Genome-wide siRNA screen identifies SMCX, EP400, and Brd4 as E2-dependent regulators of human papillomavirus oncogene expression

Jennifer A. Smith, Elizabeth A. White, Mathew E. Sowa, Maria L. C. Powell, Matthias Ottinger, J. Wade Harper, and Peter M. Howley1

Department of Pathology, Harvard Medical School, Boston, MA 02115

Contributed by Peter M. Howley, December 22, 2009 (sent for review November 30, 2009) An essentialstepinthe pathogenesis of human papillomavirus(HPV)– or specific amino acid substitutions in the E2 transactivation associated cancers is the dysregulated expression of the viral onco- domain are unable to repress E6/E7 transcription (11, 18, 19). genes. The papillomavirus E2 protein can silence the long control This suggests that E2 silencing involves the recruitment of specific region (LCR) promoter that controls viral E6 and E7 oncogene expres- factors to the LCR. Although the bromodomain protein Brd4 has sion. The mechanisms by which E2 represses oncogene expression been previously implicated in E2-mediated transcriptional re- and the cellular factors through which E2 mediates this silencing are pression of the LCR (20), other work suggests that additional largely unknown. We conducted an unbiased, genome-wide siRNA factors are also involved in E2-mediated repression (21). screen and series of secondary screens that identified 96 cellular To develop a more comprehensive understanding of E2-medi- genes that contribute to the repression of the HPV LCR. In addition ated repression of the E6/E7 promoter, we performed an unbiased, to confirming a role for the E2-binding bromodomain protein Brd4 in genome-wide siRNA screen to identify cellular genes that repress E2-mediated silencing, we identified a number of genes that have not expression from the HPV18 LCR. From more than 21,000 human previously been implicated in E2 repression, including the demethy- genes, the screen identified 96 that are involved in repression of the lase JARID1C/SMCX as well as EP400, a component of the NuA4/TIP60 HPV18 LCR. Although the majority of these cellular genes func-

histone acetyltransferase complex. Each of these genes contributes tion in concert with E2, some act independent of E2 to repress the MICROBIOLOGY independently and additively to E2-mediated silencing, indicating HPV18 LCR. Several Gene Ontology (GO) processes, including that E2 functions through several distinct cellular complexes to re- transcription, chromatin modification and DNA replication, were press E6 and E7 expression. over-represented in the list of 96 repressors. Among these genes, we validated the cellular demethylase JARID1C/SMCX and HPV | transcription | repression | LCR | demethylase EP400, a component of the NuA4/TIP60 histone acetyltransferase complex that was first identified through its ability to bind the apillomaviruses infect squamous epithelial cells and cause a adenovirus E1A protein, as mediators of E2 repression (22–25). In Pvariety of epithelial lesions (1). A subset of the more than 140 addition, we confirmed a role for the E2-binding protein Brd4 in human papillomavirus (HPV) types infect mucosal squamous transcriptional repression (20). Knock down of each of these pro- epithelia, including that of the anogenital tract; these are classified teins individually resulted in partial de-repression of the LCR. Our as either low-risk or high-risk, depending on the lesions associated screen revealed that a single cellular protein or pathway was not with infection. Infections with high-risk HPVs (i.e., HPV16 or sufficient for E2’s full transcriptional repression of the HPV18 HPV18) cause squamous intraepithelial lesions that can progress LCR. Instead, our findings support the hypothesis that E2 recruits to cancer, most notably cervical cancer. HPV is considered the and coordinates the activities of several distinct cellular pathways to major cause of human cervical cancer, the second most common down-regulate the expression of the papillomavirus oncogenes. cancer in women worldwide (2). The viral proteins E6 and E7 account for the oncogenic Results potential of high-risk HPVs in part through their ability to target Genome-Wide siRNA Screen. The initial screen used C33A cells, an and degrade p53 and Rb, respectively (1). The long control region HPV-negative human cervical carcinoma cell line engineered to (LCR) is the upstream enhancer and promoter region that drives stably express FLAG-HA–tagged bovine PV1 (BPV1) E2 from a transcription of E6 and E7. An early step in cervical carcino- bicistronic mRNA that also encodes the IL2 receptor α subunit genesis frequently involves the integration of HPV DNA into (IL2Rα) (26, 27). An E2-repressible reporter in which luciferase cellular chromosomes in a manner that disrupts the E1 and/or E2 expression is controlled by the HPV18 LCR was introduced into ORF (3–6). Because E2 is capable of repressing expression from these cells, and single cell clones were isolated and characterized the LCR, its loss leads to the increased, dysregulated expression of (28). Transfection with siRNAs targeting BPV1 E2 resulted in a the HPV oncogenes E6 and E7 (7–9). Expression of E2 in HPV- reduction of E2 protein below detection levels and a 7- to 11-fold A positive cervical cancer cells causes a growth arrest and sen- increase in luciferase activity (Fig. 1 ). Luciferase activity was not escence (10, 11) due to E2-mediated repression of E6 and E7 affected by control siRNA (siC#1) or siRNA against USP15, a expression (12, 13). The E2 protein consists of an N-terminal cellular protein not involved in E2 repression. The screen pro- transactivation domain and a C-terminal DNA binding and tocol was optimized in C33A/BE2/18LCR c4 cells using E2#3 dimerization domain (14, 15). The E2 proteins from different papillomavirus types are well conserved both functionally and at the amino acid level. The mucosal HPV LCRs contain four E2 Author contributions: J.A.S., E.A.W., and P.M.H. designed research; J.A.S., E.A.W., M.E.S., and M.L.C.P. performed research; J.A.S., M.E.S., M.O., and J.W.H. contributed new re- binding sites (E2BS) and it has been hypothesized that E2 binding agents/analytic tools; J.A.S., E.A.W., M.E.S., and P.M.H. analyzed data; and J.A.S., E.A.W., to the two promoter proximal binding sites within the LCRs and P.M.H. wrote the paper. competes with the binding of cellular transcription factors Sp1 The authors declare no conflict of interest. and TBP at the promoter (16, 17). Yet, this steric hindrance 1To whom correspondence should be addressed. E-mail: [email protected]. model cannot fully explain E2-mediated transcriptional silencing; This article contains supporting information online at www.pnas.org/cgi/content/full/ forms of E2 that can still bind the promoter but contain deletions 0914818107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0914818107 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 C33A/BE2/18LCR c4 C33A/16E2/18LCR c1 Additional Secondary Screens Identified 96 Cellular Genes that A C 30000 25000 Contribute to Repression of HPV18 LCR. Our primary screen used 20000 25000 C33A cells that expressed BPV1 E2 because repression of the 20000 15000 HPV18 LCR by BPV1 E2 has been well studied (7, 9, 17). To 15000 10000 10000 identify cellular proteins that contribute to the repression me- 5000 diated by the E2 protein from a high-risk HPV type, we deter- RLU/[Protein] 5000 RLU/[Protein] mined which of these 231 hits were also involved in HPV16 E2- 0 0 siRNA siRNA mediated repression of the HPV18 LCR. We generated the Mock siC#1 Mock siC#1

