Bromodomain protein 4 (Brd4) plays critical roles in development, cancer progression, and virus-host pathogenesis. To gain mechanistic insight into the various biological functions of Brd4, we performed a proteomic analysis to identify and characterize Brd4-associated cellular proteins. We found that the extraterminal (ET) domain, whose function has to date not been determined, interacts with NSD3, JMJD6, CHD4, GLTSCR1, and ATAD5. These ET-domain interactions were also conserved for Brd2 and Brd3, the other human BET proteins tested. We demonstrated that GLTSCR1, NSD3, and JMJD6 impart a pTEFb-independent transcriptional activation function on Brd4. NSD3 as well as JMJD6 is recruited to regulated genes in a Brd4-dependent manner. Moreover, we found that depletion of Brd4 or NSD3 reduces H3K36 methylation, demonstrating that the Brd4/NSD3 complex regulates this specific histone modification. Our results indicate that the Brd4 ET domain through the recruitment of the specific effectors regulates transcriptional activity. In particular, we show that one of these effectors, NSD3, regulates transcription by modifying the chromatin microenvironment at Brd4 target genes. Our study thus identifies the ET domain as a second important transcriptional regulatory domain for Brd4 in addition to the carboxyl-terminal domain (CTD) that interacts with pTEFb.One mechanism underlying the regulation of gene expression is the targeting of multiprotein complexes to modified histones, which then alters the chromatin microenvironment to stimulate or inhibit gene expression. The bromodomains and extraterminal (BET) domain family of proteins are characterized by the presence of two conserved domains, the tandem, amino-terminal bromodomains (BDI and BDII), which bind acetylated chromatin, and an extraterminal (ET) domain, whose function is unknown. The BET family is conserved from yeast to mammals and includes Saccharomyces cerevisiae bromodomain factor 1 (bdf1) and bromodomain factor 2 (bdf2), Drosophila melanogaster female sterile homeotic [fs(1)h], and mammalian Brd2, Brd3, Brd4, and testes/oocyte-specific BrdT/ Brd6. In yeast, deletion of bdf1 leads to a reduced growth rate and deletion of both bdf1 and bdf2 is lethal (27). Mutations of fs(1)h cause segmental abnormalities, including missing organs and homeotic transformations in the progeny of mutant females in Drosophila (13). Knockout of Brd4 or Brd2 in mice results in early embryonic lethality (18,21).The BET proteins have been shown to be important players in human disease, including viral infections and cancer. 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 papillomavirus E2 transcriptional activation functions are also mediated through Brd4 (35,41,42). With regard to human cancer, the Brd4-NUT and Brd3-NUT fusio...

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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...