The Coactivator Host Cell Factor-1 Mediates Set1 and MLL1 H3K4 Trimethylation at Herpesvirus Immediate Early Promoters for Initiation of Infection

The coactivator host cell factor-1 mediates Set1 and MLL1 H3K4 trimethylation at herpesvirus immediate early promoters for initiation of infection

Aarthi 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 Biomedical Research 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 herpes Since its identification as a coactivator for the herpesvirus simplex virus immediate early (IE) gene enhancer complex, the activators, several lines of evidence have indicated that HCF-1 transcriptional coactivator host cell factor-1 (HCF-1) has been also plays a broadly significant role in cellular transcription. The implicated in a broad range of cellular regulatory circuits. The protein (i) is a binding partner and/or coactivator for numerous protein mediates activation through multiple interactions with cellular transcription factors including those of the krupple (Sp1 transcriptional activators, coactivators, and chromatin remodel- and Krox20), Ets (GABP), ATF/CRE (CREB3), and E2F (E2F1 ing complexes. However, the mechanisms involved in HCF-1- and E2F4) families (10–16), (ii) interacts with other coactivators dependent transcriptional stimulation were undefined. By using (PGC and FHL2) (17, 18) where it has been hypothesized to a minimal HCF-1-dependent promoter and a model activator, the mediate coactivator–activator interactions, (iii) is essential for varicella zoster IE62 protein, it was determined that HCF-1 was multiple stages of cell cycle progression (19, 20), and (iv) has not required for the assembly of the RNAPII basal complex, recently been identified as a component of complexes involved which depended solely on IE62 in conjunction with the cellular in chromatin modification and remodeling (21–23). Further- factor Sp1. In contrast, HCF-1 was required for recruitment of the more, expression array studies have identified a wide range of histone methyltransferases Set1 and MLL1 (mixed-lineage target genes whose expression is affected in cells in which the leukemia 1), leading to histone H3K4 trimethylation and tran- nuclear accumulation of HCF-1 has been inhibited (24). How- scriptional activation. Similarly, in a varicella zoster virus lytic ever, although activator partners and target genes have been identified, the biochemical mechanism by which HCF-1 stimu- infection, HCF-1, Set1, and MLL1 were recruited to the viral lates transcription is undefined. genomic IE promoter, suggesting an essential role for HCF-1 in In this study, a model promoter (VZV IE62 promoter) and chromatin modification and remodeling during initiation of lytic activator (VZV IE62) were studied to determine the role of infection. The results indicate that one biological rationale for HCF-1 in recruitment of the general transcription factor com- the incorporation of the viral IE activators in the viral particle is plex and in mediating activating chromatin modifications. The BIOCHEMISTRY to recruit HCF-1/histone methyltransferase complexes and pro- results demonstrate that IE62, in conjunction with the ubiqui- mote assembly of the viral IE gene promoters into transcrip- tous factor Sp1, mediates the assembly of the initiation complex. tionally active chromatin. These studies also contribute to the Promoter occupancy of HCF-1 is not required for this assembly model whereby the induced nuclear transport of HCF-1 in but is essential for transcriptional activation via recruitment of sensory neurons may be critical to the reactivation of latent the H3K4 methyltransferases Set1 and MLL1 (mixed-lineage herpesviruses by promoting the activation of chromatin leukemia 1). Strikingly, Sp1 is required to bridge or stabilize the modifications. interaction of the IE62 activator with the HCF-1 transcriptional coactivator, suggesting that this protein may play a general role chromatin ͉ histone methyltransferase ͉ chromatin modifications ͉ in mediating HCF-1-activator interactions. Finally, the recruit- Sp1 ͉ transcription ment of HCF-1 and the Set1/MLL1 histone methyltransferases (HMTs) to the viral IE promoter during the initiation of he cellular transcriptional coactivator host cell factor-1 infection indicates that chromatin modification and remodeling ␣ T(HCF-1) was originally isolated as a component of the herpes represent critical stages in the activation of -herpesvirus tran- simplex virus (HSV) immediate early (IE) gene enhanceosome scription. The data support the model in which HCF-1- complex containing the cellular POU