USP15 C33A/16E2/18LCR c1 clonal cell line to conduct this screen. No BE2#1 BE2#3 USP15 16E2#1 16E2#2 BPV1 E2 HPV16 E2 significant change in luciferase activity was observed with siC#1 * USP15 C USP15 or siRNA against USP15 (Fig. 1 ). However, transfection with Actin siRNAs against HPV16 E2 resulted in a dramatic decrease in Actin HPV16 E2 protein levels and a 13- to 17-fold increase in luci-

12 ferase activity. To perform the secondary screen, we transfected B 10 C33A/16E2/18LCR c1 cells with each of the four individual 8 duplexes against the 231 hits and measured luciferase activity 6 72 h post-transfection. Based on these results (Dataset S2), the 4 list of candidate genes was reduced to 141 that scored in both 2 0 BPV1 E2 and HPV16 E2 repression screens. Z-score -2 To identify cellular genes that scored as hits because they either -4 directly or indirectly decreased E2 protein levels, we performed a -6 quantitative in-cell western analysis. C33A/BE2/18LCR c4 cells -8 SMARTpools transfected with individual siRNAs were fixed, permeabilized, and stained to measure E2 expression at 72 h post-transfection. Fig. 1. Genome-wide siRNA screen to identify cellular proteins involved in E2- An experimental plate from this analysis is displayed in Fig. S2. mediated repression of LCR. (A) Characterization of C33A/BE2/18LCR c4 cell line. Cells were stained with Alexa680-succinimidyl ester to label Cells were mock-transfected or transfected with the indicated siRNAs at a final lysines within cells for normalization (red channel, Fig. S2A) and concentration of 20 nM. Cell extracts were harvested 72 h post-transfection immunostained for HA to detect E2 protein (green channel, Fig. to determine relative luciferase units (RLU) (Top) or indicated protein levels B C (Bottom). The experiment was performed in triplicate. Bars indicate mean RLU S2 ). Figure S2 is a composite image of S2A and S2B. Twenty normalized to protein concentration ± SD; representative Western blots are genes negatively affected E2 expression and/or stability because shown. (B) Primary screen z-score distribution of all SMARTpools analyzed. Each their siRNA-mediated knock down led to decreased E2 protein Dharmacon SMARTpool library plate was screened in triplicate with data point levels (Dataset S2). One of these genes was IL2Rα. As the E2 and displaying the mean ± SD. Area boxed in red indicates the 511 SMARTpools with IL2Rα proteins were translated from the same mRNA via an an average z-score ≥2. (C) C33A/16E2/18LCR c1 cell line was characterized as IRES in the bicistronic reporter, this was an expected result that described in A. Bars illustrate the mean of three experiments ± SD. Asterisk validated the assay. Because decreased E2 levels could result in a fi indicates a nonspeci c band in the HA blot for HPV16 E2. de-repression of transcription from the HPV18 LCR, we elimi- nated these candidates from further analysis. Based on manual examination of the candidate list, we excluded an additional 25 siRNA as the positive control and USP15 siRNA as the negative cellular genes because only one of the four duplexes consistently control. The screen, represented in Fig. S1A, was performed with scored positive (15/25) or the intended target of the duplexes (10/ the Dharmacon human siGENOME SMARTpool library of 25) was unclear (i.e., Entrez Gene ID was discontinued, pre- 21,121 pools. Each pool comprises four independent siRNA du- dicted, hypothetical, or inferred). plexes against the target gene. Luciferase activity was measured Next, we determined which of the remaining cellular genes 72 h post-transfection, and z-scores from three replicate experi- repressed the HPV18 LCR in an E2-dependent manner and which ments for each SMARTpool were averaged and standard devia- were capable of repressing transcription from the HPV18 LCR in tions (SD) determined. A SMARTpool was considered a hit if the the absence of E2. We generated a clonal C33A cell line (C33A/ z-score was ≥2. The hit rate in the primary screen was 2.4%. Of B, 18LCR c4) that stably expressed the HPV18 LCR-luciferase the 511 hits (Dataset S1), indicated by the red box in Fig. 1 400 reporter in the absence of any papillomavirus E2 protein. Transient were classified as weak (z-scores ≥2 and <3); 102 as moderate (z- fi ≥ < ≥ transfection of a BPV1 E2 expression construct con rmed that the scores 3 and 5); and nine as strong hits (z-scores 5). Because HPV18 LCR in these cells could be repressed 3-fold by E2 (Fig. the screen measured an increase in luciferase activity and positive S3A). Introduction of siRNA against luciferase mRNA suppressed z-scores, we did not account for SMARTpools that decreased cell luciferase activity 6-fold (Fig. S3B), and luciferase levels were viability or negatively affected cellular proliferation. Thus this unaffected by siC#1 or USP15 siRNA. We introduced individual screen would have missed cellular genes that could be involved in siRNA duplexes against the 96 remaining candidate genes into repression of the HPV18 LCR, but, when knocked down, reduced these cells, and determined that 58 of the 96 were dependent on E2 cell viability. To validate hits from the genome-wide siRNA for HVP18 LCR repression and 34 functioned in an E2-inde- screen, a deconvolution secondary screen was performed in which pendent manner. The E2-dependence of four cellular proteins the four duplexes from each of the 511 SMARTpool hits were remained unclear from this analysis (Dataset S3). individually screened at a final concentration of 20 nM (Fig. S1B). Of 22 broad GO processes, the 96 cellular genes identified were From the 511 primary hits, 391 (77%) validated with at least one significantly enriched in five GO processes at a P value < 0.01 siRNA duplex, whereas 120 initial hits failed to validate with any (Table S1). Because our screen was designed to identify cellular of the individual siRNAs (Dataset S1). The majority of these proteins that play a role in transcriptional repression of the HPV18 nonvalidated targets (109 of 120) had been classified as weak in LCR, it was not surprising to find enrichment in biosynthesis, cell the primary screen. This level of nonconcordance is similar to that cycle, chromatin modification, DNA replication, and transcription. observed in other high-throughput screens using the Dharmacon library (29, 30). A total of 231 cellular genes were selected for Validation of E2-Dependent Hits in Repression of HPV18 LCR Using a further analysis (130 that confirmed with two or more duplexes, Non–High-Throughput Format. From the 58 E2-dependent hits, we and 101 that confirmed with one duplex and a z-score ≥5). focused our validation studies on three cellular proteins, namely,

