The coactivator host cell factor-1 mediates Set1 andMLL1 H3K4 trimethylation at herpesvirus immediateearly promoters for initiation of infectionAarthi Narayanan*, William T. Ruyechan†, and Thomas M. Kristie*‡

*Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 4-131, 4 Center Drive,Bethesda, MD 20892; and †Department of Microbiology and Immunology, University at Buffalo, State University of New York, 251 BiomedicalResearch Building, Buffalo, NY 14214

Communicated by Bernard Moss, National Institutes of Health, Bethesda, MD, May 9, 2007 (received for review March 23, 2007)

Originally identified as an essential component of the herpessimplex virus immediate early (IE) gene enhancer complex, thetranscriptional coactivator host cell factor-1 (HCF-1) has beenimplicated in a broad range of cellular regulatory circuits. Theprotein mediates activation through multiple interactions withtranscriptional activators, coactivators, and chromatin remodel-ing complexes. However, the mechanisms involved in HCF-1-dependent transcriptional stimulation were undefined. By usinga minimal HCF-1-dependent promoter and a model activator, thevaricella zoster IE62 protein, it was determined that HCF-1 wasnot required for the assembly of the RNAPII basal complex,which depended solely on IE62 in conjunction with the cellularfactor Sp1. In contrast, HCF-1 was required for recruitment of thehistone methyltransferases Set1 and MLL1 (mixed-lineageleukemia 1), leading to histone H3K4 trimethylation and tran-scriptional activation. Similarly, in a varicella zoster virus lyticinfection, HCF-1, Set1, and MLL1 were recruited to the viralgenomic IE promoter, suggesting an essential role for HCF-1 inchromatin modification and remodeling during initiation of lyticinfection. The results indicate that one biological rationale forthe incorporation of the viral IE activators in the viral particle isto recruit HCF-1/histone methyltransferase complexes and pro-mote assembly of the viral IE gene promoters into transcrip-tionally active chromatin. These studies also contribute to themodel whereby the induced nuclear transport of HCF-1 insensory neurons may be critical to the reactivation of latentherpesviruses by promoting the activation of chromatinmodifications.

chromatin � histone methyltransferase � chromatin modifications �Sp1 � transcription

The cellular transcriptional coactivator host cell factor-1(HCF-1) was originally isolated as a component of the herpes

simplex virus (HSV) immediate early (IE) gene enhanceosomecomplex containing the cellular POU domain protein Oct-1 andthe viral transactivator VP16 (1–6). The protein has been mostthoroughly studied in this context where it mediates the VP16transcriptional activation of the viral IE genes (7, 8). In ananalogous manner, HCF-1 also mediates the induction of therelated varicella zoster virus (VZV) IE genes by the viraltransactivators ORF10 and IE62 (8). The transcription of these�-herpesvirus genes is regulated by multiple mechanisms andfactors via complex combinatorial enhancer-promoter domains.Strikingly, HCF-1 has been shown to be essential for IE geneexpression, suggesting that it mediates a common rate-limitingstep. Most intriguingly, both HSV and VZV establish latency inthe neurons of sensory ganglia. In these cells, HCF-1 is uniquelysequestered in the cytoplasm of sensory neurons and is rapidlytransported to the nucleus upon stimulation that results in viralreactivation (9). Therefore, whereas the protein is essential forviral lytic replication, it may also be a key component in the�-herpesvirus latency reactivation cycle.

Since its identification as a coactivator for the herpesvirusactivators, several lines of evidence have indicated that HCF-1also plays a broadly significant role in cellular transcription. Theprotein (i) is a binding partner and/or coactivator for numerouscellular transcription factors including those of the krupple (Sp1and Krox20), Ets (GABP), ATF/CRE (CREB3), and E2F (E2F1and E2F4) families (10–16), (ii) interacts with other coactivators(PGC and FHL2) (17, 18) where it has been hypothesized tomediate coactivator–activator interactions, (iii) is essential formultiple stages of cell cycle progression (19, 20), and (iv) hasrecently been identified as a component of complexes involvedin chromatin modification and remodeling (21–23). Further-more, expression array studies have identified a wide range oftarget genes whose expression is affected in cells in which thenuclear accumulation of HCF-1 has been inhibited (24). How-ever, although activator partners and target genes have beenidentified, the biochemical mechanism by which HCF-1 stimu-lates transcription is undefined.

