The TAF II250 Subunit of TFIID Has Histone Acetyltransferase Activity

Cell, Vol. 87, 1261–1270, December 27, 1996, Copyright 1996 by Cell Press

The TAFII250 Subunit of TFIIDHas Histone Acetyltransferase Activity

Craig A. Mizzen,1,7 Xiang-Jiao Yang,2,7 shown that functional preinitiation complexes can beassembled in a step-wise fashion. The first step is TFIIDTetsuro Kokubo,3 James E. Brownell,1

binding, a process that may be facilitated by TFIIA. TheAndrew J. Bannister,4 Tom Owen-Hughes,5

subsequent binding of TFIIB creates a platform that isJerry Workman,5 Lian Wang,6

in turn recognized by a complex containing RNA poly-Shelley L. Berger,6 Tony Kouzarides,4

merase II and TFIIF. Further incorporation of TFIIE andYoshihiro Nakatani,2 and C. David Allis1

TFIIH completes preinitiation complex formation. Re-1Department of Biologycent studies have revealed that complexes of RNA poly-University of Rochestermerase II, general initiation factors, and cofactors mayRochester, New York 14627enter the preinitiation complex as a preassembled unit2Laboratory of Molecular Growth Regulation(reviewed by Koleske and Young, 1995; Orphanides etNational Institute of Child Healthal., 1996; Pugh, 1996). Although the assembly pathwayand Human Developmentmost relevant to the in vivo situation remains unclear,National Institutes of HealthTFIID binding to the promoter could be an importantBethesda, Maryland 20892step for promoter activation in different pathways since3Nara Institute of Science and TechnologyTFIID is the only component of the preinitiation complexNara, 630-01that is capable of binding specifically to core promoters.Japan

Control of promoter recognition by TFIID appears to4Wellcome/CRC Instituterepresent an important pathway for transcriptional regu-Cambridge Universitylation. TFIID is a target for a number of transcriptionalCambridge CB2 1QRactivators whose interactions with TFIID may enhanceUnited Kingdomthe rate of promoter binding by TFIID or stabilize TFIID–5Department of Biochemistrypromoter complexes (reviewed by Kingston and Green,and Molecular Biology1994; Struhl, 1996). TFIID itself is a multimeric proteinPennsylvania State Universitycomplex consisting of TBP and TBP-associated factorsUniversity Park, Pennsylvania 16802(TAFIIs) whose sizes range from Mr z10,000 to >200,0006Wistar Institute(for review, see Burley and Roeder, 1996). To date,Philadelphia, Pennsylvania 19104cDNAs encoding nine TAFII subunits of Drosophila TFIID(dTAFII230, 150, 110, 85, 62, 42, 28a, 28b, and 22) havebeen cloned.Although TBP alone can bind core promot-Summaryers containing TATA elements and support basal tran-scription in conjunction with other general transcriptionThe transcription initiation factor TFIID is a multimericfactors and RNA polymerase II, TAFIIs are required forprotein complex composed of TATA box–binding pro-activated transcription. Several TAFIIs have been showntein (TBP) and many TBP-associated factors (TAFIIs).to provide interaction sites for distinct activators andTAFIIs are important cofactors that mediate activatedtranscription-initiation factors. These interactions couldtranscription by providing interaction sites for distinctserve either to facilitate TFIID recruitment per se or toactivators. Here, we present evidence that humaninduce conformational alterations that affect recruit-TAFII250 and its homologs in Drosophila and yeastment or function of downstream factors (reviewed inhave histone acetyltransferase (HAT) activity in vitro.Orphanides et al., 1996; Roeder, 1996; Verrijzer andHAT activity maps to the central, most conserved por-Tjian, 1996).tion of dTAFII230 and yTAFII130. The HAT activity of

Recent studies showing capabilities for transcrip-dTAFII230 resembles that of yeast and human GCN5tional activation in yeast depleted of the TAF subunitsin that it is specific for histones H3 and H4 in vitro.of TFIID (Moqtaderi et al., 1996; Walker et al., 1996)Our findings suggest that targeted histone acetylationsuggest redundant pathways for activator responses.

at specific promoters by TAFII250 may be involved inConsistent with this, direct linkage of enhancer-bindingmechanisms by whichTFIID gains access to transcrip-domains and either TBP (Chatterjee and Struhl, 1995;tionally repressed chromatin.Klages and Strubin, 1995), TAFs (reviewed in Moqtaderiet al., 1996), or subunits within the RNA polymerase

Introduction II holoenzyme (Barberis et al., 1995) can bypass therequirement for activation domains. These lines of evi-

Transcription of protein-coding genes in eukaryotes re- dence suggest that recruitment of either factor may leadquires the orchestrated assembly of a large preinitiation to at least some transcriptional activation, although mul-complex containing a well-studied collection of general tiple interactions could be required for full activation.transcription factors, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, Central to eukaryotic transcriptional regulation areand TFIIH and RNA polymerase II, at promoters (re- how TFIID gains access to a chromatin template andviewed by Burley and Roeder, 1996; Orphanides et al., how a stable association is maintained within the chro-1996; Roeder, 1996). Reconstitution of transcription has mosomal enviroment. A potentially relevant finding is

the presence of a histone octamer-like substructure in7 These authors contributed equally to this work. TFIID (Hoffmann et al., 1996; Nakatani et al., 1996; Xie