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.0914818107 Smith et al. Downloaded by guest on September 26, 2021 Brd4, EP400, and SMCX. The SMARTpools against each of luciferase activity when compared with C33A/16E2/18LCR c1 these three proteins were strong hits in the primary screen and all cells transfected with siC#1. Taken together, these experiments four individual duplexes scored positive in the secondary screens validated the high-throughput screens and indicate that a number with either BPV1 E2 or HPV16 E2. EP400, a known component of distinct cellular proteins and pathways contribute to E2- of multiple histone acetylase complexes, was of interest because it mediated transcriptional repression of the HPV18 LCR. has been described to interact with viral proteins from adenovirus and SV40 and was found to interact with five other E2-dependent Brd4, SMCX, and EP400 Each Contribute to E2-Mediated Transcriptional hits identified in our screen (Fig. S4) (24, 25, 31, 32). SMCX was Repression of Endogenous HPV18 LCR in HeLa Cells. HeLa cells are an selected based on its role as a histone demethylase associated with HPV18-positive epithelial cell line originally derived from a cer- transcriptional repression (22, 23, 33). The E2-interacting bro- vical cancer in which E6 and E7 are expressed from the LCR and modomain protein Brd4 was selected for further analysis, as it has the E2 ORF has been disrupted (4). The continued expression of previously been implicated in E2-dependent transcriptional re- E6 and E7 is required for cell growth. Exogenous expression of E2 in HeLa cells arrests growth, inducing apoptosis and/or senescence pression (20). To validate cellular genes identified in the high- through repression of E6 and E7 transcription (7, 10, 11, 13, 34). throughput screens, individual siRNA duplexes were tested for We asked whether the cellular genes that we had identified (i.e., their effect on luciferase activity in C33A/16E2/18LCR c1 cells in Brd4, EP400, and SMCX) contributed to the ability of E2 to re- a non–high-throughput format. As shown in Fig. 2, each of the fi press transcription from the endogenous HPV18 LCR in HeLa four siRNA duplexes against Brd4, EP400, or SMCX ef ciently cells. To avoid confounding effects because of E2-induced sen- reduced target protein levels. The siRNA-mediated knock down A escence, we used HeLa/16E6/16E7 cells for these experiments. of Brd4 increased HPV16 E2 protein levels (Fig. 2 ), which is These HeLa cells had been engineered to express HPV16 E6 and consistent with data from the in-cell western analysis in which HPV16 E7 from retrovirus promoters that are not subject to E2- Brd4 knock down increased BPV1 E2 protein expression (Dataset mediated repression (35). S2). Z-scores from the screens correlated with the luciferase HeLa/16E6/16E7 cells stably transduced with an expression activity measured in the individual assays for all three genes. plasmid for HPV16 E2 or the control plasmid pOZN were trans- Those duplexes with the greatest z-scores in the screens caused fected with the indicated siRNAs and harvested 72 h post- the largest increase in luciferase activity in the non–high- transfection. The level of transcription from the HPV18 LCR was throughput experiments. Several other siRNA duplexes directed determined by quantitative PCR using primers specific for HPV18

