Brd4 is a chromatin adaptor containing tandem bromodomains binding to acetylated histone H3 and H4. Although Brd4 has been implicated in the transcriptional control of papillomavirus-encoded E2 protein, it is unclear how Brd4 regulates E2 function and whether the involvement of Brd4 in transactivation and transrepression is common to different types of E2 proteins. Using DNase I footprinting performed with in vitro reconstituted human papillomavirus (HPV) chromatin and nucleosome-free DNA templates, we found that Brd4 facilitates E2 binding to its cognate sequences in chromatin depending on bromodomains and the E2-interacting region of Brd4. Moreover, the coactivator and corepressor function of Brd4 requires at least one intact bromodomain and is mediated by its direct association with E2 proteins encoded by cancer-inducing high risk HPV-16 and HPV-18, wart-causing low risk HPV-11, and bovine papillomavirus type 1, in part through enhancing the protein stability of E2 that is normally degraded via the ubiquitin-dependent proteasome pathway. Our findings indicate that a chromatin adaptor can bridge and enhance the binding of a sequence-specific transcription factor to chromatin and further promote the stability of a labile transcription factor via direct protein-protein interaction.
"In addition, several viruses target BET proteins to regulate viral and cellular transcription. The papillomavirus E2 proteins bind to the C-terminal region of Brd4, using this interaction in tethering the viral genomes to mitotic chromosomes (29,30) and activating transcription (31,32). Retroviruses HIV and HTLV instead contain the Tat/TAR ribonucleoprotein (RNP) transactivation complex that directly interacts with CycT1 to recruit P-TEFb to RNAPII for the stimulation of viral transcripts (33). "
[Show abstract][Hide abstract] ABSTRACT: The bromodomain protein Brd4 regulates the transcription of signal-inducible genes. This is achieved by recruiting the positive
transcription elongation factor P-TEFb to promoters by its P-TEFb interaction domain (PID). Here we show that Brd4 stimulates
the kinase activity of P-TEFb for phosphorylation of the C-terminal domain (CTD) of RNA polymerase II over basal levels. The
CTD phosphorylation saturation levels, the preferences for pre-phosphorylated substrates, and the phosphorylation specificity
for Ser5 of the CTD however remain unchanged. Inhibition of P-TEFb by Hexim1 is relieved by Brd4, although no mutual displacement
with the Cyclin T-binding domain of Hexim1 was observed. Brd4 PID shows a surprising sequence motif similarity to the trans-activating Tat protein from HIV-1, which includes a core RxL motif, a polybasic cluster known as arginine-rich motif, and
a C-terminal leucine motif. Mutation of these motifs to alanine significantly diminished the stimulatory effect of Brd4 and
fully abrogated its activation potential in presence of Hexim1. Yet the protein was not found to bind Cyclin T1 as Tat, but
only P-TEFb with a dissociation constant of 0.5 μM. Our data suggest a model where Brd4 acts on the kinase subunit of P-TEFb
to relieve inhibition and stimulate substrate recognition.
Nucleic Acids Research 05/2014; 42(12). DOI:10.1093/nar/gku449 · 9.11 Impact Factor
"In line with this hypothesis, two BTB domain–containing proteins, BTBD1 and BTBD2, were identified as partners of HPV16E2 protein in a yeast two-hybrid screen . The process of E2 protein degradation is even more complex, since several cellular proteins have been shown to modulate in E2 stability as TAX1BP1 or BRD4 [87, 91, 92]. Moreover, since HPVs infect different tissue niches, it is unlikely that all E2 proteins are degraded in the same way, and to our knowledge, there are no data available on the stability of E2 proteins from cutaneous β- or μ-HPV types. "
[Show abstract][Hide abstract] ABSTRACT: Over 100 genotypes of human papillomaviruses (HPVs) have been identified as being responsible for unapparent infections or for lesions ranging from benign skin or genital warts to cancer. The pathogenesis of HPV results from complex relationships between viral and host factors, driven in particular by the interplay between the host proteome and the early viral proteins. The E2 protein regulates the transcription, the replication as well as the mitotic segregation of the viral genome through the recruitment of host cell factors to the HPV regulatory region. It is thereby a pivotal factor for the productive viral life cycle and for viral persistence, a major risk factor for cancer development. In addition, the E2 proteins have been shown to engage numerous interactions through which they play important roles in modulating the host cell. Such E2 activities are probably contributing to create cell conditions appropriate for the successive stages of the viral life cycle, and some of these activities have been demonstrated only for the oncogenic high-risk HPV. The recent mapping of E2-host protein-protein interactions with 12 genotypes representative of HPV diversity has shed some light on the large complexity of the host cell hijacking and on its diversity according to viral genotypes. This article reviews the functions of E2 as they emerge from the E2/host proteome interplay, taking into account the large-scale comparative interactomic study.
The Open Virology Journal 12/2012; 6(1):173-89. DOI:10.2174/1874357901206010173
"Binding of the E2 and NRIP complex to the E2-binding site on the HPV-16 promoter induces HPV gene expression in a hormoneindependent manner (Chang et al., 2011). Recent reports have identified several cellular proteins that directly bind E2, including TFIIB (Yao et al., 1998), Brd4 (Gagnon et al., 2009; Lee and Chiang, 2009; You et al., 2004), Tax1BP1 (Wang et al., 2009), Brm (Kumar et al., 2007), Mdm2 (Gammoh et al., 2009), and p300 (Hadaschik et al., 2003; Kruppel et al., 2008), to regulate HPV gene expression, E2 protein stability, or maintain viral episomes in HPV-infected cells (You et al., 2004). We add NRIP as an E2-binding protein and report that NRIP is a transcription cofactor of both the GR and E2 to enhance GR-and viral E2-mediated HPV gene expression in a hormone-dependent manner and a hormone-independent manner, respectively (Fig. 5G). "
[Show abstract][Hide abstract] ABSTRACT: We previously identified a gene, nuclear receptor-interaction protein (NRIP), which functions as a transcription cofactor in glucocorticoid receptor (GR) and human papillomavirus E2 (HPV E2)-driven gene expression. Here, we comprehensively evaluated the role of NRIP in HPV-16 gene expression. NRIP acts as a transcription cofactor to enhance GR-regulated HPV-16 gene expression in the presence of hormone. NRIP also can form complex with E2 that caused NRIP-induced HPV gene expression via E2-binding sites in a hormone-independent manner. Furthermore, NRIP can associate with GR and E2 to form tri-protein complex to activate HPV gene expression via GRE, not the E2-binding site, in a hormone-dependent manner. These results indicate that NRIP and GR are viral E2-binding proteins and that NRIP regulates HPV gene expression via GRE and/or E2 binding site in the HPV promoter in a hormone-dependent or independent manner, respectively.
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