domain protein Oct-1 and dependent chromatin modulation would play a critical role in the the viral transactivator VP16 (1–6). The protein has been most regulation of the viral lytic and latency reactivation cycle. thoroughly studied in this context where it mediates the VP16 Results transcriptional activation of the viral IE genes (7, 8). In an HCF-1-Dependent Coactivation of IE62 Target Genes. Previously it analogous manner, HCF-1 also mediates the induction of the was demonstrated that HCF-1 was an essential component related varicella zoster virus (VZV) IE genes by the viral transactivators ORF10 and IE62 (8). The transcription of these ␣ -herpesvirus genes is regulated by multiple mechanisms and Author contributions: A.N. and T.M.K. designed research; A.N. performed research; W.T.R. factors via complex combinatorial enhancer-promoter domains. contributed new reagents/analytic tools; A.N. and T.M.K. analyzed data; and A.N. and Strikingly, HCF-1 has been shown to be essential for IE gene T.M.K. wrote the paper. expression, suggesting that it mediates a common rate-limiting The authors declare no conflict of interest. step. Most intriguingly, both HSV and VZV establish latency in Freely available online through the PNAS open access option. the neurons of sensory ganglia. In these cells, HCF-1 is uniquely Abbreviations: HCF-1, host cell factor-1; HSV, herpes simplex virus; IE, immediate early; VZV, sequestered in the cytoplasm of sensory neurons and is rapidly varicella zoster virus; HMT, histone methyltransferase. transported to the nucleus upon stimulation that results in viral ‡To whom correspondence should be addressed. E-mail: thomas࿝[email protected]. reactivation (9). Therefore, whereas the protein is essential for This article contains supporting information online at www.pnas.org/cgi/content/full/ viral lytic replication, it may also be a key component in the 0704351104/DC1. ␣-herpesvirus latency reactivation cycle. © 2007 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0704351104 PNAS ͉ June 26, 2007 ͉ vol. 104 ͉ no. 26 ͉ 10835–10840 Downloaded by guest on September 30, 2021 Fig. 2. Recruitment of transfected and endogenous HCF-1 in the presence and absence of IE62. ChIP assays were done 48 h after transfection by using the indicated antibodies or control antibodies (HA, V5, and IgG). The signal intensities were quantitated as described in Materials and Methods and are expressed as a percentage of the extract signal. The data shown are derived from a single ChIP assay but are representative of at least two independent experiments. ϩIE62/ϪIE62 is the fold ratio of the intensities in the presence of IE62 to those in the absence of IE62. (Upper) HeLa cells were transfected with the IE62P-61 reporter plasmid (Reporter), the HA-tagged IE62 expression Fig. 1. HCF-1-dependent coactivation of a minimal IE62 target gene. (A) The plasmid (HA-IE62), and/or the V5-tagged HCF-1 expression plasmid (HCF-1-V5) IE62 promoter–reporters are schematically illustrated, depicting the binding as indicated. (Lower) HeLa cells were transfected with the IE62P-61 reporter sites for various cellular and viral factors. EC represents the VZV IE enhancer plasmid (Reporter) or cotransfected with the pCMV-IE62 expression plasmid. core that assembles the Oct-1, ORF10, and HCF-1 multiprotein complex. GA, Ext, input extract before immunoprecipitation. Sp1, CCAAT, CRE, and IE62 are the putative binding sites for GA-binding protein, Sp1, CCAAT-binding proteins, ATF/CRE factors, and VZV IE62, respec- ϩ tively. Luc, luciferase reporter gene coding region; 1, transcription initiation IE62 Recruitment of the Coactivator HCF-1 to the IE62 Minimal Model site. (B) HeLa cells were transfected with HCF-1 RNAi vector pU6-si-HCF-1 (Si) Promoter. or the control RNAi pU6-si (C). The indicated reporter genes were transfected The HCF-1 dependence of IE62-mediated activation or cotransfected 48 h later with increasing amounts of pCMV-IE62 (0, 50, 100, suggested that IE62 might directly recruit HCF-1 to the mini- and 200 ng). Reporter expression levels were normalized to the activity of the mally responsive promoter target gene. Therefore, HCF-1 pro- control vector (fold expression of reporter relative to control). The data are moter occupancy was determined by ChIP assays in the presence representative of three independent experiments. HCF-1 depletion was de- and absence of IE62. As shown in Fig. 2, both cotransfected termined to be 70% by quantitative Western blot analysis, whereas no effect V5-tagged HCF-1 (Fig. 2 Upper) and endogenous HCF-1 (Fig. on the expression of IE62 in HCF-1-depleted cells was detected. 