against E2-dependent hits (BRD8, CAPN1, CD2BP2, DEFA3, E7 mRNA and normalized to the housekeeping transcript G6PD. MICROBIOLOGY ENPP7, ENY2, EPC, LRP1, PEX14, PPIB, PRAP1, SKP1A) There was a 4-fold decrease in HPV18 E7 mRNA in the HPV16 were further analyzed in a non–high-throughput format (Fig. S5). E2-expressing cells compared with those with the control plasmid All but one of these siRNAs caused at least a 2-fold increase in pOZN, indicating that, as expected, HPV16 E2 repressed the endogenous HPV18 LCR. E2-mediated repression was alleviated by siRNA targeting HPV16 E2 (Fig. 3A). HPV18 E7 mRNA levels also increased in the cells that received siRNAs targeting Brd4, 25000 A 25000 B SMCX, or EP400, demonstrating that these cellular proteins con- 20000 20000 15000 15000 10000 10000 HeLa/16E6/16E7/16E2 HeLa/16E6/16E7/pOZN 5000 5000 A B RLU/[Protein] RLU/[Protein] 0 0 7 7 siRNA siRNA 6 6 siC#1 siC#1 5 5 16E2#2 16E2#2 Brd4-02 Brd4-03 Brd4-04 Brd4-05 4 4 Brd4 EP400-01 EP400-02 EP400-03 EP400-04 EP400 3 3 2 2 HPV16 E2 HPV16 E2 * 1 1 * Actin 0 0 Relative HPV18 E7 mRNA Relative HPV18 E7 mRNA Actin Brd4 Brd4

siRNA siC#1 siRNA siC#1 SMCX SMCX EP400 EP400 16E2#1 16E2#1 25000 C HeLa/16E6/16E7/16E2 HeLa/16E6/16E7/pOZN 20000 C D 15000 siRNA siRNA siC#1 16E2#1 Brd4 SMCX EP400 10000 siC#1 16E2#1 Brd4 SMCX EP400 HPV16 E2 HPV16 E2 5000 RLU/[Protein] Brd4 Brd4 0 siRNA SMCX SMCX siC#1

16E2#2 EP400 EP400 SMCX-01 SMCX-02 SMCX-03 SMCX-04 SMCX Actin Actin HPV16 E2 * Actin Fig. 3. Brd4, EP400, and SMCX are E2-dependent corepressors of the HPV18 oncogenes in HeLa cells. HeLa/16E6/16E7/16E2 (A and C) or HeLa/16E6/16E7/ Fig. 2. Brd4, EP400, and SMCX contribute to E2-mediated transcriptional pOZN cells (B and D) were transfected with the indicated siRNAs at a final repression of the HPV18 LCR. C33A/16E2/18LCR c1 cells were transfected with concentration of 50 nM and cells were harvested at 72 h post-transfection. the indicated siRNAs at 20 nM. Cell extracts were harvested 72 h post- (A and B) HPV18 E7 transcript levels, normalized to G6PD transcript levels, transfection to determine RLU, as well as HPV16 E2 (HA), actin, Brd4 (A), EP400 were determined by qRT-PCR. Each siRNA transfection was performed at (B), and SMCX (C) protein levels. Experiments were performed in triplicate, least three times; bars indicate the mean RNA level ± SD relative to values with each bar representing the average ± SD and representative immunoblots obtained in siC#1-transfected cells. (C and D) Cell extracts were separated by displayed. The long and short isoforms of Brd4 are indicated by the lines in A. SDS/PAGE and E2, Brd4, SMCX, EP400, and actin protein levels were visual- Asterisk indicates a nonspecific band in the HA blots for HPV16 E2. ized by immunoblot analysis. Representative immunoblots are displayed.

Smith et al. PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 tribute to repression of the endogenous HPV18 LCR in HeLa cells. 45000 HPV18 E7 mRNA levels remained largely unchanged in the sim- 40000 ilarly treated control HeLa/16E6/16E7/pOZN cells (Fig. 3B). We 35000 did observe a modest 2-fold effect with the siRNA duplex to SMCX 30000 in the control HeLa/16E6/16E7/pOZN cells, suggesting that 25000 SMCX may have E2-dependent as well as E2-independent roles in 20000 repression of the HPV18 LCR in HeLa cells. This differs from the 15000 RLU/[Protein] results in C33A cells where the siRNA duplex to SMCX did not 10000 affect transcription from the HPV18 LCR luciferase reporter in 5000 C33A/18LCR c4 cells (Dataset S3), no doubt reflecting some level 0 of cell line variability. Knock-down efficiency of the intended tar- gets was confirmed by Western blot analysis (Fig. 3 C and D). Taken siRNA fi Brd4/siC#1

together, these results further validate the genes identi ed in the Brd4 10 nM Brd4 40 nM Brd4/SMCX Brd4/EP400 siC#1 40 nM siC#1 10 nM SMCX/siC#1 EP400/siC#1 Brd4/GAPDH SMCX 10 nM SMCX 40 nM EP400 10 nM EP400 40 nM EP400/SMCX 16E2#2 10 nM GAPDH 10 nM GAPDH 40 nM SMCX/GAPDH high-throughput screens and indicate that Brd4, SMCX, and EP400 EP400/GAPDH

