminal Mycepitopes), CS2HA (2 � HA amino-ter-minal epitopes)(Rupp et al., 1994) orCS2G4 (Gal4DBD 1-147) expressionplasmids using polymerase chain reac-tion (PCR) amplification of mouse em-bryonic cDNA. Restriction sites were in-cluded during PCR amplification tofacilitate cloning and stop codons wereintroduced where appropriate. CS2G4was constructed by subcloning the Hin-dIII/EcoRI fragment from pSG424 (Sa-dowski et al., 1988) into the HindIII/EcoRI sites of CS2MT, which removedthe 6 x Myc epitopes. Five copies of theGal4 UAS were PCR amplified fromGal4E1b5CAT (Wilson-Rawls et al.,2004) and subcloned into the KpnI/

BglII sites of pGL3Promoter andpGL3Control (Promega, Madison,WI), to create 5XUASpGL3Promoterand -Control, respectively. All cloneswere sequenced for verification.

Transient Transfections andLuciferase Reporter Assays

NIH3T3 mouse fibroblast cells wereseeded at 4 � 104 cells/well in com-plete media (DMEM supplementedwith 10% Newborn Calf Serum) in 24-well tissue-culture dishes. Each wellwas transfected with a total of 400 ngof plasmid DNA using 1 �l of Lipo-fectamine (Invitrogen, Carlsbad, CA)and 4 �l of PLUS reagent (Invitrogen),according to manufacture’s protocol.Transfected cells were lysed 24 hr af-ter transfection in 100 �l/well Lucif-erase Cell Culture Lysis Buffer (Pro-mega) and subjected to a singlefreeze–thaw cycle at �80°C. Lucif-erase activity was measured for eachwell by reacting 20 �l of cell lysatewith 100 �l of Luciferase Assay Buffer(Promega) in white 96-well plates, us-ing an FLx800 microplate reader(BioTek Instruments, Inc., Winooski,VT). Sample variables were per-formed in triplicate per experimentand each experiment was repeated atleast three times. Data are presentedfrom a single representative experi-ment.

Myogenic Conversion Assayin C3H10T1/2 Cells

C3H10T1/2 mouse fibroblast cellswere seeded at 1.9 � 105 cells inDMEM supplemented with 10% fetalbovine serum in 35-mm tissue culturedishes coated with 0.1% gelatin. Uponplating, the cells were immediatelytransfected according to manufac-ture’s protocol with 6 �l of Fugene6(Roche Applied Science, Indianapolis,IN) and 2 �g of plasmid DNA andincubated for 36 hr. The culture me-dium was removed and the cells wereincubated in low mitogen containingmedia (DMEM supplemented with 2%horse serum). Myogenic conversionwas scored after 5 days by the expres-sion of skeletal specific myosin, whichwas immunolabeled using the Anti-Myosin MY32 antibody (Sigma-Al-drich, St. Louis, MO), as described in(Wilson-Rawls et al., 1999).

578 ANDERSON ET AL.

Co-immunoprecipitationsand Western Blots

NIH3T3 cells were transfected asstated above, with the exception thatcells were transfected in 60-mm plateswith 2 �g of plasmid DNA. Accordingto each experiment, Myc-epitope tagfusion proteins were co-expressedwith target hemagglutinin (HA)-epitope tag fusion proteins. Immuno-precipitations were carried out usingmouse monoclonal Anti-Myc antibody(Invitrogen) as described in Wilson-Rawls et al. (1999). Initially, the ex-pression of all epitope tagged proteinswas confirmed by Western blot usingantibodies specific. For each pulldownexperiment, Western blots were per-formed to ensure equivalent total in-put protein.

For Western blotting, proteins weretransferred to an Immobilon-P PVDFmembrane (Millipore, Billerica, MA),dried in 100% methanol and used forwestern blotting as previously de-scribed in (Wilson-Rawls et al., 1999),with the following exceptions. ForCo-IP experiments, the primary anti-body was mouse Anti-HA (Invitrogen),diluted 1:500 in 1% Carnation nonfatdry milk in Tris Buffered Saline con-taining 0.1% Tween-20 (MTBST). Thesecondary antibody was alkalinephosphate conjugated Anti-MouseIgG (Invitrogen), diluted 1:10,000 inMTBST. For Gal4/UAS experiments,mouse monoclonal RK5C1 antibody(Santa Cruz Biotechnology, SantaCruz, CA) was used as a primary an-tibody to confirm the expression ofGal4DBD fusion proteins (Westernblots not shown). The secondary anti-body was the same as for the Co-IPexperiments. Membranes were im-aged on a Molecular DynamicsSTORM chemifluorescent imager us-ing ECF substrate (GE HealthcareBio-Sciences Corp., Piscataway, NJ).

ACKNOWLEDGMENTSWe thank Dr. Anthony Firulli for pro-viding the CS2MT plasmid, Dr. SteveTapscott for providing the CS2HAplasmid, Dr. Scott Bingham for assis-tance in DNA sequencing, and Dr.James Elser. We also thank Dr. KenroKusumi, Dr. Lei Lei, Dr. Brian Ver-relli, and Megan Rowton for theirvaluable discussion.

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