2 Lower) were recruited to the IE62P-61 promoter in the presence of IE62. Although a low level of endogenous HCF-1 could be detected in the absence of IE62, this occupancy was required for the combinatorial regulation of the expression of strongly stimulated in the presence of IE62 (8.5-fold). the HSV and VZV ␣-herpesvirus IE genes by the respective viral transactivators VP16, ORF10, and IE62 (8). As illustrated in Fig. IE62-Mediated Assembly of the Basal Transcription Complex Is Not 1A, the VZV IE62 promoter contains enhancer core elements HCF-1-Dependent. To investigate the biochemical mechanism(s) that assemble the multiprotein Oct-1/HCF-1/ORF10 complex as by which IE62/HCF-1 stimulates transcription, ChIP assays were well as sites for costimulatory factors such as GABP and Sp1 done to assess the promoter occupancy of basal transcription (25). In addition, this promoter contains an IE62 binding site factors in the presence and absence of IE62 or HCF-1. As shown that responds to the viral transactivator (26, 27). As previously in Fig. 3A, in the absence of IE62, low levels of Sp1 and the basal demonstrated, IE62 stimulated the expression of an IE62 factors TBP, TFIIB, TFIIH, and RNAPII could be detected. promoter-reporter gene and this induction was severely com- However, a strong stimulation of promoter occupancy of all promised in HCF-1-depleted cells (Fig. 1B). factors was found in the presence of IE62. In particular, stim- Similarly, an IE62 promoter-reporter construct containing ulation of Sp1 occupancy in the presence of IE62 underscores the only the IE62 binding site, a TATA element, and an adjacent Sp1 cooperative DNA binding suggested previously (SI Fig. 8) (29). binding site was induced by IE62 in an HCF-1-dependent To determine whether HCF-1 was required for the assembly manner. In contrast, deletion of the TATA proximal Sp1 binding of the RNAPII transcription complex, occupancy was also site abrogated IE62-mediated induction, thereby defining the assessed in HCF-1-positive and HCF-1-depleted cells (Fig. 3B). minimal promoter requirements for IE62-mediated activation Strikingly, the depletion of HCF-1 had no effect on the recruit- and HCF-1 coactivation. As shown in supporting information ment of the RNAPII complex to the minimal promoter, indi- (SI) Fig. 8, the requirement for Sp1 for IE62 activation of the cating that the IE62-Sp1 activators were solely responsible for IEP-61 promoter may be explained by the lack of stable IE62 this assembled but transcriptionally inactive complex. binding in the absence of the Sp1 binding site. In addition to its autoregulatory function, IE62 also induces Transcriptional Induction by IE62 Results in HCF-1-Dependent Set1- transcription of the early and late VZV gene classes (28). As and MLL1-Mediated Trimethylation of Histone H3K4. Recently, shown in SI Fig. 9, promoter–reporter constructs representing HCF-1 was identified as a component of the MLL family of early and late VZV gene classes were induced in the presence of HMTs (22, 23). As HCF-1 is critical for IE62-mediated tran- IE62. In each case, depletion of HCF-1 resulted in a significant scriptional induction yet it is not required for recruitment/ reduction in the IE62-dependent induction, indicating that the assembly of the RNAPII complex, the state of selected chro- requirement for HCF-1 in IE62 transcriptional activation is a matin modifications was determined in HCF-1-positive and general one and is not restricted to the regulation of the IE genes. HCF-1-depleted cells (Fig. 4A). In the absence of IE62, the IE62

10836 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0704351104 Narayanan et al. Downloaded by guest on September 30, 2021 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 or HCF-1. ChIP assays were done by using the indicated transcription factor (TF) antibodies or control IgG. The signal intensities are expressed as a percentage of the extract signal. Intensities and fold (ϩIE62/ϪIE62) were quantitated as described in Materials and Methods. The data shown are derived from a single ChIP assay but are representative of at least two independent experiments. (A) HeLa cells were transfected with the IE62P-61 reporter plasmid or cotransfected with pCMV-IE62. (B) HeLa cells were transfected with either the HCF-1 RNAi vector pU6-si-HCF-1 (HCFϪ) or the control RNAi pU6-si (HCFϩ). The IE62P-61 reporter was cotransfected with pCMV-IE62 48 h later. %HCFϩ represents the ratio of signal intensity in the absence of HCF-1 to that in the presence of HCF-1. Ext, extract.

