Proc. Natl. Acad. Sci. USAVol. 93, pp. 1764-1769, March 1996Biochemistry

A role for the Msx-1 homeodomain in transcriptional regulation:Residues in the N-terminal arm mediate TATA binding proteininteraction and transcriptional repressionHAILAN ZHANG*t, KATRINA M. CATRON*t, AND CORY ABATE-SHEN*t§*Center for Advanced Biotechnology and Medicine, Piscataway, NJ 08854; and tGraduate Program in Molecular Genetics and Microbiology and TDepartment ofNeuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ 08854

Communicated by Herbert Weissbach, Roche Institute of Molecular Biology, Nutley, NJ, November 15, 1995

ABSTRACT In a previous study we showed that themurine homeodomain protein Msx-1 is a potent transcrip-tional repressor and that this activity is independent of itsDNA binding function. The implication of these findings isthat repression by Msx-1 is mediated through its associationwith certain protein factors rather than through its interac-tion with DNA recognition sites, which prompted investigationof the relevant protein factors. Here we show that Msx-1interacts directly with the TATA binding protein (TBP) butnot with several other general transcription factors. Thisinteraction is mediated by the Msx-1 homeodomain, specifi-cally through residues in the N-terminal arm. These sameN-terminal arm residues are required for repression by Msx-1,suggesting a functional relationship between TBP associationand transcriptional repression. This is further supported bythe observation that addition of excess TBP blocks the re-pressor action of Msx-1 in in vitro transcription assays.Finally, DNA binding activity is separable from both TBPinteraction and repression, which further shows that theseother activities of the Msx-1 homeodomain are distinct. There-fore, these findings define a role for the Msx-1 homeodomain,particularly the N-terminal arm residues in protein-proteininteraction and transcriptional repression, and implicate amore complex role overall for homeodomains in transcrip-tional regulation.

It has become increasingly evident that the regulation ofappropriate gene expression during development requires notonly specific gene activation but also restricted gene expressionwhich is achieved by the action of transcriptional repressorproteins. Accordingly, many important developmental regu-latory genes encode proteins that function as transcriptionalrepressors. Among these is the murine homeobox gene msx-1which encodes a homeodomain-containing protein that func-tions as a transcriptional repressor (1, 2). Msx-] is expressedduring murine development in many embryonic regions in-cluding the neural tube, the limb buds, and derivatives of thecranial neural crest (3-11). A common feature of these diverseexpression zones is their involvement in epithelial-mesenchymal interactions, indicating that msx-1 may be in-volved in inductive influences between these tissues (12). Itsbiological relevance is further evident from targeted disrup-tion of msx-1 which results in various developmental abnor-malities, the most deleterious of which are defects of cranio-facial structures (13).We have been studying the biochemical properties of Msx-1

in an effort to understand how it accomplishes its importantdevelopmental function. We have shown that Msx-1, similar toother homeodomain-containing proteins, exhibits sequence-specific DNA binding activity mediated by its homeodomain(1). We further showed that it functions as a potent transcrip-

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

tional repressor in both in vitro and in vivo assays (2) (K.M.C.and C.A.-S., unpublished data). However, Msx-1 (and mem-bers of the Msx family) has the distinctive property that itscognate DNA binding sites are not required for its repressoraction (2) (K.M.C. and C.A.-S., unpublished data), the impli-cation being that repression is mediated by some other mech-anism, most likely interactions with other protein factors. Herewe investigate its mechanism of repression by exploring theprotein factors with which Msx-1 interacts and the functionalconsequences of such interactions. We show that Msx-1 inter-acts directly with the TATA binding protein (TBP) via itshomeodomain and that residues in the N-terminal arm medi-ate this association. Moreover, these same residues are alsorequired for repression by Msx-1. These findings identify a rolefor the Msx-1 homeodomain in transcriptional repressionthrough its interaction with TBP.