each contribute to the ability of HPV16 E2 to inhibit transcription Brd4/EP400/SMCX from the endogenous HPV18 LCR in HeLa cells. Brd4/EP400/SMCX/siC#1 Brd4/EP400/SMCX/GAPDH Brd4, SMCX, and EP400 Have Additive Effects on E2-Dependent Transcriptional Repression of HPV18 LCR. The genome-wide siRNA HPV16 E2 screen and subsequent secondary screens illustrated that E2- mediated repression of the HPV18 LCR involves several cellular GAPDH proteins. Because knock down of any individual cellular protein did not decrease E2-mediated repression to the same extent as the Brd4 knock down of either BPV1 E2 or HPV16 E2 in C33A cells, we hypothesized that E2-mediated repression was not mediated by a single cellular protein. To determine whether Brd4, SMCX and EP400 EP400 have additive effects on E2-mediated repression, siRNAs were transfected into C33A/16E2/18LCR c1 cells singly or in SMCX combination and luciferase levels determined 72 h post-transfection Actin (Fig. 4). Neither siC#1 or GAPDH siRNA changed luciferase activity. In contrast, there was an 18-fold increase in luciferase Fig. 4. Brd4, EP400, and SMCX contribute additively to E2-mediated tran- activity when cells were transfected with 16E2#2. Consistent with scriptional repression. C33A/16E2/18LCR c1 cells were transfected with the fi previous experiments, transfection with siRNAs against Brd4, speci ed siRNAs at the concentrations indicated or a concentration of 10 nM/ fi siRNA duplex in the combined transfection assays. After 72 h, cell extracts EP400, or SMCX signi cantly increased luciferase activity but not were harvested, RLU determined and protein expression examined by to the same extent as 16E2#2 siRNA. Varying the final siRNA Western blot. The experiment shown was performed in triplicate and corre- concentration did not alter the level of knock down detectable by sponding immunoblots are displayed. Each bar indicates the mean ± SD. Western blot analysis, nor did it significantly affect the change in luciferase activity observed. When Brd4, EP400 and SMCX siRNAs were transfected into C33A/16E2/18LCR c1 cells in pair-wise BPV1 E2 and HPV16 E2 Bind SMCX and EP400. Because Brd4, EP400, combinations, the effect on luciferase activity was greater than that and SMCX contribute to E2-dependent repression of the HPV18 achieved with any of the siRNAs individually. The increase in LCR and an interaction between E2 and Brd4 is well established luciferase levels in Brd4/EP400 combined or Brd4/SMCX com- (27, 36), we asked whether SMCX and/or EP400 complex with E2. bined siRNA-transfected cells was comparable to that observed C33A cells were transfected with HA-tagged BPV1 E2 alone or in with HPV16 E2 siRNA alone. When any of these three duplexes combination with FLAG-tagged SMCX. Cell lysates were harvested was individually paired with siC#1 or GAPDH siRNA, luciferase 48 h post-transfection, and proteins were immunoprecipitated with anti-HA or anti-SMCX antibodies. Input and precipitated proteins levels were similar to those observed in cells transfected with Brd4, were separated by SDS/PAGE and visualized by immunoblot anal- EP400 or SMCX siRNA alone. Luciferase activity, and hence the ysis. Immunoprecipitation with an anti-HA antibody efficiently decrease in E2-mediated transcriptional repression, was greatest in pulled down the HA-tagged BPV1 E2 protein (Fig. 5A) as well as C33A/16E2/18LCR c1 cells simultaneously transfected with Brd4, Brd4, as expected. Some SMCX also immunoprecipitated with EP400, and SMCX siRNAs. Adding siC#1 or GAPDH siRNA did BPV1 E2, indicating that these proteins are present in a complex, not further enhance luciferase levels, confirming that the changes in fi perhaps together with other cellular proteins. SMCX was not luciferase levels are caused by the knock down of these speci c detected in the immunoprecipitates from cells transfected with the proteins. We speculate that the alleviation of E2-mediated re- control plasmid pOZN, indicating that the coimmunoprecipitation pression in cells simultaneously transfected with siRNAs against of SMCX and BPV1 E2 is specific. These results were confirmed in a Brd4, EP400, and SMCX is greater than in those transfected with reciprocal immunoprecipitation of SMCX where the presence of siRNA against HPV16 E2 because E2 levels are reduced, but BPV1 E2 was detected in cells cotransfected with both BPV1 E2 and not eliminated, by siRNA knock down and a certain level of E2- SMCX (Fig. 5B). No E2 was detected in lysates immunoprecipitated mediated repression likely remains in siRNA-treated cells. It is with control IgG. An interaction between HPV16 E2 and SMCX possible that decreasing the expression of three of the major ef- was detected in immunoprecipitates from C33A cells cotransfected fectors to E2-mediated repression results in a more dramatic phe- with FLAG-tagged HPV16 E2 and HA-tagged SMCX (Fig. S6A). notype than reduction of E2 levels alone. Western blot analysis To determine whether E2 is able to form a complex with EP400, confirmed that each siRNA efficiently knocked down its target C33A cells were transfected with HA-tagged BPV1 E2 and FLAG- protein, even when added to cells in combination (Fig. 4). The tagged EP400 (Fig. 5C). Cell lysates were harvested 48 h post- additive effect of knocking down Brd4, EP400, and SMCX suggests transfection. Immunoprecipitation with an anti-HA antibody effi- that E2 recruits multiple proteins to the HPV18 LCR to mediate ciently pulled down the HA-tagged BPV1 E2, as well as EP400, repression of its oncogenes. indicating E2 and EP400 are present in the same complex. EP400

