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

Abstract

An essential step in the pathogenesis of human papillomavirus (HPV)-associated cancers is the dysregulated expression of the viral oncogenes. The papillomavirus E2 protein can silence the long control region (LCR) promoter that controls viral E6 and E7 oncogene expression. The mechanisms by which E2 represses oncogene expression and the cellular factors through which E2 mediates this silencing are largely unknown. We conducted an unbiased, genome-wide siRNA screen and series of secondary screens that identified 96 cellular genes that contribute to the repression of the HPV LCR. In addition to confirming a role for the E2-binding bromodomain protein Brd4 in E2-mediated silencing, we identified a number of genes that have not previously been implicated in E2 repression, including the demethylase JARID1C/SMCX as well as EP400, a component of the NuA4/TIP60 histone acetyltransferase complex. Each of these genes contributes independently and additively to E2-mediated silencing, indicating that E2 functions through several distinct cellular complexes to repress E6 and E7 expression.P apillomaviruses infect squamous epithelial cells and cause a variety of epithelial lesions (1). A subset of the more than 140 human papillomavirus (HPV) types infect mucosal squamous epithelia, including that of the anogenital tract; these are classified as either low-risk or high-risk, depending on the lesions associated with infection. Infections with high-risk HPVs (i.e., HPV16 or HPV18) cause squamous intraepithelial lesions that can progress to cancer, most notably cervical cancer. HPV is considered the major cause of human cervical cancer, the second most common cancer in women worldwide (2).The viral proteins E6 and E7 account for the oncogenic potential of high-risk HPVs in part through their ability to target and degrade p53 and Rb, respectively (1). The long control region (LCR) is the upstream enhancer and promoter region that drives transcription of E6 and E7. An early step in cervical carcinogenesis frequently involves the integration of HPV DNA into cellular chromosomes in a manner that disrupts the E1 and/or E2 ORF (3-6). Because E2 is capable of repressing expression from the LCR, its loss leads to the increased, dysregulated expression of the HPV oncogenes E6 and E7 (7-9). Expression of E2 in HPVpositive cervical cancer cells causes a growth arrest and senescence (10, 11) due to E2-mediated repression of E6 and E7 expression (12, 13). The E2 protein consists of an N-terminal transactivation domain and a C-terminal DNA binding and 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 binding sites (E2BS) and it has been hypothesized that E2 binding to the two promoter proximal binding sites within the LCRs competes with the binding of cellular transcription factors Sp1 and TBP at the promoter (16,17). Yet, this steric hindrance model cannot fully explain E2-mediated transcriptional silencing; f...

The Brd4 Extraterminal Domain Confers Transcription Activation Independent of pTEFb by Recruiting Multiple Proteins, Including NSD3

“…After 10 days of selection, multiple single-cell clones of each cell line were established and maintained as subconfluent monolayers under puromycin selection. Cell lines coexpressing HPV18LCR-luciferase (p5194) and BPV1 E2TA (p#5066) have been previously described (45).…”

“…This recruitment of pTEFb by Brd4 stimulates the transcription of primary responsive genes (19). Moreover, papillomavirus E2-mediated viral transcriptional activation and repression functions are dependent on Brd4 (22,35,41,42,45,52).…”

“…Several different viruses target the individual BET proteins for a variety of purposes but often to regulate viral and cellular transcription (4,7,31,37,41,45,57,60). The papillomavirus E2 proteins bind to Brd4, and some utilize this interaction in tethering the viral genomes to mitotic chromosomes (1,3,57,59).…”

The Role of E2 Proteins in Papillomavirus DNA Replication

“…The transactivation domain mediates functional interactions with cellular transcription factors Sp1 and AP1, histone acetylase complexes containing CBP/p300 and pCAF, and with nucleosome assembly protein hNAP1 (Lee et al, 2002a;Lee et al, 2000;Li et al, 1991;Müller et al, 2002;Rehtanz et al, 2004;Thierry et al, 1992). E2-dependent activities are also modulated by interactions with Brm, a chromatin remodeling protein associated with SWI/SNF ATP-dependent chromatin remodeling complex, and with EP400, a component of the NuA4/TIP60 histone acetylase complex, and SMCX, also known as histone demethylase JARID1C , and Tax1BP1 (Kumar et al, 2007;Smith et al, 2010;Wang et al, 2009b). E2 binds to the papillomavirus oncoproteins E6 and E7, leading to the modulation of their functions (Gammoh et al, 2006;Grm et al, 2005).…”

Protein-protein Interactions: Network Analysis and Applications in Drug Discovery

“…In line with this, care should also be taken when the interaction of HPV E2 with host Brd4 is envisaged as a target for the treatment of HPV infection as was recently suggested, because of the crucial role of this PPI for viral episome maintenance and transcriptional activation [38]. Indeed, E2-Brd4 disruptors might enhance tumor development due to the repressive effect of the E2-Brd4 interaction on the expression of E6 and E7 oncogenes from the integrated viral genome [39,40].…”