In this study, a model promoter (VZV IE62 promoter) andactivator (VZV IE62) were studied to determine the role ofHCF-1 in recruitment of the general transcription factor com-plex and in mediating activating chromatin modifications. Theresults demonstrate that IE62, in conjunction with the ubiqui-tous factor Sp1, mediates the assembly of the initiation complex.Promoter occupancy of HCF-1 is not required for this assemblybut is essential for transcriptional activation via recruitment ofthe H3K4 methyltransferases Set1 and MLL1 (mixed-lineageleukemia 1). Strikingly, Sp1 is required to bridge or stabilize theinteraction of the IE62 activator with the HCF-1 transcriptionalcoactivator, suggesting that this protein may play a general rolein mediating HCF-1-activator interactions. Finally, the recruit-ment of HCF-1 and the Set1/MLL1 histone methyltransferases(HMTs) to the viral IE promoter during the initiation ofinfection indicates that chromatin modification and remodelingrepresent critical stages in the activation of �-herpesvirus tran-scription. The data support the model in which HCF-1-dependent chromatin modulation would play a critical role in theregulation of the viral lytic and latency reactivation cycle.

ResultsHCF-1-Dependent Coactivation of IE62 Target Genes. Previously itwas demonstrated that HCF-1 was an essential component

Author contributions: A.N. and T.M.K. designed research; A.N. performed research; W.T.R.contributed new reagents/analytic tools; A.N. and T.M.K. analyzed data; and A.N. andT.M.K. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

Abbreviations: HCF-1, host cell factor-1; HSV, herpes simplex virus; IE, immediate early; VZV,varicella zoster virus; HMT, histone methyltransferase.

‡To whom correspondence should be addressed. E-mail: thomas�kristie@nih.gov.

This article contains supporting information online at www.pnas.org/cgi/content/full/0704351104/DC1.

© 2007 by The National Academy of Sciences of the USA

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required for the combinatorial regulation of the expression ofthe HSV and VZV �-herpesvirus IE genes by the respective viraltransactivators VP16, ORF10, and IE62 (8). As illustrated in Fig.1A, the VZV IE62 promoter contains enhancer core elementsthat assemble the multiprotein Oct-1/HCF-1/ORF10 complex aswell as sites for costimulatory factors such as GABP and Sp1(25). In addition, this promoter contains an IE62 binding sitethat responds to the viral transactivator (26, 27). As previouslydemonstrated, IE62 stimulated the expression of an IE62promoter-reporter gene and this induction was severely com-promised in HCF-1-depleted cells (Fig. 1B).

Similarly, an IE62 promoter-reporter construct containingonly the IE62 binding site, a TATA element, and an adjacent Sp1binding site was induced by IE62 in an HCF-1-dependentmanner. In contrast, deletion of the TATA proximal Sp1 bindingsite abrogated IE62-mediated induction, thereby defining theminimal promoter requirements for IE62-mediated activationand HCF-1 coactivation. As shown in supporting information(SI) Fig. 8, the requirement for Sp1 for IE62 activation of theIEP-61 promoter may be explained by the lack of stable IE62binding in the absence of the Sp1 binding site.

In addition to its autoregulatory function, IE62 also inducestranscription of the early and late VZV gene classes (28). Asshown in SI Fig. 9, promoter–reporter constructs representingearly and late VZV gene classes were induced in the presence ofIE62. In each case, depletion of HCF-1 resulted in a significantreduction in the IE62-dependent induction, indicating that therequirement for HCF-1 in IE62 transcriptional activation is ageneral one and is not restricted to the regulation of the IE genes.

IE62 Recruitment of the Coactivator HCF-1 to the IE62 Minimal ModelPromoter. The HCF-1 dependence of IE62-mediated activationsuggested that IE62 might directly recruit HCF-1 to the mini-mally responsive promoter target gene. Therefore, HCF-1 pro-moter occupancy was determined by ChIP assays in the presenceand absence of IE62. As shown in Fig. 2, both cotransfectedV5-tagged HCF-1 (Fig. 2 Upper) and endogenous HCF-1 (Fig.2 Lower) were recruited to the IE62P-61 promoter in thepresence of IE62. Although a low level of endogenous HCF-1could be detected in the absence of IE62, this occupancy wasstrongly stimulated in the presence of IE62 (8.5-fold).