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et al., 1996). The N-terminal regions of the dTAFII42 anddTAFII62 proteins have sequence similarities with theC-terminal core domains, but not the N-terminal tails,of histones H3 and H4, respectively (Kokubo et al.,1994a). The crystal structure of the N-terminal portionsof dTAFII42/dTAFII62 reveals that they adopt the canoni-cal histone fold, consisting of two short a helices flank-ing a long, central a helix (Xie et al., 1996). Moreover, thedTAFII42/dTAFII62 complex exists as a heterotetramer,resembling the (H3/H4)2 heterotetrameric core of thehistone octamer. In addition, biochemical studies withhuman TAFIIs (hTAFIIs) suggest that TFIID contains ahistone octamer-like structure composed of two dimersof the histone H2B-like TAFII (hTAFII15/20 or dTAFII28a)attached to a tetramer of histones H3/H4-like TAFIIs(Hoffmann et al., 1996). These similarities to histonecomplexes have prompted the suggestion that TAFII

complexes may wrap and interact with promoter DNAin a nucleosome-like structure (Hoffmann et al., 1996).

Biochemical studies have demonstrated that physicalinteractions of activators with general transcription fac-tors play an important role in activated transcriptionfrom naked DNA templates (reviewed by Burley andRoeder, 1996; Orphanides et al., 1996; Roeder, 1996).

Figure 1. HeLa Nuclear Extracts Contain a High-Molecular-WeightMoreover, the recent discovery that dTAFII230/hTAFII250 Polypeptide with HAT Activitypossesses kinase activity that phosphorylates the

(A) Aliquots of HeLa cell crude nuclear extract were resolved onRAP74 subunit of TFIIF in vitro (Dikstein et al., 1996) 8% SDS–PAGE gels containing histone or BSA as indicated andsuggests that enzymatic mechanisms are also involved, processed to detect acetyltransferase activity. The arrow denotesalthough the role of phosphorylation has not been dem- an activity band in the activity gel containing histone with an appar-

ent molecular weight similar to that of hTAFII250, as shown by immu-onstrated. Additionally, various enzymatic activities,noblotting in (B).such as histone acetyltransferases (HATs), histone ki-(B) An aliquot of HeLa-cell crude nuclear extract was resolved on annases, and DNA helicases, may be required for tran-8% SDS–PAGE gel and analyzed by Western blotting using antisera

scriptional regulation within the context of nucleosomal specific for hTAFII250. The arrow denotes hTAFII250. The activityand higher-order chromatin structure that represses gels and the immunoblot were aligned using prestained moleculartranscription in general (reviewed by Owen-Hughes and weight markers, as indicated on the left with molecular weight givenWorkman, 1994; Felsenfeld, 1996). in kilodaltons.

A large number of studies have established a correla-tion between chromatin transcriptional activation, i.e.,

scription from natural chromatin templates by increas-derepression, and acetylation of highly conserved lysine

ing the accessibility of TFIID to promoters. Our dataresidues situated in the amino-terminal tails of the coreprovide additional support for the notion that targetedhistones (reviewed by Loidl, 1994; Turner and O’Neill,histone acetylation represents an important pathway in1995; Brownell and Allis, 1996). Although the mechanis-gene activation. We favor the view that eukaryotic cellstic details underlying acetylation-mediated activationhave evolved a scheme of targeted histone acetylationfrom chromatin templates remain unclear, it is generallywherein HAT activities, and potentially other chromatin-thought that neutralization of the positive charge of ly-modifying activities (for example, Roest et al., 1996; re-sine residues upon acetylation perturbs histone-DNAviewed by Kaiser and Meisterernst, 1996), are recruitedcontacts within nucleosomal and higher-order structureto specificpromoters through selective interactions withand influences histone interactions with specific nonhis-activator proteins.tone regulatory proteins (reviewed by Wolffe, 1994;

Brownell and Allis, 1996). The recent findings that theResultsyeast transcriptional adaptor-protein GCN5 (Brownell et

al., 1996) and related human proteins (hGCN5, Wang etA Large Molecular-Weight, TBP-Associatedal., 1997; hP/CAF, Yang et al.,1996) possess HAT activityPolypeptide Has HAT Activityin vitro provide strong evidence that histone acetylationWe recently demonstrated the utility of an activity gelis linked to transcriptional activation and suggest thatassay to detect proteins with HAT activity in complexhistone acetylation may be a targeted phenomenon (re-samples (Brownell and Allis, 1995; Brownell et al., 1996).viewed by Brownell and Allis, 1996; Wolffe and Pruss,In this technique, samples are resolved in SDS–PAGE1996).gels containing histones prior to detection of HAT activ-In this report, we demonstrate that, like the GCN5ity, and thus the molecular weight estimates of activeprotein family, TAFII250 and its homologs in Drosophilapolypeptides can be determined. Using this assay toand yeast (hereafter referred to collectively as TAFII250)characterize a crude HeLa nuclear extract, we identifiedhave HAT activity in vitro. This finding suggests thata polypeptide with a molecular weight of approximatelyacetylation of histone amino termini at or near core pro-

moters by TAFII250 could be involved in facilitating tran- 200 kDa that possessed strong HAT activity (Figure 1A).