MATERIALS AND METHODSPlasmids. The msx-l expression plasmids used in this study

are listed in Table 1. The pGEM series used for in vitrotranscription/translation contained the full-length msx-l cod-ing sequence cloned into the Kpn I and HindlIl sites ofpGEM-7Zf(+) (Promega). The mutations listed in Table 1were introduced by overlapping PCR mutagenesis as described(15). The pGEX series used in the glutathione S-transferase(GST) interaction assays contained full-length msx-1 or theindicated truncated regions cloned into the BamHI and SmaI sites (pGEX-MsxA2-5) or BamHI and EcoRI sites (pGEX-MsxA1) of pGEX-2T (Pharmacia). The truncations weregenerated by PCR amplification using oligonucleotides thatcorresponded to the appropriate 5' and 3' sequences and whichcontained restriction sites for cloning into pGEX. The trun-cated sequences encode the amino acids listed in Table 1. ThepM2 series encoding Gal4/Msx fusion polypeptides used intransient transfection analysis were constructed by subcloningthe appropriate msx-J sequences from pGEM into the corre-sponding sites of pM2 as described (2). Three of the pDS56series used to express msx-1 proteins as hexahistidine fusionsin E. coli were previously described: pDS56-msx-1 (2) andpDS56-myc-msx and pDS56-myc-msxHD (14). As described inref. 14, the latter two plasmids contain sequences encoding aheterologous epitope from the c-Myc protein which is recog-nized by an anti-Myc monoclonal antibody. pDS56-msx-A wasconstructed by subcloning the appropriate sequence frompGEM-msx-A into pDS56. The sequence of each msx-J plas-mid was verified using the Sequenase Version 2.0 kit (UnitedStates Biochemical). The pET-TBP and pGEX-TBP plasmids(both of which encode full-length human TBP) were generous

Abbreviations: TBP, TATA binding protein; GST, glutathione S-transferase; GTF, general transcription factor.§To whom reprint requests should be addressed at: Center forAdvanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway,NJ 08854-5638.

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Table 1. The Msx-1 expression plasmids

Name Plasmid

Msx pGEM-7zf(+)-MsxMsx-A pGEM-7zf(+)-Msx(K168A, R170A, F173A)Msx-B pGEM-7zf(+)-Msx(L181A, F185A)Msx-C pGEM-7zf(+)-Msx(R183A, K184A, R186A, Q187A)Msx-D pGEM-7zf(+)-Msx(R196A)Msx-E pGEM-7zf(+)-Msx(1212A, Q215A, N216A)Gal4/Msx pM2-Gal4-MsxGal4/Msx-A pM2-Gal4-Msx(K168A, R170A, F173A)Gal4/Msx-B pM2-Gal4-Msx(L181A, F185A)Gal4/Msx-C pM2-Gal4-Msx(R183A, K184A, R186A, Q187A)Gal4/Msx-D pM2-Gal4-Msx(R196A)Gal4/Msx-E pM2-Gal4-Msx(1212A, Q215A, N216A)GST-Msx pGEX-2T-MsxGST-MsxAl pGEX-2T-Msx(1-165)GST-MsxA2 pGEX-2T-Msx(1-225)GST-MsxA3 pGEX-2T-Msx(166-225)GST-MsxA4 pGEX-2T-Msx(166-297)GST-MsxA5 pGEX-2T-Msx(226-297)rMsx* pDS56-MsxrMsx-A pDS56-Msx(K168A, R170A, F173A)rMyc-Msx pDS56-Myc-MsxrMyc-MsxHD pDS56-Myc-Msx(157-233)The construction of plasmids is described in Materials and Methods

and in refs. 1 (rMsx) and 14 (rMyc-Msx and rMyc-MsxHD). Msx refersto the full-length coding sequence unless otherwise indicated.*r indicates plasmids used to produce recombinant proteins in Esch-erichia coli strain MC15.

gifts of Kam Yeung, Dongmin Ma, and Danny Reinberg[University of Medicine and Dentistry of New Jersey(UMDNJ)]. Purified yeast TBP (used in in vitro transcriptionassays) was a gift of Juan Inostroza and Danny Reinberg(UMDNJ).

Protein Purification. The pGEX-msx-1 plasmids encodingGST fusion proteins were expressed in E. coli strain BL21.Protein expression was induced by addition of isopropyl 3-D-thiogalactoside (1 mM), and the GST-Msx-1 fusion proteinswere isolated from sonicated cell lysates by immobilization onglutathione/agarose beads (Sigma) as described (16). TheGST-fusion proteins referred to throughout the text are thosebound to the glutathione/agarose beads. The pDS56-msx-1plasmids encoding hexahistidine fusion proteins were purifiedfrom guanidine-extracted cell lysates by nickel affinity chro-matography as detailed in refs. 1 and 15. Protein purity was-50-70% as determined by Coomassie blue staining of anSDS/polyacrylamide gel (H.Z. and C.A.-S., unpublished data).