IE62-Mediated Assembly of the Basal Transcription Complex Is NotHCF-1-Dependent. To investigate the biochemical mechanism(s)by which IE62/HCF-1 stimulates transcription, ChIP assays weredone to assess the promoter occupancy of basal transcriptionfactors in the presence and absence of IE62 or HCF-1. As shownin Fig. 3A, in the absence of IE62, low levels of Sp1 and the basalfactors TBP, TFIIB, TFIIH, and RNAPII could be detected.However, a strong stimulation of promoter occupancy of allfactors was found in the presence of IE62. In particular, stim-ulation of Sp1 occupancy in the presence of IE62 underscores thecooperative DNA binding suggested previously (SI Fig. 8) (29).

To determine whether HCF-1 was required for the assemblyof the RNAPII transcription complex, occupancy was alsoassessed in HCF-1-positive and HCF-1-depleted cells (Fig. 3B).Strikingly, the depletion of HCF-1 had no effect on the recruit-ment of the RNAPII complex to the minimal promoter, indi-cating that the IE62-Sp1 activators were solely responsible forthis assembled but transcriptionally inactive complex.

Transcriptional Induction by IE62 Results in HCF-1-Dependent Set1-and MLL1-Mediated Trimethylation of Histone H3K4. Recently,HCF-1 was identified as a component of the MLL family ofHMTs (22, 23). As HCF-1 is critical for IE62-mediated tran-scriptional induction yet it is not required for recruitment/assembly of the RNAPII complex, the state of selected chro-matin modifications was determined in HCF-1-positive andHCF-1-depleted cells (Fig. 4A). In the absence of IE62, the IE62

Fig. 1. HCF-1-dependent coactivation of a minimal IE62 target gene. (A) TheIE62 promoter–reporters are schematically illustrated, depicting the bindingsites for various cellular and viral factors. EC represents the VZV IE enhancercore that assembles the Oct-1, ORF10, and HCF-1 multiprotein complex. GA,Sp1, CCAAT, CRE, and IE62 are the putative binding sites for GA-bindingprotein, Sp1, CCAAT-binding proteins, ATF/CRE factors, and VZV IE62, respec-tively. Luc, luciferase reporter gene coding region; �1, transcription initiationsite. (B) HeLa cells were transfected with HCF-1 RNAi vector pU6-si-HCF-1 (Si)or the control RNAi pU6-si (C). The indicated reporter genes were transfectedor cotransfected 48 h later with increasing amounts of pCMV-IE62 (0, 50, 100,and 200 ng). Reporter expression levels were normalized to the activity of thecontrol vector (fold expression of reporter relative to control). The data arerepresentative of three independent experiments. HCF-1 depletion was de-termined to be 70% by quantitative Western blot analysis, whereas no effecton the expression of IE62 in HCF-1-depleted cells was detected.

Fig. 2. Recruitment of transfected and endogenous HCF-1 in the presenceand absence of IE62. ChIP assays were done 48 h after transfection by using theindicated antibodies or control antibodies (HA, V5, and IgG). The signalintensities were quantitated as described in Materials and Methods and areexpressed as a percentage of the extract signal. The data shown are derivedfrom a single ChIP assay but are representative of at least two independentexperiments. �IE62/�IE62 is the fold ratio of the intensities in the presence ofIE62 to those in the absence of IE62. (Upper) HeLa cells were transfected withthe IE62P-61 reporter plasmid (Reporter), the HA-tagged IE62 expressionplasmid (HA-IE62), and/or the V5-tagged HCF-1 expression plasmid (HCF-1-V5)as indicated. (Lower) HeLa cells were transfected with the IE62P-61 reporterplasmid (Reporter) or cotransfected with the pCMV-IE62 expression plasmid.Ext, input extract before immunoprecipitation.