Histone Acetylation by TAFII2501263

Figure 2. Anti-TBP Antibodies Immunoprecipitate HAT Activity

(A and B) Immunoprecipitations were performed from COS whole-Figure 3. Recombinant Drosophila TAFII230 Possesses HAT Activitycell extracts (A) and HeLa-cell nuclear extracts (B) with the indicated(A) FLAG epitope–tagged, affinity-purified recombinant dTAFII230antibodies. After extensive washing, the resulting immune com-and FLAG–dTAFII62 were resolved on 8% SDS gels containing his-plexes were assayed for their ability to acetylate either free histonestone or BSA as indicated and processed to detect acetyltransferaseor BSA. The anti-CBP antibody served as a positive control sinceactivity.both CBP and the associated P/CAF have HAT activity (see text).(B) Identical dTAFII230 and dTAFII62 samples were electrophoresedThe anti-E1A antibody served as a negative control since E1A isin a parallel SDS–PAGE gel and immunoblotted with antisera to thenot detectable in these cell lines.FLAG epitope.(C) The anti-TBP immunoprecipitate was resolved on an 8% SDS–(C) Identical dTAFII230 and dTAFII62 samples were electrophoresedPAGE gel and analyzed by Western blotting using antisera specificin a parallel SDS–PAGE gel and stainedwith Coomassie blue. Arrowsfor hTAFII250. The arrow denotes hTAFII250.in (A), (B), and (C) indicate the position of full-length dTAFII230.

This activity appeared to be specific for histone and extracts was routinely more than 6-fold greater thanwas not due to autoacetylation, since activity was not background (Figure 2A).detected when a sample was analyzed on a gel con- HAT activityassociated with anti-TBP immunoprecipi-taining BSA. Interestingly, we did not detect activities tates was approximately 5-fold greater than backgroundresembling hGCN5 (apparent molecular weight 60 kDa) when immunoprecipitates were prepared from whole-or hP/CAF (apparent molecular weight 95 kDa) in crude cell extracts (Figure 2A) and from nuclear extracts (Fig-HeLa nuclear extract, suggesting that the total activities ure 2B). Moreover, the HAT activity associated with TBPof these known HATs in this sample are lower or that was specific to histones, since BSA was not acetylatedthese proteins are not renatured as readily in the assay by anti-TBP immunoprecipitates under similar conditionsprocedure. Due to the similarity in molecular weight, we (Figure 2B). As expected, Western blot analysis con-speculated that the active species might be hTAFII250, firmed that hTAFII250 was present in the anti-TBP immu-the 250 kDa (nominal) subunit of human TFIID (Hisatake noprecipitate (Figure 2C). Together, these results sug-et al., 1993; Ruppert et al., 1993). Immunoblot analysis of gest that HAT activity may be attributable to hTAFII250the nuclear extract using an hTAFII250-specific antisera and further suggest that this activity is preserved in(Figure 1B) demonstrated that the immunoreactive band TFIID.possessed electrophoretic mobility similar to that of thedetected HAT, further supporting the hypothesis that Recombinant dTAFII230 Has HAT ActivityhTAFII250 has HAT activity in this assay. To rigorously test the hypothesis that TAFII250 has HAT

To further test this hypothesis and to determine if HAT activity, we expressed dTAFII230 as a FLAG epitope–activity could be detected in TAFs, we used antibodies tagged protein in Sf9 cells using baculovirus-mediatedto TBP to immunoprecipitate TBP–TAF complexes from transfection and assayed the affinity-purified recombi-COS whole-cell extracts and HeLa-cell nuclear extracts nant protein for HAT activity. Recombinant dTAFII230and assayed the immunoprecipitates for HAT activity. displayed acetyltransferase activity when assayed withTo monitor the specificity of this procedure, two controls a histone-containing gel but not when the gel containedwere employed. First, an E1A-specific antibody was BSA (Figure 3A). The absence of HAT activity in a nonho-used as a negative control. Second, a CBP-specific anti- mologous recombinant protein, FLAG–dTAFII62, pre-body was used as a positive control, since CBP per se pared by identical procedures indicated that HAT activ-(Ogryzko et al., 1996) and its associated hP/CAF (Yang ity was a property of dTAFII230 rather than contaminantset al., 1996) have HAT activity. from baculovirus-infected Sf9 cells. Similar amounts of

The results of these immunoprecipitation-HAT assays dTAFII62 and dTAFII230 were detected at their respectiveare shown in Figure 2. A low level of HAT activity was molecular weights by both immunoblot analysis withassociated with anti-E1A immunoprecipitates prepared antisera to the FLAG epitope (Figure 3B) and Coomassiefrom whole-cell extract (Figure 2A) and nuclear extract blue staining (Figure 3C). Furthermore, Coomassie blue(Figure 2B). This represents background activity since staining revealed that the recombinant proteins werewe found several other antibodies against nuclear pro- highly purified; only low levels of a small set of ex-teins precipitated similar amounts of activity (data not traneous proteins were detected. These data demon-shown). In contrast, the amount of HAT activity associ- strate that HAT activity is associated with recombinant

dTAFII230 under the conditions employed.ated with anti-CBP immunoprecipitates from whole-cell