In Vitro Transcription/Translation. The pGEM-msx-1 plas-mids were linearized with HindIll and RNA was synthesizedusing T7 RNA polymerase according to the manufacturer'sinstructions (Promega). Proteins were translated in vitro usinga rabbit reticulocyte lysate system as per the directions of themanufacturer (Promega). Radiolabeled proteins were pre-pared by including L-[35S]methionine (> 1000 Ci/mmol; 1 Ci =37 GBq; Amersham) in the reaction mixture, and translationefficiency was determined by SDS/polyacrylamide gel elec-trophoresis followed by autoradiography. Nonradiolabeledproteins used for gel retardation assays were synthesized inparallel with radiolabeled ones to verify translation efficiency.

Antibodies. Anti-Myc is a monoclonal antibody directedagainst a 10 amino acid epitope of the c-Myc protein (see ref.14). Anti-Msx-1 is a rabbit polyclonal antiserum directedagainst the purified Msx-1 homeodomain and has been de-scribed (2). The anti-TBP antibodies are monoclonal antibod-ies directed against human TBP; SL27-2-640 used in Fig. 1Cand SL39-2-573 used in Fig. 2 were generous gifts of NouriaHernandez (Cold Spring Harbor Laboratory).

RESULTS

Msx-1 Interacts with TBP But Not with Other GeneralTranscription Factors (GTFs). Two previous findings sug-gested that the repressor action of Msx-1 was mediated at leastin part through its direct interaction with one or more of theGTFs (2). (i) Msx-1 repressed transcription in vitro in a systemconsisting of purified GTFs. (ii) Msx-1 associated with aDNA-protein complex containing several GTFs includingTBP, TFIIA, and TFIIB. To determine whether Msx-1 didindeed associate directly with one or more of these proteins,we performed in vitro interaction assays using a GST-Msx-1(GST-Msx) fusion protein and 35S-labeled GTFs (Fig. 1A). Ofthe various GTFs tested, including TBP, TFIIA, TFIIB, TFIIE,and a subunit of TFIIH, GST-Msx interacted only with TBP(Fig. IA). Comparison of the "Input" TBP with the TBPbound to GST-Msx suggests that the interaction between theseproteins is relatively efficient (i.e., 20%) (Fig. 1A). Theinteraction was also specific since TBP associated with GST-Msx but not with GST alone (Fig. 1A). Finally, the interactionbetween Msx-1 and TBP occurred in the absence of DNA (Fig.1A) and was not abrogated by addition of either ethidiumbromide or micrococcal nuclease (H.Z. and C.A.-S. unpub-lished data) using standard conditions (18).The potential for TBP and Msx-1 to form a protein complex

was further investigated using two additional in vitro interac-tion strategies. First, we performed the complementary GSTinteraction assay using a GST-TBP fusion protein and 35S-labeled Msx-1 (Fig. 1B). This complementary approach re-vealed that 35S-labeled Msx-1 associated specifically with GST-TBP and that the efficiency was similar to that observedbetween GST-Msx and 35S-labeled TBP (compare Fig. 1 A andB). The second approach was a coimmmunoprecipitation assayin which we tested the ability of 35S-labeled TBP to beprecipitated along with an epitope-tagged version of Msx-1(Myc-Msx) by the anti-epitope antibody (aMyc) (Fig. 1C). Asshown in Fig. iC, 35S-labeled TBP was precipitated by theanti-Myc antibody (aMyc) in the presence but not in theabsence of Myc-Msx (compare lanes 4 and 7). In contrast,35S-labeled TBP was not precipitated by control IgG (aCon-trol) either in the absence (lane 3) or the presence (lane 6) ofMyc-Msx, whereas it was precipitated by an anti-TBP antibody(aTBP) in the absence of Myc-Msx (lane 5). Together with theGST interaction assays, these data demonstrate that Msx-1interacts efficiently and specifically with TBP in vitro.Msx-1 Interacts with TFIID as well as TBP. In cells, the

majority of TBP is found as a component of the multiproteincomplex termed TFIID. Therefore an important issue inevaluating the potential significance of the Msx-1-TBP asso-ciation was to ascertain that Msx-1 also interacted with TBP inthe TFIID complex. To determine whether this was the case,we performed in vitro interaction assays using GST-Msx and achromatographic fraction from HeLa nuclear extracts that isenriched for TFIID [i.e., the 1 M KCl fraction from phospho-cellulose chromatography (19)]. As shown in Fig. 2, GST-Msxinteracted with TBP in the TFIID fraction. The interaction wasspecific since GST alone did not bind TBP in the TFIIDcomplex and was relatively efficient (i.e., >20%) as indicatedby the amount of bound TBP relative to the Input (Fig. 2). Thisfinding shows that Msx-1 associates with TBP as it is normallyfound as a component of TFIID.

