Background: Brd4 plays a central role in cellular growth control and cancer development. Results: Brd4 depletion leads to chromatin decondensation, while dissociation of Brd4 from chromatin triggers severely fragmented chromatin morphology. Conclusion: Brd4 is crucial for maintaining normal chromatin structure. Significance: The mechanistic insight into Brd4 function will contribute to understanding how perturbing this bromodomain protein function can lead to oncogenic progression.

bReplication of the human papillomavirus (HPV) DNA genome relies on viral factors E1 and E2 and the cellular replication machinery. Bromodomain-containing protein 4 (Brd4) interacts with viral E2 protein to mediate papillomavirus (PV) genome maintenance and viral transcription. However, the functional role of Brd4 in the HPV life cycle remains to be clearly defined. In this study, we provide the first look into the E2-Brd4 interaction in the presence of other important viral factors, such as the HPV16 E1 protein and the viral genome. We show that Brd4 is recruited to actively replicating HPV16 origin foci together with HPV16 E1, E2, and a number of the cellular replication factors: replication protein A70 (RPA70), replication factor C1 (RFC1), and DNA polymerase ␦. Mutagenesis disrupting the E2-Brd4 interaction abolishes the formation of the HPV16 replication complex and impairs HPV16 DNA replication in cells. Brd4 was further demonstrated to be necessary for HPV16 viral DNA replication using a cell-free replication system in which depletion of Brd4 by small interfering RNA (siRNA) silencing leads to impaired HPV16 viral DNA replication and recombinant Brd4 protein is able to rescue viral DNA replication. In addition, releasing endogenous Brd4 from cellular chromatin by using the bromodomain inhibitor JQ1(؉) enhances HPV16 DNA replication, demonstrating that the role of Brd4 in HPV DNA replication could be uncoupled from its function in chromatin-associated transcriptional regulation and cell cycle control. Our study reveals a new role for Brd4 in HPV genome replication, providing novel insights into understanding the life cycle of this oncogenic DNA virus. Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that replicate in differentiating cutaneous and mucosal epithelia (1). They are one of the most prevalent sexually transmitted pathogens in the world. High-risk HPVs are known etiological agents of cervical, anogenital, and head and neck cancers (2), with HPV16 being responsible for over 50% of cervical cancer cases worldwide (3-5).HPVs specifically infect basal epithelial cells. HPV genome replication occurs during two different stages of the viral life cycle. In the infected basal epithelial cells, the viral genomes replicate an average of once per cell cycle during S phase, in synchrony with the host DNA replication (6). This allows the viral genome to be maintained as stable episomes at 50 to 100 copies per cell. This stage of DNA replication ensures a persistent infection in the basal layer of the epidermis. Terminal differentiation of infected cells triggers vegetative viral DNA replication, producing viral genomes, which can then be assembled into virions and be released from the surface of differentiated epithelium (7).Replication of the HPV genome is carried out by viral E1 and E2 proteins in combination with various components of the cellular DNA replication machinery (7). E2 binds to several consensus E2 binding sites near the HPV origin of replication (Ori) and recruits E1 to the...

BRD4 is implicated in the pathogenesis of a number of different cancers. It is also the target of translocation t(15;19) that accounts for the highly aggressive NUT midline carcinoma (NMC). We discovered that t(15;19) NMC cells display the ability to grow into stem cell-like spheres and express an exceptionally high level of the stem cell marker, SOX2. The BRD4-NUT fusion oncogene resulting from t(15;19) translocation is required for the abnormal activation of SOX2, which drives the stem cell-like proliferation and cellular transformation in NMC cells. SOX2 knockdown phenocopies the effects of BRD4-NUT inhibition, whereas ectopic SOX2 expression rescues the phenotype. The BRD4-NUT induced abnormal SOX2 activation was observed in multiple NMC cell lines as well as in NMC primary tumors. We further demonstrate that BRD4-NUT oncoprotein recruits p300 to stimulate transcription activation, and that inhibition of p300 represses SOX2 transcription in NMC cells. These studies identify this stem cell marker as a novel BRD4-NUT target that supports the highly aggressive transforming activity of t(15;19) carcinomas. Our study provides new mechanistic insights for understanding how alteration of BRD4 function by BRD4-NUT oncogene leads to the highly malignant NMC carcinoma. Because abnormal stem cell self-renewal is frequently observed during tumor formation and metastasis, the aberrant stem cell-like proliferation associated with BRD4 dysregulation observed in NMC carcinoma may have implications for studying the oncogenic mechanism of other BRD4-associated tumors.