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responsive promoter was marked by the repressive dimethyl-H3K9. However, in the presence of IE62, the activatingtrimethyl-H3K4 was significantly enhanced, whereas dimethyl-H3K9 was significantly reduced. In addition, IE62 specificallyresulted in significant recruitment of the H3K4 HMTs Set1 andMLL1, relative to the HMT Set7/9. Interestingly, IE62-mediatedoccupancy by MLL1 increased only 4-fold. In contrast, IE62

resulted in a 30-fold increase in the promoter occupancy by Set1,suggesting that IE62 preferentially recruits this H3K4 HMT.

Most significantly, in the absence of HCF-1 (Fig. 4B), therecruitment of Set1 and MLL1 and the resulting H3K4 trim-ethylation were all severely reduced (9.5%, 18.4%, and 5.7% ofHCF-1�, respectively). This reduction was not due to reducedlevels of the critical protein components in HCF-1-depleted cells

Fig. 3. Assembly of the RNAPII complex by IE62-Sp1. Recruitment of Sp1 and factors of the RNAPII initiation complex in the presence and absence of IE62 orHCF-1. ChIP assays were done by using the indicated transcription factor (TF) antibodies or control IgG. The signal intensities are expressed as a percentage ofthe extract signal. Intensities and fold (�IE62/�IE62) were quantitated as described in Materials and Methods. The data shown are derived from a single ChIPassay but are representative of at least two independent experiments. (A) HeLa cells were transfected with the IE62P-61 reporter plasmid or cotransfected withpCMV-IE62. (B) HeLa cells were transfected with either the HCF-1 RNAi vector pU6-si-HCF-1 (HCF�) or the control RNAi pU6-si (HCF�). The IE62P-61 reporter wascotransfected with pCMV-IE62 48 h later. %HCF� represents the ratio of signal intensity in the absence of HCF-1 to that in the presence of HCF-1. Ext, extract.

Fig. 4. HCF-1-dependent Set1- and MLL1-mediated H3K4 trimethylation. Recruitment of HMTs and H3K4 trimethylation in the presence and absence of IE62or HCF-1. ChIP assays were done by using the indicated antibodies or control IgG. The signal intensities are expressed as a percentage of the extract signal.Intensities and fold (�IE62/�IE62) were quantitated as described in Materials and Methods. The data shown are derived from a single ChIP assay but arerepresentative of at least two independent ChIP assays. The results of the real-time PCR are graphed as a percentage of the input sample. m, d, and t representmono-, di-, and trimethylation, respectively. (A) HeLa cells were transfected with the IE62P-61 reporter plasmid or cotransfected with pCMV-IE62. ChIP assayswere done 48 h later by using the indicated modified histone, HMT, or control IgG. (B) HeLa cells were transfected with either the HCF-1 RNAi vector pU6-si-HCF-1(HCF�) or the control RNAi pU6-si (HCF�). The reporter plasmid pGL3-IE62P-61 was cotransfected with pCMV-IE62 48 h later. %HCF� represents the ratio ofsignal intensity in the absence of HCF-1 to that in the presence of HCF-1. Ext, extract.

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as demonstrated by quantitative Western blot analysis (SI Fig.10). Thus, recruitment of the coactivator HCF-1 results in Set1-and MLL1-dependent H3K4 trimethylation.

Sp1 Is Required to Bridge/Stabilize the IE62-HCF-1 Interaction. Sp1 isclearly an important component of the IE62-HCF-1-mediatedtranscriptional induction as shown by the removal of the TATAproximal Sp1 site, which abrogates stable IE62 binding (SI Fig.8) and by the increased promoter occupancy by Sp1 in thepresence of IE62 (Fig. 3). Interestingly, both IE62 and HCF-1interact directly with Sp1 (12, 29), leading to the possibility that,in addition to cooperative DNA binding interactions with IE62,Sp1 may also function to mediate or stabilize the interaction ofIE62 with HCF-1. As shown in Fig. 5A, both endogenous HCF-1and Sp1 were coimmunoprecipitated with IE62 (lane 4). How-ever, RNAi-mediated depletion of Sp1 reduced the coimmuno-precipitation of HCF-1 to background levels (Fig. 5B, comparelanes 4 and 5 with lane 6). Thus, Sp1 is required to bridge orstabilize the interaction of the activator IE62 with its coactivatorHCF-1.

