Jianxin You

Interaction of the Bovine Papillomavirus E2 Protein with Brd4 Tethers the Viral DNA to Host Mitotic Chromosomes

The papillomavirus E2 protein tethers viral genomes to host mitotic chromosomes to ensure genome maintenance. We have identified the bromodomain protein Brd4 as a major cellular interacting partner of the bovine papillomavirus E2. Brd4 associates with mitotic chromosomes and colocalizes with E2 on mitotic chromosomes. The site of E2 binding maps to the C-terminal domain of Brd4. Expression of this C-terminal Brd4 domain functions in a dominant-negative manner to abrogate the colocalization of E2 with Brd4 on mitotic chromosomes, to block association of the viral episomes with Brd4, and to inhibit BPV-1 DNA-mediated cellular transformation. Brd4 also associates with HPV16 E2, indicating that Brd4 binding may be a shared property of all papillomavirus E2 proteins. The interaction of E2 with Brd4 is required to ensure the tethering of viral genomes to the host mitotic chromosomes for persistence of viral episomes in PV-infected cells.

Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Interacts with Bromodomain Protein Brd4 on Host Mitotic Chromosomes

ABSTRACT The latency-associated nuclear antigen (LANA) of Kaposis sarcoma-associated herpesvirus (KSHV) is required for viral episome maintenance in host cells during latent infection. Two regions of the protein have been implicated in tethering LANA/viral episomes to the host mitotic chromosomes, and LANA chromosome-binding sites are subjects of high interest. Because previous studies had identified bromodomain protein Brd4 as the mitotic chromosome anchor for the bovine papillomavirus E2 protein, which tethers the viral episomes to host mitotic chromosomes (J. You, J. L. Croyle, A. Nishimura, K. Ozato, and P. M. Howley, Cell 117:349-360, 2004, and J. You, M. R. Schweiger, and P. M. Howley, J. Virol. 79:14956-14961, 2005), we examined whether KSHV LANA interacts with Brd4. We found that LANA binds Brd4 in vivo and in vitro and that the binding is mediated by a direct protein-protein interaction between the ET (extraterminal) domain of Brd4 and a carboxyl-terminal region of LANA previously implicated in chromosome binding. Brd4 associates with mitotic chromosomes throughout mitosis and demonstrates a strong colocalization with LANA and the KSHV episomes on host mitotic chromosomes. Although another bromodomain protein, RING3/Brd2, binds to LANA in a similar fashion in vitro, it is largely excluded from the mitotic chromosomes in KSHV-uninfected cells and is partially recruited to the chromosomes in KSHV-infected cells. These data identify Brd4 as an interacting protein for the carboxyl terminus of LANA on mitotic chromosomes and suggest distinct functional roles for the two bromodomain proteins RING3/Brd2 and Brd4 in LANA binding. Additionally, because Brd4 has recently been shown to have a role in transcription, we examined whether Brd4 can regulate the CDK2 promoter, which can be transactivated by LANA.

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.

Bromodomain protein Brd4 associated with acetylated chromatin is important for maintenance of higher-order chromatin structure.

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. Chromatin structure organization is crucial for regulating many fundamental cellular processes. However, the molecular mechanism that regulates the assembly of higher-order chromatin structure remains poorly understood. In this study, we demonstrate that Brd4 (bromodomain-containing protein 4) protein participates in the maintenance of the higher-order chromatin structure. Brd4, a member of the BET family of proteins, has been shown to play important roles in cellular growth control, cell cycle progression, and cancer development. We apply in situ single cell chromatin imaging and micrococcal nuclease (MNase) assay to show that Brd4 depletion leads to a large scale chromatin unfolding. A dominant-negative inhibitor encoding the double bromodomains (BDI/II) of Brd4 can competitively dissociate endogenous Brd4 from chromatin to trigger severely fragmented chromatin morphology. Mechanistic studies using Brd4 truncation mutants reveal that the Brd4 C-terminal domain is crucial for maintaining normal chromatin structure. Using bimolecular fluorescence complementation technology, we demonstrate that Brd4 molecules interact intermolecularly on chromatin and that replacing Brd4 molecules by BDI/II causes abnormal nucleosome aggregation and chromatin fragmentation. These studies establish a novel structural role of Brd4 in supporting the higher chromatin architecture.

Brd4-independent transcriptional repression function of the papillomavirus E2 proteins

ABSTRACT The papillomavirus E2 protein is a critical viral regulatory protein with transcription, DNA replication, and genome maintenance functions. We have previously identified the cellular bromodomain protein Brd4 as a major E2-interacting protein and established that it participates in tethering bovine papillomavirus type 1 E2 and viral genomes to host cell mitotic chromosomes. We have also shown that Brd4 mediates E2-dependent transcriptional activation, which is strongly inhibited by the disruption of E2/Brd4 binding as well as by short hairpin RNA (shRNA) knockdown of Brd4 expression levels. Since several mutants harboring single amino acid substitutions within the E2 transactivation domain that are defective for both transcriptional transactivation and Brd4 binding are also defective for transcriptional repression, we examined the role of Brd4 in E2 repression of the human papillomavirus E6/E7 promoter. Surprisingly, in a variety of in vivo assays, including transcription reporter assays, HeLa cell proliferation and colony reduction assays, and Northern blot analyses, neither blocking of the binding of E2 to Brd4 nor shRNA knockdown of Brd4 affected the E2 repression function. Our study provides evidence for a Brd4-independent mechanism of E2-mediated repression and suggests that different cellular factors must be involved in E2-mediated transcriptional activation and repression functions.

