Genome-Wide Sirna Screen Identifies SMCX, EP400, and Brd4 As E2
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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.