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.0914818107 Smith et al. Downloaded by guest on September 26, 2021 HA (BPV1 E2) IP HA (BPV1 E2) IP acetyl transferase component of the NuA4/TIP60 complex, spe- A _ C _ fi SMCX +_ + BPV1 E2 _+ + ci cally acetylates histone H2A on K5, K14 of histone H3, as well as BPV1 E2 ++ EP400 ++ K5, K8, K12, and K16 of histone H4 (38). These are the same acetylated lysine residues that Brd4 binds preferentially during Bound Bound Input Input Bound Input Input Input Bound Input Bound Bound interphase and mitosis (39). Evidence suggests that histone acety- BPV1 E2 BPV1 E2 lation may function as a recruitment signal for proteins involved in Brd4 EP400 E2-mediated repression of the LCR (20); it is reasonable to spec- Actin ulate that one role of the NuA4/TIP60 complex in E2-mediated SMCX transcriptional repression may be to facilitate Brd4 recruitment to Actin the HPV18 LCR. Although the NuA4/TIP60 complex is frequently associated SMCX IP IgG IP with transcriptional activation, EP400 has been shown to func- B _ _ _ SMCX _ +++_ _ +_ + tion as a transcriptional repressor, at least partly by antagonizing BPV1 E2 + + ++ TIP60 function (40–42). EP400 represses specific subsets of cellular genes in an adenovirus E1A-dependent manner (43, 44). Bound Bound Input Bound Input Bound Input Bound Input Input Bound Input Input Bound Input Bound Here we show that E2 and EP400 coimmunoprecipitate with one SMCX another (Fig. 5C and Fig. S6B). We propose that the papil- lomavirus E2 protein may alter EP400 complex formation and BPV1 E2 direct EP400 as well as other components of the NuA4/TIP60 Actin complex to novel targets within an infected cell. Relocation of these proteins to the HPV18 LCR could result in transcriptional Fig. 5. SMCX and EP400 form a complex with E2. (A and B) C33A cells were inhibition of the E6 and E7 oncogenes. transfected with expression plasmids for HA-BPV1 E2, FLAG-SMCX, or the The second protein selected for more detailed study was corresponding parental plasmids. At 48 h post-transfection, cells were har- SMCX, also known as JARID1C. SMCX possesses demethylase vested and proteins immunoprecipitated with the indicated antibodies (HA, activity against tri- and di-methylated H3K4 (22, 23). The asso- SMCX, IgG). Total cellular proteins and bound proteins were separated by ciation of SMCX with DNA-binding proteins, including REST, is SDS/PAGE and visualized by immunoblot analysis using antibodies against HA (detection of BPV1 E2), SMCX, Brd4, and actin. (C) C33A cells were thought to contribute to the transcriptional repression of other

transfected with expression plasmids for HA-BPV1 E2, FLAG-EP400, or the genes (23). In addition, SMCX inhibits the transcriptional activity MICROBIOLOGY corresponding control plasmids and cell extracts were harvested 48 h post- of Smad3 in a manner independent of its enzymatic activity (33). transfection. Proteins were immunoprecipitated and visualized as described Here, we show that this demethylase and transcriptional repressor in A, with an anti-HA antibody used for detection of BPV1 E2 and an anti- is involved in E2-mediated repression of the papillomavirus EP400 antibody used for detection of EP400. oncogenes and establish that E2 and SMCX are found in the same complex (Fig. 5 A and B and Fig. S6A). We hypothesize that E2 recruits SMCX to the HPV18 LCR to maintain a transcriptionally also coimmunoprecipitated with HPV16 E2 in cotransfected cells inactive promoter. In support of this idea, specific nucleosome B (Fig. S6 ). We conclude that E2 forms a complex or complexes with structures along the HPV16 and HPV18 LCRs have been shown SMCX and EP400, and note that these interactions may be direct or to play a role in E6 and E7 repression (45). require additional cellular proteins. Finally, our third target for further investigation was the E2- interacting protein Brd4, which has previously been implicated in Discussion several aspects of the papillomavirus replication cycle —initially in To investigate the mechanisms by which the papillomavirus E2 E2-mediated genome maintenance and subsequently in E2’stran- protein controls expression of E6 and E7, we performed an un- scriptional activation function (27, 46). Its role in E2-mediated biased, genome-wide siRNA screen to identify cellular genes that transcriptional repression was first documented in biochemical fi contribute to repression of the LCR promoter. We identi ed 511 studies by Wu et al., which indicated that Brd4 was necessary and SMARTpools that, when introduced into cells, resulted in de- sufficient for E2-mediated repression of the HPV11 LCR (20). B repression of the HPV18 LCR (Fig. 1 and Dataset S1). We then Previous work from our laboratory suggested that there are Brd4- performed a series of secondary screens to focus on genes whose independent mechanisms for E2-mediated transcriptional re- involvement in transcriptional repression was conserved among pression (21). In these studies, neither shRNA-mediated knock different E2 proteins and did not decrease E2 protein levels down of Brd4 nor expression of the C-terminal domain (CTD) of (Dataset S2). This left 96 cellular genes that contribute, either Brd4 restored LCR activity in the presence of E2. In the present directly or indirectly, to repression of the HPV18 LCR. Using study, we confirm a role for Brd4 in repression of the HPV onco- C33A/18LCR cells not expressing any papillomavirus E2 protein, genes and show that it is one of several cellular proteins necessary we determined that 58 genes contributed to repression of the for E2-mediated transcriptional repression. Although the siRNA- HPV18 LCR in an E2-dependent manner and that 34 functioned mediated knock down of Brd4 resulted in a partial alleviation of in the absence of E2; the E2-dependence of 4 of the candidate repression of the HPV18 LCR, it did not abolish E2-mediated genes remained unclear (Dataset S3). repression. Taken together, these studies suggest Brd4-dependent We focused our subsequent studies on three E2-dependent cel- as well as Brd4-independent mechanisms of E2-mediated trans- lular genes, Brd4, EP400, and SMCX. A protein interactome con- criptional repression. sisting of the E2-dependent candidate corepressors suggested a There are two reasons that may explain why the previous study central role for EP400 in E2-mediated transcriptional repression from our laboratory did not identify a Brd4-dependent con- (Fig. S4). A member of the SWI2/SNF2 family of chromatin- tribution to E2-mediated transcriptional repression (21). First, remodeling proteins, EP400 was first identified through its inter- the shRNA-mediated knock down used in the former experi- action with adenovirus E1A and was subsequently shown to bind ments was not as effective at knocking down Brd4 when com- SV40 large T antigen (25, 31, 32). EP400 is a component of the pared with the Brd4 siRNAs used in this study. Second, the NuA4/TIP60 histone acetylase complex (24, 25, 37). Three other Brd4-CTD has been shown to function in a dominant negative members of NuA4 (i.e., BRD8, EPC1, and YEATS/GAS41) were manner in other E2-mediated functions, and the former study hits in our primary screen, indicating that this complex is involved in assumed that it would also function as a dominant negative if E2-mediated repression of the HPV18 LCR. TIP60, the histone Brd4 contributed to E2-mediated transcriptional repression. We