Interaction of Msx-1 with TBP Requires the Homeodomain.To define the regions of Msx-1 that mediate its interaction withTBP, we produced a series of truncated polypeptides contain-ing various combinations of its three main regions-i.e., theN-terminal region, the homeodomain, and the C-terminalregion (Fig. 3A and Table 1). These were produced as GSTfusion polypeptides and tested in parallel with GST-Msx fortheir ability to interact with 35S-labeled TBP in in vitrointeraction assays (Fig. 3). The truncated GST-Msx polypep-

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C

B Msx

A TBP TFIIA TFIIB TFIIEp34 TFIIEp56 TFIIHp62

x x x x x xE) CA CD C coD cC CC

CU C) U.

(0 U 0.*

) U 0. U) U 0.*

) U 0. U)

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35S-TBP

- - - + + - - Myc-Msx- + + Myc-MsxHD

+ - - + - + - aControl- + - - + - + (xMyc- - + - - - - aTBP

1 2 3 4 5 6 7 8 9

FIG. 1. Msx-1 interacts with TBP but not other GTFs. (A and B) GST interaction assays. (A) The indicated proteins [i.e., TBP, TFIIA (uncleaveda and ,B subunits), TFIIB, TFIIEp34, TFIIEp56, or TFIIHp62] were synthesized in vitro and radiolabeled with [35S]methionine. The GST orGST-Msx proteins (6 jig of each) were incubated with the 35S-labeled proteins in buffer A [20 mM Tris HCl, pH 7.9/0.2 mM EDTA/0.1 M NaCl/1mM dithiothreitol/1 mM phenylmethylsulfonyl fluoride/0.2% Nonidet P-40 (NP-40)]. (B) 35S-labeled Msx was synthesized in vitro and incubatedwith GST or GST-TBP (6 ,ug of each) in buffer A. (A and B) Proteins were incubated for 1 hr at 4°C. The agarose beads were then collected bycentrifugation, washed three times with buffer A containing 0.4% NP-40, and bound proteins were eluted by boiling in SDS sample buffer. Proteinswere resolved by SDS/polyacrylamide gel electrophoresis and visualized by autoradiography. Input represents 20% (1 ,ul) of the total 35S-labeledprotein (5 jil) used in the interaction assays. Markers show the position of 14C-labeled protein molecular size standards (NEN). (C)Coimmunoprecipitation assay. Reaction mixtures containing 35S-labeled TBP (5 jil) were incubated with (+) or without (-) (nonradiolabeled)Myc-Msx (1 ,ug) or Myc-MsxHD (3 ,ug) in 1OS buffer as in ref. 28. Samples were precipitated with (+) or without (-) control IgG (aControl),anti-Myc IgG (aMyc), or anti-TBP IgG (caTBP) essentially as described (17) with the exception that Pansorbin cells (Calbiochem) were used forall precipitations. Lane 1 shows the position of 14C-labeled protein molecular size standards (Markers). Lane 2 (Input) represents 20% (1 jil) ofthe total 35S-labeled TBP (5 ,ul) used in the interaction assay. The arrow designates the mobility of 35S-labeled TBP.

tides that contained the homeodomain (i.e., GST-MsxA2,GST-MsxA3, and GST-MsxA4) interacted with TBP, whereasthose that lacked the homeodomain (i.e., GST-MsxAl andGST-MsxA5) did not interact with TBP (Fig. 3). Thus, thehomeodomain is required for the Msx-1-TBP association. Thisobservation is further supported by results of co-immunopre-cipitation assays which demonstrate specific interaction be-tween 35S-labeled TBP and an epitope-tagged version of theMsx-1 homeodomain (Myc-MsxHD) (Fig. 1C, lanes 8 and 9).Taken together, these findings reveal a role for the homeodo-main as a protein interaction domain that mediates associationof Msx-1 with TBP.Although the N-terminal region did not interact with TBP

on its own (see GST-MsxAl), it facilitated the interaction ofthe homeodomain with TBP since a GST-fusion polypeptidethat contained both Msx-1 regions interacted more efficientlywith TBP as compared with the protein containing only the

TFIID fraction

ax- x0 5

0 C. I

FIG. 2. Msx-1 interacts with TFIID. GST interaction assay was

performed using GST or GST-Msx (2 jig of each) and a chromato-graphic fraction enriched for TFIID from HeLa nuclear extracts (200jig). The bound proteins were resolved by SDS/polyacrylamide gelelectrophoresis and the bound TBP was visualized by Western blotanalysis using anti-TBP antibody. The antigen-antibody complexeswere visualized using a chemiluminescent reagent (Amersham). Con-trol TBP shows the mobility of purified TBP. Input represents 10% (20jig) of the total HeLa nuclear extract sample used in the interactionassay.

homeodomain (Fig. 3, compare GST-MsxA2 and GST-MsxA3). It seems likely that the role of the N-terminal regionis to augment the stability of the Msx-1-TBP interaction. Thismay explain our previous finding in which we did not detect astable complex formed between the Msx homeodomain andGTFs in gel retardation assays (2).