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Substrate Specificity of RecombinantdTAFII230 HAT ActivityTo characterize the recombinant dTAFII230 HAT activityfurther, we performed HAT assays in solution with puri-fied protein. Acetylation of a HeLa core histone mix-ture and individual HeLa core histones, purified byreverse-phase high pressure liquid chromatography(HPLC), was measured by a filter-binding assay and byfluorography (Figure 4). As a positive control, we as-sayed a crude extract of recombinant hGCN5 expressedin bacteria. Extracts of uninduced bacteria and purifieddTAFII62 were used as negative controls for hGCN5 anddTAFII230, respectively. Liquid-scintillation counting ofreactants retained by P81 filters (Figure 4A) showed thatboth hGCN5 and dTAFII230, in contrast to uninducedbacterial extract or dTAFII62, catalyzed efficient [3H]-acetate transfer to the HeLa core histone mixture. Inagreement with the activity gel assay (Figure 3), acetateincorporation into BSA was not observed with eitherhGCN5 or dTAFII230, nor was acetylation of proteinsendogenous to the enzyme preparations detected.Given that the amino-terminal portions of dTAFII42 anddTAFII62 adopt structures similar to the histone fold andcan form a (dTAFII42/dTAFII62)2 heterotetramer resem-bling the (H3/H4)2 tetramer of nucleosomes (Xie et al.,1996), we tested these TAFs as substrates for acetyla-tion. Consistent with the lack of sequences related tothe histone amino-terminal tails in dTAFII42/dTAFII62,they were not acetylated by dTAFII230 or hGCN5 (datanot shown).

Histone substrates acetylated by dTAFII230 and hGCN5in vitro were identified by fluorography. As shown in theupper portion of Figure 4B, H3 and H4 were the majoracetate acceptors when the HeLa core histone mixture(lane 1) was incubated with dTAFII230. H3 and H4 wereacetylated by dTAFII230 regardless of whether they werepresented individually (lanes 4 and 5) or as part of themixture. Although H2A was acetylated by dTAFII230when presented individually (lane 2), little or no H2Aacetylation was detected in the core histone mixture.H2B was not acetylated by dTAFII230 when presented Figure 4. Substrate Specificities of Drosophila TAFII230 and Human

GCN5individually (lane 3) or as part of the mixture. No acetyla-tion was detected in a control reaction where H4 (or H3, (A) Acetylation of proteins endogenous to enzyme preparations (no

substrate) BSA and HeLa core histones by recombinant dTAFII230data not shown) and [3H]-acetyl-CoA were incubatedand hGCN5 in vitro was assessed by measuring 3H-acetate incorpo-without enzyme (lane 6). When an equimolar mixtureration using a filter-binding assay. Uninduced bacterial extract (unin-of H2A, H2B, H3, and H4, reconstituted from purifiedduced) and recombinant dTAFII62 were used as negative controls

fractions (lane 7), was employed as substrate, acetyla- for hGCN5 and dTAFII230, respectively.tion of H3 and H4 was indistinguishable from that seen (B) Specificity of HeLa core histone acetylation by dTAFII230 (twoin the histone mixture prior to purification. upper panels) and hGCN5 (two lower panels) was determined by

The dual H3 and H4 specificity of dTAFII230 differed fluorography of acetylated reaction products resolved on SDS–PAGE gels. Fluorograms are shown aligned with the correspondingfrom a strong preference for only H3 displayed byCoomassie blue–stained gels as indicated. Lane 1 shows unfraction-hGCN5. As shown in the lower panels of Figure 4B,ated HeLa core histone mixture; lanes 2–5, reverse-phase HPLC-H3 was the preferred substrate for hGCN5 under allpurified HeLa histones H2A, H2B, H3, and H4, respectively; lane 6,

conditions tested (see also Wang et al., 1997). Acetyla- reverse-phase HPLC-purified HeLa H4 incubated with [3H]-acetyl-tion of H2A, H2B, and H4 by hGCN5 was not detected CoA but without added enzyme; and lane 7, equimolar mixture ofin the histone mixture (lane 1) or when presented individ- HeLa core histones reconstituted from RP-HPLC–purified fractions.ually (lanes 2, 3, and 5, respectively). Based on approxi- The positions of the individual histones in the gels are indicated at

the left of the figure. The lack of H4 acetylation by hGCN5 comparedmate measurements, we estimate that the specific activ-to that described by Yang et al. (1996) may be related to differencesity of hGCN5 (dpm 3H-acetate transferred/pmol enzyme)in the recombinant proteins employed.is two to four times that of dTAFII230 (data not shown).

Site Specificity of Recombinant dTAFII230closely correlated with distinct biological processes (seeHAT ActivitySobel et al., 1995; Turner and O’Neill, 1995). To deter-Utilization of specific acetylation sites within the core

histone amino termini is remarkably nonrandom and mine whether specific residues modified by dTAFII230

Histone Acetylation by TAFII2501265

site for both hGCN5 and dTAFII230. Interestingly, thissame residue has recently been shown to be the pre-ferred site of H3 acetylation by yeast GCN5 (yGCN5) invitro (Kuo et al., 1996). While little acetylation of theunacetylated H4 peptide was seen with hGCN5, thispeptide was a good substrate for dTAFII230, in agree-ment with the results obtained with intact histones (Fig-ure 4B).