Recruitment of HCF-1 and Chromatin Modifications at the VZV IE62Promoter During VZV Lytic Infection. The data presented here usinga model IE62-responsive promoter argue that HCF-1-mediatedmodifications would be represented at the viral IE promoterduring the early stages of infection. Therefore, cells were in-fected with VZV, and 4 h after infection, the occupancy of theviral IE62 promoter by IE62, Sp1, HCF-1, Set1/MLL1 HMTs,and trimethyl-H3K4 was assessed. As shown in Fig. 6, resultscomparable with those obtained by using the transfected modelpromoter were found at the viral genomic IE promoter, includ-ing clear occupancy of IE62, Sp1, HCF-1, Set1, and MLL1. Asanticipated, based on the HCF-1 and Set/MLL occupancy, thepromoter exhibited a strong trimethyl-H3K4 signal. In contrast,the IE62 coding region did not reveal any significant HCF-1occupancy or Set1/MLL1-mediated H3K4 trimethylation.

The requirement of HCF-1 in mediating these modificationsduring a viral infection was similarly addressed by infection of acell culture that had been partially depleted of HCF-1. In thesecells, a 52% HCF-1 depletion resulted in similar decreases inHCF-1, Set1, MLL1, and trimethyl-H3K4 signals at the viral IEpromoter, whereas no significant change in the promoter occu-pancy by the viral IE62 protein was found (data not shown). The

results indicate that HCF-1-mediated chromatin modulation ofthe IE gene promoter domain is likely to play a critical role inthe initiation of the viral infectious cycle.

DiscussionNumerous lines of evidence have indicated that HCF-1 is acellular coactivator of broad significance. In this study, the VZVIE62 activator and an IE62-responsive model promoter wereused to define the functional role of HCF-1 in transcriptionalregulation. As shown in the model depicted in Fig. 7, HCF-1 wasnot required for the recruitment of the RNAPII basal factorcomplex, which depended on the presence of the viral IE62activator in concert with the ubiquitous cellular activator Sp1.The ability of IE62 to mediate the formation of this complex isconsistent with in vitro studies indicating that IE62 interactsdirectly with TBP and TFIIB (26) and enhances the binding ofTBP/TFIID (29). Additionally, the requirement for Sp1 forstable IE62 promoter occupancy underscores the importance offactors such as Sp1, which has been observed in studies ofIE62-mediated activation (30). However, despite the formationof this initiation complex, IE62-Sp1 was unable to stimulatetranscription in the absence of HCF-1.

Recruitment of the coactivator HCF-1 to the model promoterby the IE62-Sp1 activators results in Set1 and MLL1 HMTpromoter occupancy, H3K4 trimethylation, and transcriptionalactivation. H3K4 trimethylation is a hallmark of transcriptionalactivation and probably serves as a signal for the recruitment ofremodeling and activation components (31–35). Therefore, al-though HCF-1 may play additional roles, one primary functionof this coactivator is to promote chromatin modifications char-acteristic of activated transcription. Interestingly, in the absenceof the IE62 activator, the model promoter is marked withdimethyl-H3K9 modifications, a repressive mark that precludesH3K4 trimethylation. It remains unclear whether the recruit-ment of HCF-1/Set1/MLL1 prevents the accumulation of thesemarks or whether there are additional components, such as anH3K9 demethylase, that may also be required for HCF-1-

Fig. 5. Sp1 is required to mediate IE62–HCF-1 interaction. HA-IE62 immu-noprecipitates (IP-IE62) of HeLa cell extracts were subjected to Western blotanalysis with HA (HA-IE62), HCF-1, and Sp1 antisera. (A) HeLa cells weretransfected with the HA-IE62 expression plasmid (HA-IE62) or control vector(�). (B) HeLa cells were transfected with Sp1 RNAi (RNAi-Sp1) or control RNAi(RNAi-C) and subsequently retransfected with the HA-IE62 expression plas-mid. *, HCF-1 background level (compare lanes 4 and 6); **, 78% depletion ofSp1 as determined by quantitative Western blot analysis.