Residues in the N-Terminal Arm of the Msx-1 Homeodo-main Mediate TBP Interaction and Transcriptional Repres-sion. To define specific residues within the homeodomainrequired for its interaction with TBP, we produced a series ofMsx-1 polypeptides that contained clustered alanine substitu-tions within various homeodomain segments (Fig. 4A andTable 1). These segments correspond to the well characterizedstructural subdivisions of the homeodomain-namely, a-heli-ces I, II, and III and the N-terminal arm (reviewed in ref. 20).Alanine substitutions were made in the context of full-lengthMsx-1 to account for the contribution of the N-terminalregion toward TBP interaction (see Fig. 3) and also toexamine the effect of these amino acid replacements onother functional properties of Msx-1 (see below). As shownin Fig. 4B, an Msx-l polypeptide that contained substitutionsof residues in the N-terminal arm (Msx-A) exhibited asignificant reduction in its ability to associate with GST-TBP(compare Msx with Msx-A). In contrast, substitutions ofother residues throughout the Msx-1 homeodomain hadminimal effect on its interaction with TBP (Fig. 4 A and B).These results show that residues within the N-terminal armof the homeodomain are primarily responsible for mediatingthe interaction of Msx-1 with TBP. This finding is noteworthyin light of the well-known significance of this homeodomainsegment for mediating the functional properties of homeo-domain-containing proteins in vivo (21, 22). In fact, it hasbeen proposed from homology modeling that the orientationof the N-terminal arm residues allows for their appropriateinteractions with other protein factors (22).We next considered whether a relationship existed between

the ability of Msx-1 to interact with TBP and its action as atranscriptional repressor. Thus, we examined whetherpolypeptides containing alanine substitutions within the ho-meodomain had altered transcriptional properties relative toMsx-1 (Fig. 4A and C). Transcriptional activity was evaluatedin transient transfection assays using expression plasmids that

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A

GST-Msx

GST-Msx Fusion Polypeptides

GST1 166 225 297

M Homeodomain _

TBPInteraction

1 165

GST-Msx21 go] | -1 166 225

GST-MsxA2 i |Homeodomain1 66 225

GST-Msxk3 RwHomeodomain|166 225 297

GST-Msx.14 ^ Homeodomain | l226 297

GST-MsxA5 imt I

B < < < c <x xc x x x

u) U 0) U) U) () 0)() i

2 2 2 2

_ . .

2' CAa o a7 o a 0

FIG. 3. Interaction of Msx-1 with TBP requires the homeodomain.(A) Diagram of the GST-Msx fusion polypeptides showing the regionsof Msx-1 contained in each of the truncated polypeptides (designatedGST-MsxA1-A5). The position of the homeodomain is shown, and theN-terminal and C-terminal regions are designated by the light stippledboxes. The amino acids corresponding to these regions are shown bythe numbers (also listed in Table 1). The dark stippled box shows theGST fusion. The degree of interaction of each GST-Msx polypeptidewith TBP is designated by + [ + + + indicates a strong interaction (i.e.,>20%), + + indicates a relatively weaker interaction, and - indicatesno interaction]. Experiments were performed a minimum of threetimes and the results are the estimate from these experiments;representative data are shown in B. (B) GST interaction assays were

performed by mixing 35S-labeled TBP (5 ,ul) with GST or indicatedGST-Msx polypeptide (6 ,ug of each). The bound protein was resolvedby SDS/polyacrylamide gel electrophoresis and visualized by autora-diography. The lane labeled "Input" represents 20% (1 ,u) of the total35S-labeled TBP (5 ,l) used in the interaction assay. The arrow

designates the mobility of 35S-labeled TBP.