Putative Catalytic Domain of dTAFII230and yTAFII130To determine the portion of the dTAFII230 moleculeresponsible for HAT activity, we expressed a seriesof C-terminal deletion mutants as depicted in Figure6A. A HAT-activity gel analysis employing equimolaramounts of these proteins (Figure 6B) revealed that thethree mutants bearing the largest deletions, N545, N596,and N885, did not possess detectable HAT activity. Incontrast, mutant proteins N1140 and N1480 and alsofull-length dTAFII230 displayed HAT activity. These re-sults suggest that sequences involved in histone ace-tylation by dTAFII230 are situated between residues 1and 1140. Furthermore, sequences between residues885 and 1140 are critical for HAT activity, owing to possi-ble roles in either catalysis or protein folding.

To further define the HAT catalytic domain, we ana-lyzed the yeast homolog. yTAFII130 has 52% sequencesimilarity with the N-terminal region of dTAFII230 (aa1–1370) and lacks the region corresponding to theC-terminus, including the bromodomains (see Figure6A). The full-length recombinant yTAFII130 displays HATFigure 5. Site Specificity of Drosophila TAFII230 and Human GCN5activity both in the activity gel and liquid assays (data(A and B) Acetylation of histone amino-terminal peptides by hGCN5not shown).For mapping theHAT domain,yTAFII130 was(A) and dTAFII230 (B) was assessed by measuring 3H-acetate incor-

poration using the filter-binding assay. For each peptide substrate, divided into the three overlapping fragments, namely Nincubations without (open bars) and with (closed bars) enzyme were (aa 1–450), M (aa 354–817), and C (aa 690–1066) (Figureperformed in parallel. Acetylation of proteins endogenous to the 7A), each of which was expressed in and purified fromenzyme preparations was assessed by incubations without peptides

E. coli. HAT-activity gel analysis revealed that only theand is shown in the columns marked (E).M region, representing the central portion of yTAFII130,(C) The structures of the peptidesused in (A) and (B) are shown. Siteshad HAT activity (Figure 7B). Significantly, this region iswhere e-N-acetyllysine was incorporated during peptide synthesis in

order to mimic sites that are acetylated in vivo are indicated by highly conserved in dTAFII230 and hTAFII250, aligning(Ac). All peptides were MAP reagents except the diacetyl-(9/14)-H3 to residues 499–1003 of dTAFII230, which are includedpeptide (denoted by the asterisk), which was synthesized with a in the active mutant protein N1140 (see Figure 6B).C-terminal cysteine.

Among various acetyltransferases including theGCN5protein family, cytoplasmic HAT1, and a-N-acetyltrans-

are the same as those modified by GCN5 (Kuo et al., ferases, putative acetyl-CoA binding sites (Lu et al.,1996), we assessed the ability of dTAFII230 and hGCN5 1996) are conserved (Kleff et al., 1995; Borrow et al.,to acetylate histone amino-terminal peptides synthe- 1996; Parthun et al., 1996; Reifsnyder et al., 1996). How-sized with and without acetyl groups on the e-amino ever, multiple-alignment analysis (Lawrence et al., 1993)groups of specific lysines. The peptide sequences and showed no significant sequence similarity within resi-positions of acetate groups incorporated during synthe- dues 1 to 1140 of dTAFII230 (Figure 6) and 354 to 817sis are depicted in Figure 5C. of yTAFII130 (Figure 7) to the putative acetyl-CoA binding

Acetylation of these peptides by hGCN5 and dTAFII- sites. Moreover, comparison of these regions with pro-230 are shown in Figures 5A and 5B, respectively. Two tein-sequence databases (Altschul et al., 1990) showedcolumns, representing assays performed without and no obvious sequence similarity to any other proteins.with enzyme addition, are presented for each peptide These results lead us to suggest that the HAT catalyticassayed. In agreement with the results described above domain of TAFII250 may represent a second type of HATfor intact histone, significant levels of acetylation by domain.hGCN5 and dTAFII230 were observed for the unace-tylated H3 peptide. Note that although the diacetyl-(9/

Discussion18)-H3 peptide was also a good substrate for bothhGCN5 and dTAFII230, neither enzyme acetylated the

HAT Activity Is Intrinsic to TAFII250diacetyl-(9/14)-H3 peptide significantly. Comparison ofIn this report, we demonstrate that recombinantthe acetylation of the diacetyl-(9/14)-H3 and diacetyl-

(9/18)-H3 peptides suggests that Lys14 is a preferred dTAFII230 and yTAFII130 and natural hTAFII250 possess

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Figure 6. HAT Activity Maps to the N-termi-nal Portion of dTAFII230

(A) The uppermost diagram depicts structuraland functional domains found in dTAFII230.The TBP-binding domain (Kokubo et al.,1994b; Nishikawa et al., 1997), the two proteinkinase domains (Dikstein et al., 1996), theRAP74 binding domain (Ruppert and Tjian,1995), and the two bromodomains (Kokuboet al., 1993; Weinzierl et al., 1993) are indi-cated. (G) denotes the glycine residue at posi-tion 743 equivalent to the site of the ts-13mutation in hamster TAFII250 that causes G1arrest in cell-cycle progression (Hayashida etal., 1994). C-terminal deletion mutants thatwere tested for HAT activity in the activity gelassay are depicted in the lower portion. Openbars denote proteins that did not possessHAT activity, and closed barsdenote proteinswith HAT activity.(B) Equimolar amounts of the C-terminal–deletion mutants depicted in (A) and full-length dTAFII230 were compared using theactivity gel assay.(C) Equivalent amounts of the C-terminal–deletion mutants depicted in (A) and testedfor HAT activity in (B) are shown followingSDS–PAGE and Coomassie blue staining.