Regulation of Aurora B Expression by the Bromodomain Protein Brd4

ABSTRACT The bromodomain protein Brd4 plays critical roles in cellular proliferation and cell cycle progression. In this study, we investigated the involvement of Brd4 in cell cycle regulation and observed aberrant chromosome segregation and failures in cytokinesis in cancer cells as well as in primary keratinocytes in which Brd4 has been knocked down by RNA interference. Suppression of Brd4 protein levels in proliferating cells decreased Aurora B protein and transcript levels and abolished its chromosomal distribution. In contrast, exogenous Brd4 expression stimulated Aurora B promoter reporter activity and upregulated endogenous Aurora B expression. Aurora B kinase is a chromosomal passenger protein that is essential for chromosome segregation and cytokinesis. Either overexpression of Aurora B or its inactivation can induce defects in centrosome function, spindle assembly, chromosome alignment, and cytokinesis in various cancer cells. The impaired regulation of Aurora B expression in human cells by Brd4 knockdown or overexpression coincided with mitotic catastrophe and multinucleation that are typically observed when Aurora B is inactivated or overexpressed. Overall, our data suggest that Brd4 is essential for the maintenance of the cell cycle progression mediated at least in part through the control of transcription of the Aurora B kinase cell cycle regulatory gene.

Inhibition of E2 Binding to Brd4 Enhances Viral Genome Loss and Phenotypic Reversion of Bovine Papillomavirus-Transformed Cells

ABSTRACT The bovine papillomavirus E2 protein tethers the viral genomes to mitotic chromosomes in dividing cells through binding to the C-terminal domain (CTD) of Brd4. Expression of the Brd4-CTD competes the binding of E2 to endogenous Brd4 in cells. Here we extend our previous study that identified Brd4 as the E2 mitotic chromosome receptor to show that Brd4-CTD expression released the viral DNA from mitotic chromosomes in BPV-1 transformed cells. Furthermore, stable expression of Brd4-CTD enhanced the frequency of morphological reversion of BPV-1 transformed C127 cells resulting in the complete elimination of the viral DNA in the resulting flat revertants.

Merkel Cell Polyomavirus Large T Antigen Disrupts Host Genomic Integrity and Inhibits Cellular Proliferation

ABSTRACT Clonal integration of Merkel cell polyomavirus (MCV) DNA into the host genome has been observed in at least 80% of Merkel cell carcinoma (MCC). The integrated viral genome typically carries mutations that truncate the C-terminal DNA binding and helicase domains of the MCV large T antigen (LT), suggesting a selective pressure to remove this MCV LT region during tumor development. In this study, we show that MCV infection leads to the activation of host DNA damage responses (DDR). This activity was mapped to the C-terminal helicase-containing region of the MCV LT. The MCV LT-activated DNA damage kinases, in turn, led to enhanced p53 phosphorylation, upregulation of p53 downstream target genes, and cell cycle arrest. Compared to the N-terminal MCV LT fragment that is usually preserved in mutants isolated from MCC tumors, full-length MCV LT shows a decreased potential to support cellular proliferation, focus formation, and anchorage-independent cell growth. These apparently antitumorigenic effects can be reversed by a dominant-negative p53 inhibitor. Our results demonstrate that MCV LT-induced DDR activates p53 pathway, leading to the inhibition of cellular proliferation. This study reveals a key difference between MCV LT and simian vacuolating virus 40 LT, which activates a DDR but inhibits p53 function. This study also explains, in part, why truncation mutations that remove the MCV LT C-terminal region are necessary for the oncogenic progression of MCV-associated cancers.

Recruitment of Brd4 to the Human Papillomavirus Type 16 DNA Replication Complex Is Essential for Replication of Viral DNA

ABSTRACT Replication 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 δ. 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.

Bromodomain Protein Brd4 Plays a Key Role in Merkel Cell Polyomavirus DNA Replication

Merkel cell polyomavirus (MCV or MCPyV) is the first human polyomavirus to be definitively linked to cancer. The mechanisms of MCV-induced oncogenesis and much of MCV biology are largely unexplored. In this study, we demonstrate that bromodomain protein 4 (Brd4) interacts with MCV large T antigen (LT) and plays a critical role in viral DNA replication. Brd4 knockdown inhibits MCV replication, which can be rescued by recombinant Brd4. Brd4 colocalizes with the MCV LT/replication origin complex in the nucleus and recruits replication factor C (RFC) to the viral replication sites. A dominant negative inhibitor of the Brd4-MCV LT interaction can dissociate Brd4 and RFC from the viral replication complex and abrogate MCV replication. Furthermore, obstructing the physiologic interaction between Brd4 and host chromatin with the chemical compound JQ1(+) leads to enhanced MCV DNA replication, demonstrating that the role of Brd4 in MCV replication is distinct from its role in chromatin-associated transcriptional regulation. Our findings demonstrate mechanistic details of the MCV replication machinery; providing novel insight to elucidate the life cycle of this newly discovered oncogenic DNA virus.