encoded Gal4/Msx-1 fusion proteins and using a luciferasereporter plasmid containing Gal4 DNA binding sites (Fig. 4Aand C). We have previously established that the Gal4/Msx-1fusion polypeptide (Gal4/Msx) has similar transcriptionalrepressor properties as Msx-1 (ref. 2; K.M.C. and C.A.-S.,unpublished data). Therefore, the Gal4 binding domain was

used to ensure appropriate nuclear localization of the mutatedMsx-1 polypeptides [since substitutions in the homeodomainmay effect nuclear localization (K.M.C. and C.A.-S., unpub-lished data)] and to direct the proteins equivalently to thepromoter through the Gal4 DNA sites.As seen previously, Gal4/Msx was an effective transcrip-

tional repressor when tested in NIH 3T3 cells (Fig. 4C). Incontrast, the Gal4/Msx polypeptide containing alanine sub-stitutions in the N-terminal arm (Gal4/Msx-A) exhibitedsignificantly reduced repressor activity as compared withGal4/Msx (Fig. 4C). A similar reduction in repressor activitywas also observed using an Msx-A expression plasmid thatlacked the Gal4 DNA binding domain (K.M.C. and C.A.-S.,unpublished data). The Gal4/Msx polypeptide containingsubstitutions of conserved residues in helix I (Gal4/Msx-B)also had reduced repressor activity although to a lesser extentthan Gal4/Msx-A, whereas the transcriptional properties ofthe other Gal4/Msx polypeptides (Gal4/Msx-C, Gal4/Msx-D, and Gal4/Msx-E) were not altered relative to Gal4/Msx (Fig. 4C). These findings demonstrate three features ofMsx-1 repression: (i) that the homeodomain contributes

directly to repression in the context of the full-length protein;(ii) that specific residues within the homeodomain, primarilythose in the N-terminal arm, are required for repression; and(iii) that the residues required for repression are the same asthose that are primarily required for interaction with TBP.Therefore, one component of transcriptional repression byMsx-1 is mediated by the homeodomain through its inter-action with TBP.The implication of these findings is that the Msx-1 homeo-

domain may serve additional roles in transcriptional regulationother than binding to DNA. To explore the relationshipbetween TBP interaction, repression, and DNA binding activ-ity, we tested the binding properties of the alanine-substitutedMsx-1 polypeptides in gel retardation assays (Fig. 4D). Assayswere performed using in vitro translated Msx-1 polypeptidesand an oligonucleotide containing the Msx-1 consensus DNAsite (1). As shown in Fig. 4D, Msx-1 interacted with theconsensus DNA site in gel retardation assays, whereas theDNA binding activities of several of the alanine-substitutedMsx-1 polypeptides were either reduced (Msx-D) or abolished(Msx-A, Msx-B, Msx-E) (Fig. 4D). This finding was notsurprising since many of the alanine-substituted residues me-diate DNA-protein contact (e.g., Msx-A and Msx-E) or sta-bilize the DNA-protein interaction (e.g., Msx-B) (23). How-ever, it is noteworthy that some of the mutants that abolishedDNA binding activity did not abolish repression or TBPinteraction (Fig. 4A). In particular, an Msx-1 polypeptide(Msx-E) that contains substitutions in the DNA recognitionhelix (helix III) did not exhibit DNA binding activity but stillinteracted with TBP and repressed transcription (Fig. 4A).Therefore, DNA binding activity is not absolutely required forrepression or TBP interaction, indicating that these activitiesare separable and distinct.

Repression by Msx-1 in Vitro Is Alleviated by TBP. Tofurther explore the relationship between TBP interactionand transcriptional repression, we employed an in vitrotranscription assay to determine whether TBP affected theability of Msx-1 to repress transcription. In vitro transcriptionassays were performed using HeLa nuclear extracts as asource of GTFs and with purified recombinant Msx-1 andTBP. The reporter plasmid contained the major late pro-moter driving expression of a G-less cassette (24) whichprovides high levels of basal transcriptional activity in vitro(Fig. 5, lanes 1 and 2). As previously observed, increasingamounts of Msx-1 resulted in repression of the reporterplasmid in the in vitro assays (Fig. 5, lanes 5 and 6). Incontrast, Msx-A, which contains alanine substitutions ofresidues in the N-terminal arm of the homeodomain, did notrepress transcription of the reporter plasmid (Fig. 5, lanes 9and 10). Therefore, the N-terminal arm residues mediaterepression by Msx-1 in vitro as well as in vivo. Finally, theaddition of exogeneous TBP alleviated the repressor activityof Msx-1 in the in vitro transcription assays (Fig. 5, lanes 7and 8), although its addition did not affect the levels of basaltranscription (Fig. 5, lanes 3 and 4). This observation furtherimplicates a functional relationship between TBP interactionand repression by Msx-1 and paves the way for future studiesto explore this functional relationship and the role of otherprotein factors in this process.