HAT activity and show that the HAT activity of dTAFII230 The Bromodomains of dTAFII230 Areis specific for H3 and H4 in vitro. Several independent Dispensable for HAT Activitylines of evidence suggest that these observations are Since the HAT activities of GCN5 and dTAFII230 bothnot artifactual. Recombinant dTAFII230 displayed HAT show a strong preference for H3 as a substrate in vitroactivity in both the liquid and activity gel assays. The and since the only significant homology between theseactivity gel assay, which involves denaturation of sam- proteins is found in their bromodomains (one in hGCN5ples in 1% SDS (with boiling) prior to fractionation and and two in dTAFII230; see Figure 6A), we tested whethersubsequent renaturation within the gel matrix, strongly the bromodomain may play a role in histone recognitionsuggests that the HAT activity detected is intrinsic to or binding. Deletion of the C-terminal 588 amino acidsdTAFII230. Polypeptides potentially associated with of dTAFII230 removes both of the bromodomains butdTAFII230 following affinity purification are expected to does not affect HAT activity in vitro (N1480 in Figure 6)dissociate and separate from the dTAFII230 polypeptide and does not alter the H3/H4 specificity of dTAFII230during electrophoresis, and their reassociation during (data not shown). Thus, it is unlikely that the bromodo-renaturation is prevented by entrapment within the gel main is involved in histone recognition or acetate trans-matrix. fer by dTAFII230. This is supported by our finding that

However, one might argue the possibility that HAT yTAFII130, which lacks bromodomains, has HAT activ-activity is catalyzed by polypeptides tightly associated ity in vitro (Figure 7). Moreover, the bromodomain inwith dTAFII230 in complexes that resist dissociation in yGCN5 is dispensable for HAT activity in vitro (Candau1% SDS. The following results indicate that this is not et al., 1997).the case. The use of stronger denaturing conditions (4% A function has not yet been demonstrated for theSDS or 4% SDS plus 8 M urea) or dTAFII230 purified bromodomain in any protein, but it has been suggestedfurther by size-exclusion chromatography in buffer con-

to mediate protein–protein interactions (Haynes et al.,taining 6 M guanidine hydrochloride gave results similar

1992; Tamkun et al., 1992). Given that the bromodomainto those shown in Figure 3 (data not shown). Moreover,

is required for yGCN5 function in yeast (Marcus et al.,the active deletion mutants of dTAFII230 (N1140 and1994), a role in targeting the HAT activity of GCN5 toN1480 in Figure 6) and of yTAFII130 ([M] in Figure 7)appropriate chromatin loci hasbeen proposed (Brownellshow the expected molecular weights on both Coomas-and Allis, 1996). However, our finding that both yTAFII130sie blue–stained gels and HAT-activity gels. Since theand the dTAFII230 mutants lacking the bromodomainmutants of dTAFII230 and yTAFII130 were expressed inpossess HAT activity in vitro argues against a role ininsect cells and bacteria, respectively, and since thesubstrate recognition.yTAFII130 mutants were purified using buffer containing

6 M guanidine hydrochloride, it is unlikely that the HATSubstrate Specificity of the dTAFII230 HATactivity of both sets of mutantproteins is due to contami-The dual H3/H4 substrate specificity of dTAFII230 is simi-nating polypeptides. Finally, our analyses of HeLa nu-lar to that described previously for hGCN5 and P/CAFclear extract (Figure 1) and anti-TBP immunopre-(Yanget al., 1996) and yGCN5 (Kuoet al., 1996). Similarly,cipitates (Figure 2) provide independent support that

hTAFII250 also has HAT activity. we have found that dTAFII230, like hGCN5 (Yang et al.,

Histone Acetylation by TAFII2501267

that efficient acetylation of chromatin substrates byHATs like GCN5 may require multimeric HAT complexescontaining subunits capable of transiently exchangingwith nucleosomal histones (Roth and Allis, 1996). It isan intriguing possibility that the histone-like features ofTAFIIs described above may play a role in facilitatingchromatin acetylation by TAFII250.

Possible Significance of TAFII250 HAT ActivitySeveral lines of evidence suggest that acetylation orremoval of core histone amino termini enhances tran-scription-factor binding to nucleosomal DNA (Lee et al.,1993; Vettesse-Dadey et al., 1994). Nucleosome assem-bly in vitro represses transcription of plasmids in vitrounless TBP-promoter or TFIID–promoter complexes areformed prior to nucleosome assembly (Workman andRoeder, 1987; Meisterernst et al., 1990), suggesting thatnucleosomes inhibit binding of TBP or TFIID to natu-ral (chromatin) templates. Moreover, experimental evi-dence suggests that the amino termini of the core his-tones mediate this inhibition. Imbalzano et al. (1994)reported that TBP binding to a TATA box in nucleosomal

