Bromodomain Protein 4 Mediates the Papillomavirus E2 Transcriptional Activation Function

Abstract

The papillomavirus E2 regulatory protein has essential roles in viral transcription and the initiation of viral DNA replication as well as for viral genome maintenance. Brd4 has recently been identified as a major E2-interacting protein and, in the case of the bovine papillomavirus type 1, serves to tether E2 and the viral genomes to mitotic chromosomes in dividing cells, thus ensuring viral genome maintenance. We have explored the possibility that Brd4 is involved in other E2 functions. By analyzing the binding of Brd4 to a series of alanine-scanning substitution mutants of the human papillomavirus type 16 E2 N-terminal transactivation domain, we found that amino acids required for Brd4 binding were also required for transcriptional activation but not for viral DNA replication. Functional studies of cells expressing either the C-terminal domain of Brd4 that can bind E2 and compete its binding to Brd4 or short interfering RNA to knock down Brd4 protein levels revealed a role for Brd4 in the transcriptional activation function of E2 but not for its viral DNA replication function. Therefore, these studies establish a broader role for Brd4 in the papillomavirus life cycle than as the chromosome tether for E2 during mitosis.The papillomaviruses (PVs) are small DNA viruses that are etiologic agents for papillomas and warts in a variety of higher vertebrates including humans. Specific human papillomaviruses (HPVs) have been associated with some human cancers, most notably cervical cancer (58). The papillomaviruses establish long-term, persistent infections of squamous epithelial cells, and the viral life cycle is tightly linked with the differentiation program of the host cell (21). In the infected dividing basal cells of the epithelium, the viral DNA is maintained as a stable plasmid. Vegetative viral DNA replication occurs only in the more differentiated squamous epithelial cells. Bovine papillomavirus (BPV) DNA remains extrachromosomal in transformed rodent cells, a system that has served as a useful model for studying viral genome maintenance (28).The papillomavirus E2 protein has important roles in regulating viral transcription, in enhancing E1-dependent viral DNA replication, and in genome maintenance (21). E2 is a DNA binding protein that was first identified as a transcriptional activator (46). Subsequent studies established that E2 can also repress some genes, depending upon the location of its cognate binding sites within the promoter region (49). Indeed, E2 functions to repress the promoter directing the E6 and E7 viral oncogenes in the cancer-associated HPV type 16 (HPV16) and HPV18 genomes (38). For viral genome replication, E2 binds the viral helicase E1 and guides it to the origin of replication in the process of initiating origin-dependent viral DNA replication (6, 34). For genome maintenance, E2 has been shown to associate with mitotic chromosomes and, in doing so, to anchor the viral genomes to the host chromosomes during mitosis (4,22,31,35,44). The structure of E2 resembles that of a prototypic tr...

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

“…Anti-GFP (Clontech) and endogenous Brd4 immunoprecipitations (anti-Brd4 antibody [41]) were also performed similarly from 10-cm plates, except that the lysates were precleared with protein A-Sepharose CL-4B (GE Healthcare) for 1 h at 4°C. The supernatant was subsequently incubated overnight at 4°C with antibody.…”

“…Given the involvement of JMJD6, NSD3, and CHD4 in histone modification and chromatin remodeling, we next asked whether any of the ET domain-associated proteins are important for the transcriptional activation functions of Brd4. We analyzed the effect of siRNA-mediated depletion of GLTSCR1, ATAD5, NSD3, JMJD6, and CHD4 on the transcriptional activation of the bovine papillomavirus (BPV1) long control region (LCR) by the viral E2 protein, whose transcriptional activation function is mediated by Brd4 (35,41,42). We generated a transcriptional reporter assay system in which the luciferase gene is under the control of the BPV1 LCR.…”

“…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).…”

“…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 fusion oncoproteins are causally linked to a lethal form of undifferentiated carcinoma (15)(16)(17).…”

“…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).…”