DISCUSSIONThis study demonstrates a functional association between ahomeodomain protein, Msx-1, and the core component of thegeneral transcription complex, TBP. We show that this inter-action is mediated by the homeodomain and through specificresidues in the N-terminal arm. Moreover, these same residuesare required for repression by Msx-1 which shows a relation-ship between TBP interaction and transcriptional repression.We further demonstrate that TBP interaction and repression

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A 1 165 225 297

:-: Homeodomain -- -::---.--

N-Term Arm Hliex Helix 11 Helix III

Msx NRKPRrPFITAQLLALRKFRQKQYLSAIASRAEFSSSLSLTEQVKIW NRRAKAKRILc

Msx-A

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BMsx Msx-A Msx-B Ms)

13 a.

X CL0 U) Q.lU) 0 .UL U) 0 (Jm

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(C

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cc

D Msx-E U.a)

0. 2

Scc

Q U) U)'

.:'lM.. 4 m C-) cm Lux ec A A Aen (n u( 0 t e

CD a a 0 O (5

FIG. 4. Residues in the N-terminal arm of the Msx-1 homeodomain mediate both TBP interaction and transcriptional repression. (A) Theschematic diagram shows the position of the homeodomain in the context of full-length Msx-1. Below is shown the amino acid sequence of thisregion indicating the positions of the N-terminal arm (N-Term Arm) and helices I, II, and III. Alanine substitutions within the homeodomain inMsx A-E are shown (also described in Table 1). The summary to the right shows the relationship between the degree of TBP interaction, the relativefold transcriptional repression (assayed in the context of the Gal4 binding domain), and relative DNA binding activity. The summary is derivedfrom the data shown in B-D. Relative activity is designated by + (+ + + or + + + + indicates a strong interaction and/or activity, + + indicatesa relatively weak interaction, and - indicates no interaction and/or activity). (B) GST interaction assays were performed by mixing the indicated35S-labeled Msx polypeptide (5 ,lI) with GST or GST-TBP (6 ,tg of each). The bound protein was resolved by SDS/polyacrylamide gelelectrophoresis and visualized by autoradiography. Input represents 20% (1 ulI) of the total 35S-labeled Msx protein (5 ,l) used in each interactionassay. Experiments were performed a minimum of three times; a representative assay is shown. (C) Transient transfection assays were performedin NIH 3T3 cells using the Gal4/Msx expression system exactly as described (2). The pM2-msx expression plasmids (100 ng) encoded the indicatedMsx polypeptides (Gal4/Msx A-E) as fusion proteins with the Gal4 DNA binding domain (described in Table 1). Expression of the Gal4/Msxpolypeptides was verified by Western blot analysis using an anti-Gal4 monoclonal antibody and protein expression was found to be comparable(2). The reporter plasmid (2 ,ug) contained five copies of the corresponding Gal4 DNA binding site (5XGal4) situated upstream of a simian virus40 (SV40) promoter and the luciferase gene as in ref. 2. Data are expressed as the relative fold difference in luciferase enzymatic activity andrepresent the average of three independent experiments; the bars show the standard error between these experiments. (D) Gel retardation analysiswas performed as in ref. 1 using the Msx-1 consensus DNA site as described. The indicated Msx proteins (Msx A-E) were prepared by in vitrotranslation; since these were nonradiolabeled, translation efficiency was verified in parallel with 35S-labeled Msx proteins. DNA binding assays wererepeated a minimum of three times; a representative assay is shown. NA, no added protein; Lysate, addition of unprogrammed rabbit reticulocytelysate. Each lane contains 1 Al of a 50-Al translation reaction mixture.