Figure 7. HAT Activity Maps to the Central Portion of yTAFII130 DNA is inhibited unless human SWI/SNF and ATP are(A) The uppermost diagram depicts the structural and functional present. Significantly, SWI/SNF and ATP are not re-domains found in yTAFII130. The TBP binding domain (T. Kokubo, quired for TBP binding when nucleosomes contain hy-J. Nishikawa, and Y. N., unpublished data) is indicated. (G) denotes peracetylated histones. Similarly, Godde et al. (1995)the glycine residue at position 561 equivalent to the site of the ts-

demonstrated that TBP binding occurs only when the13 mutation in hamster TAFII250 that causes G1-arrest in cell-cycleTATA box is positioned within linker DNA at the edgeprogression (Hayashida et al., 1994). The three deletion mutantsof nucleosomes in which the core histone amino terminithat were tested for HAT activity in the activity gel assay are depicted

in the lower portion. Open bars denote proteins without HAT activity, are removed by proteolysis (presumably mimicking ace-and closed bars denote proteins with HAT activity. tylation). These authors found that TBP does not bind(B) Equimolar amounts of the deletion mutants depicted in (A) were nucleosomes containing intact core histones under anycompared using the activity gel assay. Repeated analyses confirmed

of the conditions tested. Taken together, these studiesthat the minor density visible in lane (C) is not an activity band butdemonstrate that core histone amino termini regulateis an artifact related to the crease apparent in the dried gel. Thethe accessibility of the TATA box for binding by TBP.arrowhead indicates the position of the mutant protein M.

(C) Equivalent amounts of the deletion mutants depicted in (A) and Our data suggest that acetylation of nucleosomes attested for HAT activity in (B) are shown following SDS–PAGE and promoters by TAFII250 may be part of a process actingCoomassie blue staining. The N-terminal fragment (aa 1–450) mi- to enhance the exposure of promoter elements for bind-grates with an apparent molecular weight larger than expected,

ing by TFIID. Acetylation could positively regulate pro-owing to physical properties of its amino acid sequence. The arrow-moter binding by TFIID directly by alleviating maskinghead indicates the position of the mutant protein (M).of binding sites by histone amino-terminal tails or indi-rectly by increasing promoter exposure through acetyl-

1996) and yGCN5 (J. B. and C. D. A., unpublished data), ation-induced changes in nucleosome conformationacetylates nucleosomal histones weakly in vitro. This is (Norton et al., 1990; Bauer et al., 1994) or effects onin contrast to the ability of P/CAF to acetylate H3 in core higher-order chromatin structure (Garcia-Ramirez et al.,particles (Yang et al., 1996). 1992; 1995). This proposed role is not necessarily limited

Acetylation-site utilization determined by assays to promoters containing TATA elements, since TFIIDwith synthetic histone amino-terminal peptides revealed plays a role in the recognition of other initiation elementsoverall similarity in site utilization by hGCN5 and (e.g., the initiator) in TATA-less promoters (reviewed bydTAFII230. Significantly, the experiments with H3 pep- Orphanides et al., 1996; Roeder, 1996; Verrijzer andtides containing e-N-acetyllysine at distinct positions Tjian, 1996). The possible existence of histone octamer-revealed that Lys-14 but not Lys-9 of H3 is a preferred like TAFII complexes in TFIID (Hoffmann et al., 1996; Xiesite of acetylation by both hGCN5 (Kuo et al, 1996) and et al., 1996) suggests the possibility that acetylationdTAFII230 (Figure 5). In lower eukaryotes, acetylation at of nucleosomes by TAFII250 may facilitate nucleosomeLys-9 is associated with deposition of newly synthesized displacement by TAFII complexes or facilitate exchangeH3 in vivo (Sobel et al., 1995), whereas Lys-14 of H3 is of histone and TAFII proteins.preferentially acetylated by yGCN5 in vitro (Kuo et al., It seems likely that knowledge of any enzymatic activi-1996). Thus, preferential modification of Lys-14 by ties associated with TAFIIs, such as the kinase activitydTAFII230 is consistent with a role in transcription-asso- (Dikstein et al., 1996) or the HAT activity we describeciated acetylation, as suggested previously for yGCN5 here for TAFII250, will factor significantly into under-(Brownell et al., 1996). standing TAFII function in the native context. Our finding

The basis for the observed site and substrate specific- that HAT activity is associated with a component of theity of dTAFII230 (or any other known HAT) with free his- preinitiation complex suggests that chromatin-modi-

fying activities associated with other components of thetones is not understood. Recently, it has been proposed

Cell1268

at 48C. The supernatant constituted whole-cell extract. HeLa-celltranscriptional apparatus are likely to have importantnuclear extracts were prepared according to Dignam et al., 1983.roles in transcriptional activation from chromatin tem-

Antibodies were added to 1 ml of whole-cell extract or 100 ml ofplates.nuclear extract and incubated at 48C for 2 hr. A 50:50 mix of ProteinA–Sepharose/Protein G–Sepharose (15 ml each) was added, and the

Experimental Procedures mixture rotated slowly overnight at 48C. Immune complexes werepelleted by gentle centrifugation and washed three times with 1 ml