Fig. 6. HCF-1–HMT occupancy and chromatin modifications at the viral IE62promoter during VZV infection. BS-C-1 cells were infected with VZV as de-scribed. Four hours after infection, ChIP assays were done by using theindicated antisera or control IgG. The signal intensities of the PCR products arerepresented as percent extract for the promoter (IE62P) and control codingdomains (IE62C). Intensities and fold (�IE62/�IE62) were quantitated as de-scribed in Materials and Methods. The data shown are derived from a singleChIP assay but are representative of at least two independent experiments.The results of the real-time PCR are graphed as a percentage of the inputsample. H3K4-t, trimethyl-H3K4; Ext, extract.

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mediated transcriptional activation. Recent studies have dem-onstrated the cooperative role of these enzymes in transcrip-tional activation by removal of repressive H3K9 methylation (36,37). It would therefore be of interest to determine whetherHCF-1 might coordinately recruit an H3K9 demethylase inconjunction with the Set1/MLL1 H3K4 HMTs.

In this study, the significance of the results obtained by usingthe model promoter and IE62 activator is shown by the recruit-ment of HCF-1 and the Set1/MLL1 HMTs to the genomic viralIE promoter during early infection. The data highlight thebiological relevance of chromatin modulation for the initiationof a herpesvirus infection and complement the study of Huanget al. (38), which implicated Set1 as an important regulatorycomponent of the HSV viral lytic cycle. For both HSV and VZV,the IE transactivators are packaged in the virus and releasedupon initial infection. The requirement for these activators andtheir ability to recruit the HCF-1/HMT complexes to the IEpromoters may be necessary to prevent or circumvent theassembly of these regions into repressive chromatin.

Most significantly, �-herpesviruses establish latency in sen-sory neurons. In these cells, the coactivator HCF-1 is uniquelysequestered in the cytoplasm of the cell but is rapidly relocalizedto the nucleus upon stimuli that result in viral reactivation. In themodel, the viral transactivators are not present during latency,and the relocalized HCF-1 is recruited to the viral IE promotersby alternative transcription factors that recognize the complexpromoter–enhancer domains (9). The data presented here en-rich this model by suggesting that recruitment of HCF-1/HMTcomplexes to the latent viral IE promoters would promotechromatin remodeling and viral IE gene expression. In thisrespect, the ability of Sp1 to bridge or stabilize the interaction ofHCF-1 with the IE62 activator places new emphasis on thisfactor whose cooperative interactions with numerous transcrip-tion factors may be important to a more general targeting/recruitment of HCF-1.

Materials and MethodsCell Culture and Virus. HeLa and BS-C-1 cells were maintainedaccording to standard procedures. VZV (Ellen) was obtainedfrom American Type Culture Collection (Manassas, VA). VZV

viral infections were done by overlaying cell-associated virus onnaive BS-C-1 cells. Cells were harvested for ChIP assays 4 h afterinfection.

Reporter Assays. Luciferase reporter genes contained ORF29,ORF28, and gI VZV promoter sequences cloned into pGL3-basic (SI Fig. 9). IE62P-61 and IE62P-39 were derived fromIE62PR that contained IE62 promoter sequences from �269 to�73. pCMV-IE62 expresses the IE62 activator under control ofthe CMV IE promoter and has been previously described (8).For all reporter assays, 4 � 104 HeLa cells were transfected with300 ng of pU6-Si-HCF-1 or pU6-Si control RNAi using Fu-GENE (Roche Diagnostics Corporation) according to the man-ufacturer’s recommendations. At 48 h after transfection, thecells were cotransfected with the reporter constructs and in-creasing amounts of pCMV-IE62, or control vector. The re-porter gene activity was measured 24 h later by using a Dual-Luciferase assay kit (Promega, Madison, WI) in a luminometer(Berthold, Bad Wildbad, Germany). All activity units werenormalized by protein concentration and by the activity of thecontrol vector. The data presented are representative of threeindependent experiments.