responsive promoter was marked by the repressive dimethyl- resulted in a 30-fold increase in the promoter occupancy by Set1, H3K9. However, in the presence of IE62, the activating suggesting that IE62 preferentially recruits this H3K4 HMT. trimethyl-H3K4 was significantly enhanced, whereas dimethyl- Most significantly, in the absence of HCF-1 (Fig. 4B), the H3K9 was significantly reduced. In addition, IE62 specifically recruitment of Set1 and MLL1 and the resulting H3K4 trim- resulted in significant recruitment of the H3K4 HMTs Set1 and ethylation were all severely reduced (9.5%, 18.4%, and 5.7% of MLL1, relative to the HMT Set7/9. Interestingly, IE62-mediated HCF-1ϩ, respectively). This reduction was not due to reduced occupancy by MLL1 increased only 4-fold. In contrast, IE62 levels of the critical protein components in HCF-1-depleted cells BIOCHEMISTRY

Fig. 4. HCF-1-dependent Set1- and MLL1-mediated H3K4 trimethylation. Recruitment of HMTs and H3K4 trimethylation in the presence and absence of IE62 or 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 (ϩIE62/ϪIE62) were quantitated as described in Materials and Methods. The data shown are derived from a single ChIP assay but are representative 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 represent mono-, di-, and trimethylation, respectively. (A) HeLa cells were transfected with the IE62P-61 reporter plasmid or cotransfected with pCMV-IE62. ChIP assays were 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 (HCFϪ) or the control RNAi pU6-si (HCFϩ). The reporter plasmid pGL3-IE62P-61 was cotransfected with pCMV-IE62 48 h later. %HCFϩ represents the ratio of signal intensity in the absence of HCF-1 to that in the presence of HCF-1. Ext, extract.

Narayanan et al. PNAS ͉ June 26, 2007 ͉ vol. 104 ͉ no. 26 ͉ 10837 Downloaded by guest on September 30, 2021 Fig. 5. Sp1 is required to mediate IE62–HCF-1 interaction. HA-IE62 immunoprecipitates (IP-IE62) of HeLa cell extracts were subjected to Western blot analysis with HA (HA-IE62), HCF-1, and Sp1 antisera. (A) HeLa cells were transfected with the HA-IE62 expression plasmid (HA-IE62) or control vector (Ϫ). (B) HeLa cells were transfected with Sp1 RNAi (RNAi-Sp1) or control RNAi Fig. 6. HCF-1–HMT occupancy and chromatin modiﬁcations at the viral IE62 (RNAi-C) and subsequently retransfected with the HA-IE62 expression plas- promoter during VZV infection. BS-C-1 cells were infected with VZV as de- mid. *, HCF-1 background level (compare lanes 4 and 6); **, 78% depletion of scribed. Four hours after infection, ChIP assays were done by using the Sp1 as determined by quantitative Western blot analysis. indicated antisera or control IgG. The signal intensities of the PCR products are represented as percent extract for the promoter (IE62P) and control coding domains (IE62C). Intensities and fold (ϩIE62/ϪIE62) were quantitated as de- as demonstrated by quantitative Western blot analysis (SI Fig. scribed in Materials and Methods. The data shown are derived from a single 10). Thus, recruitment of the coactivator HCF-1 results in Set1- ChIP assay but are representative of at least two independent experiments. and MLL1-dependent H3K4 trimethylation. The results of the real-time PCR are graphed as a percentage of the input sample. H3K4-t, trimethyl-H3K4; Ext, extract. Sp1 Is Required to Bridge/Stabilize the IE62-HCF-1 Interaction. Sp1 is clearly an important component of the IE62-HCF-1-mediated transcriptional induction as shown by the removal of the TATA results indicate that HCF-1-mediated chromatin modulation of proximal Sp1 site, which abrogates stable IE62 binding (SI Fig. the IE gene promoter domain is likely to play a critical role in 8) and by the increased promoter occupancy by Sp1 in the the initiation of the viral infectious cycle. presence of IE62 (Fig. 3). Interestingly, both IE62 and HCF-1 interact directly with Sp1 (12, 29), leading to the possibility that, Discussion in addition to cooperative DNA binding interactions with