Smith et al. PNAS Early Edition | 5of6 Downloaded by guest on September 26, 2021 now believe that the Brd4-CTD does not function as a dominant therapeutic approaches for HPV-associated malignant and pre- negative for Brd4’s role in E2-mediated repression of the HPV18 malignant lesions. LCR, and this is supported by the recent work of Yan et al. (47). Our study establishes that the papillomavirus E2 protein uses Experimental Methods multiple cellular proteins to inhibit expression of its oncogenes. Please refer to SI Text for detailed explanations of experimental methods, Several of these control transcription through histone mod- including the following: cell culture, cell lines and DNA plasmids; siRNAs and ification. Network analysis provides evidence for interactions transfections; quantitation of luciferase activity; analysis of protein expres- among the top 96 cellular candidates that we identified, perhaps sion; siRNA screens; quantitative in-cell Western analysis; protein–protein best reflected by the finding that four members of the NuA4/TIP60 interaction network analysis; gene ontology analysis; quantitative real time- histone acetylase complex are involved in E2-mediated repression. PCR; and transient DNA transfections and immunoprecipitations. Although this study has focused on the role of E2 in regulating HPV18 LCR transcriptional activity in C33A and HeLa human ACKNOWLEDGMENTS. We thank S. Iwase and Y. Shi (Harvard Medical School) cervical cancer cell lines, we plan to extend these studies to normal for providing anti-SMCX antibody as well as SMCX expression constructs, D. human squamous epithelial cells and to examine the role of these DiMaio (Yale University) for HeLa/16E6/16E7 cells, and D. Livingston (Harvard proteins in E2’s functions in the context of virus/host cell inter- Medical School) for the EP400 expression construct. We are grateful to mem- bers of our laboratories, as well as to B. Gewurz, G. Hoffman, C. Shamu, D. actions during the course of infection and epithelial cell differ- Wrobel, S. Rudnicki, S. Johnston, and N. Moerke for helpful discussions and entiation. It will also be informative to determine whether these feedback on various aspects of this project. This work was supported by proteins affect other parts of the papillomavirus replication cycle. National Institutes of Health Grants T32CA009361 (to J.A.S. and M.L.C.P.), A deeper understanding of the cellular proteins and pathways individual National Research Service Award F32AI080075 (to E.A.W.), involved in silencing the expression of the papillomavirus E6 and AG011085 and GM054137 (to J.W.H.), and R01CA116720 and P01CA050661 E7 oncogenes will provide new insights into the development of (to P.M.H.).