ChIP Assays. Unless otherwise indicated, 1.3 � 106 HeLa cellswere transfected with 1 �g of the appropriate reporter plasmids(IE62P-61 or IE62P-39) or cotransfected with 2 �g of the IE62expression plasmid (pCMV-IE62). For HCF-1 recruitment, 2 �gof HA-tagged IE62 (pCMV-HA-IE62) and 4 �g of V5-taggedHCF-1 (pHCF-1-V5) (18) were cotransfected with the IE62P-61reporter. For depletion of HCF-1, cells were transfected with 3�g of pU6-HCF-RNAi or pU6-control RNAi and were retrans-fected with reporter and IE62 expression plasmids 48 h later.ChIP assays were done essentially as previously described (39–41). Cell lysates were sonicated to obtain DNA fragmentsranging from 300 to 700 bp. Chromatin from 7 � 106 cells wasused for each immunoprecipitation with the following antibod-ies: IE62 (29); HCF-1 (2); IgG, RNAPII, dimethyl-H3K4,trimethyl-H3K4, monomethyl-H3K9, and dimethyl-H3K9 (cat-alog nos. 12-370, 05-952, 07-030, 05-745, 07-450, and 07-441,respectively; Upstate Biotechnology, Lake Placid, NY); Sp1,TBP, TFIIB, TFIIH, and HA (catalog nos. SC-59, SC-273,SC-274, SC-6857/6859, and SC-805, respectively; Santa CruzBiotechnology, Santa Cruz, CA); MLL1 and Set1 (catalog nos.1408/1289 and 1193, respectively; Bethyl Laboratories, Mont-gomery, TX); and Set7/9 (catalog no. 13731; Abcam, Cambridge,MA). Immunoprecipitates were washed and eluted, and thecross-linking was reversed. Recovered DNA was subjected tostandard PCR with dilutions of input DNA to ensure linearity,resolved in ethidium bromide agarose gels, and the signalintensities were quantitated by using a 4000MM Image Station(Kodak, Rochester, NY). The signal intensities of individualbands were calculated as a percentage of the intensity of theinput extract after subtraction of the appropriate backgroundantibody controls. Where indicated, samples were also subjected,in triplicate, to real-time PCR using SYBR Green (Qiagen,Valencia, CA) on a Prism 7900 system (Applied Biosystems,Foster City, CA) and analyzed with SDS 2.2.2 software. The datapresented are the quantity means. All ChIP data shown arederived from a single ChIP assay but are representative of at leasttwo independent experiments. The location and sequence ofprimer sets used are shown in SI Fig. 11.

Coimmunoprecipitations. HeLa cells (1.3 � 106) were transfectedwith 5 �g of pHA-IE62 expression plasmid or control vectorDNA. For depletion of Sp1, 5 �g of pU6-Sp1 vector (Panomics,

Fig. 7. Model of HCF-1-dependent IE62-mediated transcriptional activation.The role of HCF-1 in IE62-mediated transcriptional activation is schematicallyrepresented. TBP, TATA binding protein; PI, preinitiation assembly.

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Redwood City, CA) or control RNAi was transfected on day 1and retransfected on day 2 with 2.5 �g of pU6-Sp1 or control and48 h later pHA-IE62 expression vector. Extracts were made 48h later as described in ref. 18, sonicated briefly, and clarified bycentrifugation. Protein extract was incubated for 2 h at 4°C withHA-Sepharose beads, washed five times with binding buffer,eluted in SDS sample buffer, and resolved in 4–20% Tris-glycinegels. Western blot analyses of resolved extracts and immuno-precipitates were done with the indicated antibodies (HA,HCF-1 AB2131, and Sp1), developed for chemiluminescence by

using Super Signal Dura (Pierce, Rockford, IL), and quantitatedby using a Kodak 4000MM Image Station.

We thank J. Vogel, Yu Liang G. Kolb, H. Peng, and A. McBride fordiscussions and advice; J. Vogel, T. Pierson, P. Sharp, and B. Moss forcritical reading of the manuscript; and members of the Laboratory ofViral Diseases for helpful discussions. This study was supported by theLaboratory of Viral Diseases and the Division of Intramural Research,National Institute of Allergy and Infectious Diseases, National Institutesof Health (T.M.K.) and by National Institute of Allergy and InfectiousDiseases Grant AI18449 (to W.T.R.).

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