IE62, Numerous lines of evidence have indicated that HCF-1 is a Sp1 may also function to mediate or stabilize the interaction of cellular coactivator of broad significance. In this study, the VZV IE62 with HCF-1. As shown in Fig. 5A, both endogenous HCF-1 IE62 activator and an IE62-responsive model promoter were and Sp1 were coimmunoprecipitated with IE62 (lane 4). How- used to define the functional role of HCF-1 in transcriptional ever, RNAi-mediated depletion of Sp1 reduced the coimmuno- regulation. As shown in the model depicted in Fig. 7, HCF-1 was precipitation of HCF-1 to background levels (Fig. 5B, compare not required for the recruitment of the RNAPII basal factor lanes 4 and 5 with lane 6). Thus, Sp1 is required to bridge or complex, which depended on the presence of the viral IE62 stabilize the interaction of the activator IE62 with its coactivator activator in concert with the ubiquitous cellular activator Sp1. HCF-1. The ability of IE62 to mediate the formation of this complex is consistent with in vitro studies indicating that IE62 interacts Recruitment of HCF-1 and Chromatin Modifications at the VZV IE62 directly with TBP and TFIIB (26) and enhances the binding of Promoter During VZV Lytic Infection. The data presented here using TBP/TFIID (29). Additionally, the requirement for Sp1 for a model IE62-responsive promoter argue that HCF-1-mediated stable IE62 promoter occupancy underscores the importance of modifications would be represented at the viral IE promoter factors such as Sp1, which has been observed in studies of during the early stages of infection. Therefore, cells were in- IE62-mediated activation (30). However, despite the formation fected with VZV, and4hafterinfection, the occupancy of the of this initiation complex, IE62-Sp1 was unable to stimulate viral IE62 promoter by IE62, Sp1, HCF-1, Set1/MLL1 HMTs, transcription in the absence of HCF-1. and trimethyl-H3K4 was assessed. As shown in Fig. 6, results Recruitment of the coactivator HCF-1 to the model promoter comparable with those obtained by using the transfected model by the IE62-Sp1 activators results in Set1 and MLL1 HMT promoter were found at the viral genomic IE promoter, includ- promoter occupancy, H3K4 trimethylation, and transcriptional ing clear occupancy of IE62, Sp1, HCF-1, Set1, and MLL1. As activation. H3K4 trimethylation is a hallmark of transcriptional anticipated, based on the HCF-1 and Set/MLL occupancy, the activation and probably serves as a signal for the recruitment of promoter exhibited a strong trimethyl-H3K4 signal. In contrast, remodeling and activation components (31–35). Therefore, al- the IE62 coding region did not reveal any significant HCF-1 though HCF-1 may play additional roles, one primary function occupancy or Set1/MLL1-mediated H3K4 trimethylation. of this coactivator is to promote chromatin modifications char- The requirement of HCF-1 in mediating these modifications acteristic of activated transcription. Interestingly, in the absence during a viral infection was similarly addressed by infection of a of the IE62 activator, the model promoter is marked with cell culture that had been partially depleted of HCF-1. In these dimethyl-H3K9 modifications, a repressive mark that precludes cells, a 52% HCF-1 depletion resulted in similar decreases in H3K4 trimethylation. It remains unclear whether the recruit- HCF-1, Set1, MLL1, and trimethyl-H3K4 signals at the viral IE ment of HCF-1/Set1/MLL1 prevents the accumulation of these promoter, whereas no significant change in the promoter occu- marks or whether there are additional components, such as an pancy by the viral IE62 protein was found (data not shown). The H3K9 demethylase, that may also be required for HCF-1-

10838 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0704351104 Narayanan et al. Downloaded by guest on September 30, 2021 viral infections were done by overlaying cell-associated virus on naive BS-C-1 cells. Cells were harvested for ChIP assays4hafter infection.