are not dependent upon the DNA binding activity of the Msx-1homeodomain. Therefore, homeodomains may serve more

complex roles in transcriptional regulation, which may includemediating interactions with protein components of the generaltranscription complex and transcriptional repression.The findings of the present study include Msx-1 among the

growing number of transcriptional regulatory proteins thathave been shown to interact with TBP (17, 25-32). Althoughthe propensity of TBP to associate with so many differentproteins makes it difficult to ascertain the significance of itsinteractions with individual factors, close inspection of thesevarious protein-TBP interactions reveals two relevant themes.The first is that almost invariably the TBP interaction domainof these various factors is a transcriptional regulatory region.Msx-1 shares this feature since the residues that mediate TBP

association also mediate transcriptional repression. The sec-

ond theme that emerges is that the interaction of TBP withthese various transcriptional regulatory proteins accounts forsome but not all of their transcriptional activity. The implica-tion is that TBP serves as a common bridge for these varioustranscription factors to contact or associate with other pro-teins, in turn allowing the contribution of their additionalfunctional domains for activation or repression. This is par-ticularly relevant for Msx-1 since the N-terminal region pro-vides a significant component of its transcriptional repressor

activity (2). In fact, this region is effective when isolated fromthe rest of the protein, when targeted to the promoter by a

heterologous DNA binding site (2). In contrast, the homeodo-main is only modestly effective on its own but in the context offull-length Msx-1 provides a significant component of its

Msx-B

TBPInteraction

Msx-C

Msx-D

Msx-E

FoldRepression

++++

+++1-

++

DNABinding

+++

++++

++

'A_A,-A

A- -4-

AA

P-AR

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Proc. Natl. Acad. Sci. USA 93 (1996) 1769

Basal TBP

TBP

Msx Mlsx-A

is.-'* X#& :

1 2 3 4 5 6 7 8 9 10

FIG. 5. Repression by Msx-1 in vitro is alleviated by TBP. In vitrotranscription assays were performed exactly as described (2) usingHeLa nuclear extracts (50 jig). The reporter plasmid contained theadenovirus major late promoter driving expression of a G-less cassette(pML-G-less) (1.25 jig). Transcription reactions contained no addi-tional protein (lanes 1 and 2) or varying amounts (as shown by thetriangles) of TBP (0.2 or 0.4 ,LM), Msx (1.2 or 2.4 ,uM), or Msx-A (1.2or 2.4 ,uM). Transcription reactions were resolved on a 6% polyacryl-amide/urea gel and visualized by autoradiography. Assays were re-

peated a minimum of three times; a representative assay is shown.

repressor action (ref. 2; K.M.C. and C.A.-S., unpublisheddata). Moreover, as shown herein, the N-terminal regionaugments the interaction of the homeodomain with TBP.Therefore, the homeodomain-TBP interaction may serve totarget Msx-1 to the vicinity of the general transcription com-plex, thereby allowing it to utilize its N-terminal region intranscriptional repression. Presumably the action of the N-terminal region is mediated through its interactions withprotein factors other than TBP. It will be of interest todetermine which factors are targeted by this region of Msx-1.The major finding of the present study is that the Msx-1

homeodomain has two additional functions that have not beenpreviously described. Therefore, distinct from its action as aDNA binding domain, the Msx-1 homeodomain functions as a

protein interaction domain for mediating association with TBPand also as a transcriptional repressor region. It is noteworthythat Msx-1 (and members of the Msx family) which are

relatively unique in their potential to repress transcriptionsite-independently are more effective at interacting with TBPthan other homeodomain proteins (H.Z. and C.A.-S., unpub-lished data). Given the paradox that homeodomains exhibitoverlapping DNA binding specificities in vitro yet are respon-sible for mediating functional specificity in vivo (21, 22, 33-36),it has long been suspected that homeodomains may serve otherroles in transcriptional regulation besides binding to DNA.Here we provide direct evidence that this is indeed the case forMsx-1. In fact, what is particularly intriguing about the currentfindings is that the N-terminal arm residues within the Msx-1homeodomain that mediate these activities correspond toresidues of other homeodomains that have been shown tomediate functional specificity in vivo (21, 22). Therefore, thisstudy provides both biochemical evidence and a potentialmechanism of action to support the critical role of N-terminalarm residues in mediating homeodomain function.

Note. While this manuscript was being reviewed, Um et al.(37) reported that Eve homeodomain protein interacts withTBP.

We are grateful to Juan Inostroza, Dongmin Ma, Kam Yeung, andDanny Reinberg for their generous advice, support, and the gift of theplasmids encoding GTFs and Fred Mermelstein for helpful discussion.We acknowledge the assistance of Jun Yang in making the Msx-1mutants. We thank Nouria Hernandez for the gift of the anti-TBPantibodies. We thank all members of the Abate-Shen laboratory formany helpful discussions and especially Denise Toolan for preparationof the manuscript. This work was supported by a grant to C.A.-S. fromthe National Institutes of Health (RO1-HD29446) and a Center for

Advanced Biotechnology and Medicine graduate fellowship award toH.Z. C.A.-S. is a recipient of a National Science Foundation YoungInvestigator award.

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