1. Howley PM, Lowy DR (2007) Papillomaviruses. Fields Virology, eds Knipe DM, 25. Fuchs M, et al. (2001) The p400 complex is an essential E1A transformation target. Cell Howley PM (Lippincott, Williams &Wilkins, Philadelphia), 5th Ed, pp 2299–2354. 106:297–307. 2. zur Hausen H (2009) Papillomaviruses in the causation of human cancers—a brief 26. Yee C, Krishnan-Hewlett I, Baker CC, Schlegel R, Howley PM (1985) Presence and historical account. Virology 384:260–265. expression of human papillomavirus sequences in human cervical carcinoma cell lines. 3. Dürst M, Kleinheinz A, Hotz M, Gissmann L (1985) The physical state of human papillomavirus Am J Pathol 119:361–366. type 16 DNA in benign and malignant genital tumours. JGenVirol66:1515–1522. 27. You J, Croyle JL, Nishimura A, Ozato K, Howley PM (2004) Interaction of the bovine 4. Schwarz E, et al. (1985) Structure and transcription of human papillomavirus sequences papillomavirus E2 protein with Brd4 tethers the viral DNA to host mitotic chromosomes. in cervical carcinoma cells. Nature 314:111–114. Cell 117:349–360. 5. Schneider-Gädicke A, Schwarz E (1986) Different human cervical carcinoma cell lines 28. Ottinger M, et al. (2009) Cell-type specific transcriptional activities among different show similar transcription patterns of human papillomavirus type 18 early genes. papillomavirus long control regions and their regulation by E2. Virology 395:161–171. EMBO J 5:2285–2292. 29. Brass AL, et al. (2008) Identification of host proteins required for HIV infection 6. Baker CC, et al. (1987) Structural and transcriptional analysis of human papillomavirus through a functional genomic screen. Science 319:921–926. type 16 sequences in cervical carcinoma cell lines. J Virol 61:962–971. 30. Simpson KJ, et al. (2008) Identification of genes that regulate epithelial cell migration 7. Thierry F, Yaniv M (1987) The BPV1-E2 trans-acting protein can be either an activator using an siRNA screening approach. Nat Cell Biol 10:1027–1038. or a repressor of the HPV18 regulatory region. EMBO J 6:3391–3397. 31. Howe JA, Bayley ST (1992) Effects of Ad5 E1A mutant viruses on the cell cycle in 8. Bernard BA, et al. (1989) The human papillomavirus type 18 (HPV18) E2 gene product relation to the binding of cellular proteins including the retinoblastoma protein and is a repressor of the HPV18 regulatory region in human keratinocytes. J Virol 63: cyclin A. Virology 186:15–24. 4317–4324. 32. Lill NL, Tevethia MJ, Eckner R, Livingston DM, Modjtahedi N (1997) p300 Family 9. Thierry F, Howley PM (1991) Functional analysis of E2-mediated repression of the members associate with the carboxyl terminus of simian virus 40 large tumor antigen. HPV18 P105 promoter. New Biol 3:90–100. J Virol 71:129–137. 10. Hwang ES, et al. (1993) Inhibition of cervical carcinoma cell line proliferation by the 33. Kim TD, Shin S, Janknecht R (2008) Repression of Smad3 activity by histone introduction of a bovine papillomavirus regulatory gene. J Virol 67:3720–3729. demethylase SMCX/JARID1C. Biochem Biophys Res Commun 366:563–567. 11. Dowhanick JJ, McBride AA, Howley PM (1995) Suppression of cellular proliferation by 34. DeFilippis RA, Goodwin EC, Wu L, DiMaio D (2003) Endogenous human papillomavirus the papillomavirus E2 protein. J Virol 69:7791–7799. E6 and E7 proteins differentially regulate proliferation, senescence, and apoptosis in 12. Desaintes C, Demeret C, Goyat S, Yaniv M, Thierry F (1997) Expression of the HeLa cervical carcinoma cells. JVirol77:1551–1563. papillomavirus E2 protein in HeLa cells leads to apoptosis. EMBO J 16:504–514. 35. Psyrri A, et al. (2004) Role of the retinoblastoma pathway in senescence triggered by 13. Francis DA, Schmid SI, Howley PM (2000) Repression of the integrated papillomavirus repression of the human papillomavirus E7 protein in cervical carcinoma cells. Cancer E6/E7 promoter is required for growth suppression of cervical cancer cells. J Virol 74: Res 64:3079–3086. 2679–2686. 36. Abbate EA, Voitenleitner C, Botchan MR (2006) Structure of the papillomavirus DNA- 14. Giri I, Yaniv M (1988) Structural and mutational analysis of E2 trans-activating proteins tethering complex E2:Brd4 and a peptide that ablates HPV chromosomal association. of papillomaviruses reveals three distinct functional domains. EMBO J 7:2823–2829. Mol Cell 24:877–889. 15. McBride AA, Romanczuk H, Howley PM (1991) The papillomavirus E2 regulatory 37. Ikura T, et al. (2000) Involvement of the TIP60 histone acetylase complex in DNA proteins. J Biol Chem 266:18411–18414. repair and apoptosis. Cell 102:463–473. 16. Dong G, Broker TR, Chow LT (1994) Human papillomavirus type 11 E2 proteins repress 38. Kimura A, Horikoshi M (1998) Tip60 acetylates six lysines of a specific class in core the homologous E6 promoter by interfering with the binding of host transcription histones in vitro. Genes Cells 3:789–800. factors to adjacent elements. J Virol 68:1115–1127. 39. Dey A, Chitsaz F, Abbasi A, Misteli T, Ozato K (2003) The double bromodomain 17. Dostatni N, et al. (1991) The functional BPV-1 E2 trans-activating protein can act as a protein Brd4 binds to acetylated chromatin during interphase and mitosis. Proc Natl repressor by preventing formation of the initiation complex. Genes Dev 5:1657–1671. Acad Sci USA 100:8758–8763. 18. Goodwin EC, Naeger LK, Breiding DE, Androphy EJ, DiMaio D (1998) Transactivation- 40. Tyteca S, Vandromme M, Legube G, Chevillard-Briet M, Trouche D (2006) Tip60 and competent bovine papillomavirus E2 protein is specifically required for efficient p400 are both required for UV-induced apoptosis but play antagonistic roles in cell repression of human papillomavirus oncogene expression and for acute growth cycle progression. EMBO J 25:1680–1689. inhibition of cervical carcinoma cell lines. J Virol 72:3925–3934. 41. Ueda T, et al. (2007) Critical role of the p400/mDomino chromatin-remodeling ATPase 19. Nishimura A, et al. (2000) Mechanisms of human papillomavirus E2-mediated repression in embryonic hematopoiesis. Genes Cells 12:581–592. of viral oncogene expression and cervical cancer cell growth inhibition. JVirol74: 42. Mattera L, et al. (2009) The p400/Tip60 ratio is critical for colorectal cancer cell 3752–3760. proliferation through DNA damage response pathways. Oncogene 28:1506–1517. 20. Wu SY, et al. (2006) Brd4 links chromatin targeting to HPV transcriptional silencing. 43. Flinterman MB, et al. (2007) p400 function is required for the adenovirus E1A- Genes Dev 20:2383–2396. mediated suppression of EGFR and tumour cell killing. Oncogene 26:6863–6874. 21. Schweiger MR, Ottinger M, You J, Howley PM (2007) Brd4-independent transcriptional 44. Tworkowski KA, et al. (2008) Adenovirus E1A targets p400 to induce the cellular repression function of the papillomavirus e2 proteins. JVirol81:9612–9622. oncoprotein Myc. Proc Natl Acad Sci USA 105:6103–6108. 22. Iwase S, et al. (2007) The X-linked mental retardation gene SMCX/JARID1C defines a 45. Stünkel W, Bernard HU (1999) The chromatin structure of the long control region of human family of histone H3 lysine 4 demethylases. Cell 128:1077–1088. papillomavirus type 16 represses viral oncoprotein expression. JVirol73:1918–1930. 23. Tahiliani M, et al. (2007) The histone H3K4 demethylase SMCX links REST target genes 46. Schweiger MR, You J, Howley PM (2006) Bromodomain protein 4 mediates the to X-linked mental retardation. Nature 447:601–605. papillomavirus E2 transcriptional activation function. J Virol 80:4276–4285. 24. Doyon Y, Selleck W, Lane WS, Tan S, Côté J (2004) Structural and functional 47. Yan J, Li Q, Lievens S, Tavernier J, You J (2010) Abrogation of the Brd4-P-TEFb positive conservation of the NuA4 histone acetyltransferase complex from yeast to humans. transcription complex by the papillomavirus E2 protein contributes to the viral Mol Cell Biol 24:1884–1896. oncogene repression. J Virol 84:76–87.

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