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 from IE62PR that contained IE62 promoter sequences from Ϫ269 to ϩ73. pCMV-IE62 expresses the IE62 activator under control of the CMV IE promoter and has been previously described (8). For all reporter assays, 4 ϫ 104 HeLa cells were transfected with 300 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, the cells were cotransfected with the reporter constructs and increasing amounts of pCMV-IE62, or control vector. The reporter 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 were normalized by protein concentration and by the activity of the control vector. The data presented are representative of three independent experiments. Fig. 7. Model of HCF-1-dependent IE62-mediated transcriptional activation. 6 The role of HCF-1 in IE62-mediated transcriptional activation is schematically ChIP Assays. Unless otherwise indicated, 1.3 ϫ 10 HeLa cells represented. TBP, TATA binding protein; PI, preinitiation assembly. were transfected with 1 ␮g of the appropriate reporter plasmids (IE62P-61 or IE62P-39) or cotransfected with 2 ␮g of the IE62 expression plasmid (pCMV-IE62). For HCF-1 recruitment, 2 ␮g mediated transcriptional activation. Recent studies have dem- of HA-tagged IE62 (pCMV-HA-IE62) and 4 ␮g of V5-tagged onstrated the cooperative role of these enzymes in transcrip- HCF-1 (pHCF-1-V5) (18) were cotransfected with the IE62P-61 tional activation by removal of repressive H3K9 methylation (36, reporter. For depletion of HCF-1, cells were transfected with 3 37). It would therefore be of interest to determine whether ␮g of pU6-HCF-RNAi or pU6-control RNAi and were retrans- HCF-1 might coordinately recruit an H3K9 demethylase in conjunction with the Set1/MLL1 H3K4 HMTs. fected with reporter and IE62 expression plasmids 48 h later. In this study, the significance of the results obtained by using ChIP assays were done essentially as previously described (39–

the model promoter and IE62 activator is shown by the recruit- 41). Cell lysates were sonicated to obtain DNA fragments BIOCHEMISTRY ϫ 6 ment of HCF-1 and the Set1/MLL1 HMTs to the genomic viral ranging from 300 to 700 bp. Chromatin from 7 10 cells was IE promoter during early infection. The data highlight the used for each immunoprecipitation with the following antibod- biological relevance of chromatin modulation for the initiation ies: IE62 (29); HCF-1 (2); IgG, RNAPII, dimethyl-H3K4, of a herpesvirus infection and complement the study of Huang trimethyl-H3K4, monomethyl-H3K9, and dimethyl-H3K9 (cat- et al. (38), which implicated Set1 as an important regulatory alog nos. 12-370, 05-952, 07-030, 05-745, 07-450, and 07-441, component of the HSV viral lytic cycle. For both HSV and VZV, respectively; Upstate Biotechnology, Lake Placid, NY); Sp1, the IE transactivators are packaged in the virus and released TBP, TFIIB, TFIIH, and HA (catalog nos. SC-59, SC-273, upon initial infection. The requirement for these activators and SC-274, SC-6857/6859, and SC-805, respectively; Santa Cruz their ability to recruit the HCF-1/HMT complexes to the IE Biotechnology, Santa Cruz, CA); MLL1 and Set1 (catalog nos. promoters may be necessary to prevent or circumvent the 1408/1289 and 1193, respectively; Bethyl Laboratories, Mont- assembly of these regions into repressive chromatin. gomery, TX); and Set7/9 (catalog no. 13731; Abcam, Cambridge, Most significantly, ␣-herpesviruses establish latency in sen- MA). Immunoprecipitates were washed and eluted, and the sory neurons. In these cells, the coactivator HCF-1 is uniquely cross-linking was