Preparation of Recombinant Proteins Lysis Buffer IPH. After the final wash, the buffer was aspirated downDrosophila TAFII230 (Kokubo et al., 1993) and TAFII62 (Kokubo et to 30 ml. 1.25 ml of 20 mg/ml histones and 1 ml of 3H-acetyl-CoAal., 1994a) were expressed as FLAG-tagged proteins in Sf9 cells via were added, and a HAT assay was performed at 308C. Histonebaculovirus FLAG fusions. FLAG fusions were affinity purified with acetylation was measured using the P-81 filter assay describedM2-agarose (Kodak-IBI) according to the protocols of the manufac- above.turers. Buffer B (20 mM Tris-HCl [pH 8.0], 0.5 mM MgCl2, 10% glyc- Antibodies against CBP (PharMingen), hTAFII250, and E1A (Santaerol, and 0.1% NP-40) supplemented with 10 mg/ml aprotinin, 10 Cruz Biotechnology) are commercially available. Antisera to TBPmg/ml leupeptin, 1 mg/ml pepstatin A, 1 mM DTT, and 0.5 M KCl have been described previously (Pruzan et al., 1992). Other antisera(or, in some cases, 0.15 M KCl) was used for extraction and column tested were directed against Sp1 (S. Jackson), MDM2 (A. Levine),binding and washing. Fusion proteins were eluted with buffer B, myc (J. Pines), DNA-PK catalytic subunit (S. Jackson), hBRM (C.supplemented as above and containing 0.1 mg/ml FLAG peptide. Muchardt), and E7 (Santa Cruz).

For yTAFII130 expression, cDNAs corresponding to the yTAFII130portions shown in Figure 7A were amplified by PCR and subcloned Other Proceduresinto E. coli expression plasmid pET28a or 6His-pET5a. Recombinant Concentrations of wild-type and mutant TAFs employed in assaysproteins were affinity purified with Ni21-NTA-agarose (Qiagen) ac- were normalized according to Coomassie blue staining of 8% SDS–cording to the protocol of the manufacturer, except that Buffer PAGE minigels. FLAG epitope–tagged proteins were also character-B/0.5M KCl/6M guanidine was used for chromatography. ized by immunoblotting with M2 monoclonal antisera to the FLAG

hGCN5 was expressed in E. coli strain JM109 as the 6 3 His tag (Kodak). Western blots were visualized using goat anti-mousefusion of the 59-BglII-39–EcoRI fragment from the original phage secondary antibodies conjugated to alkaline phosphatase followingclone (Candau et al., 1996) inserted into the pRSET vector (In- reaction with NBT and BCIP.vitrogen) at BamHI–EcoRI sites. Crude hGCN5 was prepared bysonicating IPTG-induced bacterial pellets in 20% sucrose, 50 mM AcknowledgmentsTris [pH 8.0], 0.3 M NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, andthe supernatant prepared by centrifugation at 12,000 3 g for 30 min We thank Dr. Alex Vassilev for providing recombinant baculoviruses,used without further purification. Dr. Lou Schiltz for technical advice and for developing assay condi-

tions used in some of our experiments, Dr. Sharon Roth for histoneamino-terminal peptides, and Dr. Anthony Annunziato for prepara-Preparation of Substrates for HAT Assays

Crude core histones were prepared from isolated chicken erythro- tions of HeLa histones. We are grateful to Edwin Smith and Drs.Jeff Hayes, Martin Gorovsky, and Mark J. Swanson for their helpfulcyte nuclei (Olins et al., 1976) or HeLa-cell nuclei (Annunziato and

Seale, 1983) by 0.4 N H2SO4 acid extraction and 5% perchloric acid discussions and valuable comments. Y. N. and X.-J. Y. are grate-ful to Drs. A. S. Levine and B. H. Howard for their supportprecipitation. Purified HeLa core histone fractions were prepared

by reverse-phase HPLC of acid extractson a 4.6 3 200 mmBrownlee and encouragement. This research was supported by grants toC. D. A. (NIH-GM53512), T. Kouzarides. (Cancer Research campaignAquapore RP-300 column (Applied Biosystems) eluted with an ace-

tonitrile gradient in 0.1% TFA. Carboxy-terminal cysteine histone SP2081/0301), and S. B. (NSF-MCB9317243 and a Junior FacultyResearch Award from the American Cancer Society). Correspon-amino-terminal peptides and histone amino-terminal MAP peptides

(Tam, 1988) were obtained from the protein core facility at Baylor dence should be addressed to C. D. A.College of Medicine (Houston, TX).

Received October 18, 1996; revised November 7, 1996.

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performed essentially as described (Brownell and Allis, 1995). Crude Annunziato, A.T., and Seale, R.L. (1983). Histone deacetylation iscore histones (8 mg) or purified HeLa histones (2 mg) and enzyme required for the maturation of newly replicated chromatin. J. Biol.samples were incubated for 10 min at 308C in a final volume of 50 Chem. 258, 12675–12684.ml of buffer A (50 mM Tris-HCl [pH 8.0], 10% (v/v) glycerol, 1 mM Barberis, A., Pearlberg, J., Simkovich, N., Farrell, S., Reinagel, P.,DTT, 1mM PMSF, and 0.1 mM EDTA). Reactions were initiated by Bamdad, C., Sigal, G., and Ptashne, M. (1995). Contact with a com-the addition of [3H]-acetyl-CoA (100 nCi, 6.1 Ci/mmol; ICN) to a final ponent of the polymerase II holoenzyme suffices for gene activation.concentration of 0.328 mM. HAT activity was determined by liquid- Cell 81, 359–368.scintillation counting of aliquots of reactions spotted on P-81 filters

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5 mM EDTA; 0.5% (v/v) NP-40; and 0.1 mM PMSF, aprotinin, leupep- Brownell, J.E., and Allis, C.D. (1996). Special HATs for special occa-sions: linking histone acetylation to chromatin assembly and genetin, and pepstatin). The lysis mixture was incubated on ice for 20

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