reversed. Recovered DNA was subjected to sequestered in the cytoplasm of the cell but is rapidly relocalized standard PCR with dilutions of input DNA to ensure linearity, to the nucleus upon stimuli that result in viral reactivation. In the resolved in ethidium bromide agarose gels, and the signal model, the viral transactivators are not present during latency, intensities were quantitated by using a 4000MM Image Station and the relocalized HCF-1 is recruited to the viral IE promoters (Kodak, Rochester, NY). The signal intensities of individual by alternative transcription factors that recognize the complex bands were calculated as a percentage of the intensity of the promoter–enhancer domains (9). The data presented here en- input extract after subtraction of the appropriate background rich this model by suggesting that recruitment of HCF-1/HMT antibody controls. Where indicated, samples were also subjected, complexes to the latent viral IE promoters would promote chromatin remodeling and viral IE gene expression. In this in triplicate, to real-time PCR using SYBR Green (Qiagen, respect, the ability of Sp1 to bridge or stabilize the interaction of Valencia, CA) on a Prism 7900 system (Applied Biosystems, HCF-1 with the IE62 activator places new emphasis on this Foster City, CA) and analyzed with SDS 2.2.2 software. The data factor whose cooperative interactions with numerous transcrip- presented are the quantity means. All ChIP data shown are tion factors may be important to a more general targeting/ derived from a single ChIP assay but are representative of at least recruitment of HCF-1. two independent experiments. The location and sequence of primer sets used are shown in SI Fig. 11. Materials and Methods Cell Culture and Virus. HeLa and BS-C-1 cells were maintained Coimmunoprecipitations. HeLa cells (1.3 ϫ 106) were transfected according to standard procedures. VZV (Ellen) was obtained with 5 ␮g of pHA-IE62 expression plasmid or control vector from American Type Culture Collection (Manassas, VA). VZV DNA. For depletion of Sp1, 5 ␮g of pU6-Sp1 vector (Panomics,

Narayanan et al. PNAS ͉ June 26, 2007 ͉ vol. 104 ͉ no. 26 ͉ 10839 Downloaded by guest on September 30, 2021 Redwood City, CA) or control RNAi was transfected on day 1 using Super Signal Dura (Pierce, Rockford, IL), and quantitated and retransfected on day 2 with 2.5 ␮g of pU6-Sp1 or control and by using a Kodak 4000MM Image Station. 48 h later pHA-IE62 expression vector. Extracts were made 48 h later as described in ref. 18, sonicated briefly, and clarified by We thank J. Vogel, Yu Liang G. Kolb, H. Peng, and A. McBride for centrifugation. Protein extract was incubated for2hat4°Cwith discussions and advice; J. Vogel, T. Pierson, P. Sharp, and B. Moss for HA-Sepharose beads, washed five times with binding buffer, critical reading of the manuscript; and members of the Laboratory of Viral Diseases for helpful discussions. This study was supported by the eluted in SDS sample buffer, and resolved in 4–20% Tris-glycine Laboratory of Viral Diseases and the Division of Intramural Research, gels. Western blot analyses of resolved extracts and immuno- National Institute of Allergy and Infectious Diseases, National Institutes precipitates were done with the indicated antibodies (HA, of Health (T.M.K.) and by National Institute of Allergy and Infectious HCF-1 AB2131, and Sp1), developed for chemiluminescence by Diseases Grant AI18449 (to W.T.R.).

1. Kristie TM (2006) in Human Herpesviruses: Biology, Therapy, and Immuno- 22. Wysocka J, Myers MP, Laherty CD, Eisenman RN, Herr W (2003) Genes Dev prophylaxis, eds Arvin A, Campadielli-Fiume G, Roizman B, Whitley R, 17:896–911. Yamanishi K (Cambridge Univ Press, Cambridge, UK), pp 395–452. 23. Yokoyama A, Wang Z, Wysocka J, Sanyal M, Aufiero DJ, Kitabayashi I, Herr 2. Kristie TM, Pomerantz JL, Twomey TC, Parent SA, Sharp PA (1995) J Biol W, Cleary ML (2004) Mol Cell Biol 24:5639–5649. Chem 270:4387–4394. 24. Khurana B, Kristie TM (2004) J Biol Chem 279:33673–33683. 3. Kristie TM, Sharp PA (1993) J Biol Chem 268:6525–6534. 25. Moriuchi H, Moriuchi M, Cohen JI (1995) J Virol 69:4693–4701. 4. Wilson AC, Cleary MA, Lai JS, LaMarco K, Peterson MG, Herr W (1993) Cold 26. Perera LP (2000) J Biol Chem 275:487–496. Spring Harb Symp Quant Biol 58:167–178. 27. Perera LP, Mosca JD, Sadeghi-Zadeh M, Ruyechan WT, Hay J (1992) Virology 5. Wilson AC, LaMarco K, Peterson MG, Herr W (1993) Cell 74:115–125. 191:346–354. 6. Wysocka J, Herr W (2003) Trends Biochem Sci 28:294–304. 28. Ruyechan WT, Peng H, Yang M, Hay J (2003) J Med Virol 70(Suppl 7. Luciano RL, Wilson AC (2002) Proc Natl Acad Sci USA 99:13403–13408. 1):S90–S94. 8. Narayanan A, Nogueira ML, Ruyechan WT, Kristie TM (2005) J Biol Chem 29. Peng H, He H, Hay J, Ruyechan WT (2003) J Biol Chem 278:38068–38075. 280:1369–1375. 30. Ruyechan WT (2006) in Alpha Herpesviruses, ed Sandri-Goldin RM (Caister 9. Kristie TM, Vogel JL, Sears AE (1999) Proc Natl Acad Sci USA 96:1229–1233. Academic, Norfolk, VA), pp 1–20. 10. Delehouzee S, Yoshikawa T, Sawa C, Sawada J, Ito T, Omori M, Wada T, 31. Heintzman ND, Stuart RK, Hon G, Fu Y, Ching CW, Hawkins RD, Barrera Yamaguchi Y, Kabe Y, Handa H (2005) Genes Cells 10:717–731. LO, Van Calcar S, Qu C, Ching KA, et al. (2007) Nat Genet 39:311–318. 11. Freiman RN, Herr W (1997) Genes Dev 11:3122–3127. 32. Kouzarides T (2007) Cell 128:693–705. 12. Gunther M, Laithier M, Brison O (2000) Mol Cell Biochem 210:131–142. 33. Ruthenburg AJ, Allis CD, Wysocka J (2007) Mol Cell 25:15–30. 13. Knez J, Piluso D, Bilan P, Capone JP (2006) Mol Cell Biochem 288:79–90. 34. Sims RJ, III, Chen CF, Santos-Rosa H, Kouzarides T, Patel SS, Reinberg D 14. Lu R, Yang P, Padmakumar S, Misra V (1998) J Virol 72:6291–6297. (2005) J Biol Chem 280:41789–41792. 15. Luciano RL, Wilson AC (2003) J Biol Chem 278:51116–51124. 35. Wysocka J, Swigut T, Xiao H, Milne TA, Kwon SY, Landry J, Kauer M, Tackett 16. Vogel JL, Kristie TM (2000) EMBO J 19:683–690. AJ, Chait BT, Badenhorst P, et al. (2006) Nature 442:86–90. 17. Lin J, Puigserver P, Donovan J, Tarr P, Spiegelman BM (2002) J Biol Chem 36. Metzger E, Wissmann M, Yin N, Muller JM, Schneider R, Peters AH, Gunther 277:1645–1648. T, Buettner R, Schule R (2005) Nature 437:436–439. 18. Vogel JL, Kristie TM (2006) Proc Natl Acad Sci USA 103:6817–6822. 37. Wissmann M, Yin N, Muller JM, Greschik H, Fodor BD, Jenuwein T, Vogler 19. Goto H, Motomura S, Wilson AC, Freiman RN, Nakabeppu Y, Fukushima K, C, Schneider R, Gunther T, Buettner R, et al. (2007) Nat Cell Biol 9:347–353. Fujishima M, Herr W, Nishimoto T (1997) Genes Dev 11:726–737. 38. Huang J, Kent JR, Placek B, Whelan KA, Hollow CM, Zeng PY, Fraser NW, 20. Julien E, Herr W (2003) EMBO J 22:2360–2369. Berger SL (2006) J Virol 80:5740–5746. 21. Guelman S, Suganuma T, Florens L, Swanson SK, Kiesecker CL, Kusch T, 39. Lee TI, Johnstone SE, Young RA (2006) Nat Protocols 1:729–748. Anderson S, Yates JR, III, Washburn MP, Abmayr SM, et al. (2006) Mol Cell 40. Oberley MJ, Tsao J, Yau P, Farnham PJ (2004) Methods Enzymol 376:315–334. Biol 26:871–882. 41. Wells J, Farnham PJ (2002) Methods 26:48–56.

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