The Experts below are selected from a list of 16116 Experts worldwide ranked by ideXlab platform

Keiko Ozato - One of the best experts on this subject based on the ideXlab platform.

  • DNA Damage Induces Dynamic Associations of BRD4/P-TEFb With Chromatin and Modulates Gene Transcription in a BRD4-Dependent and -Independent Manner
    Frontiers in molecular biosciences, 2020
    Co-Authors: Yawei Song, Keiko Ozato, Jinping Jia, Mingze Yao, Xiaoshan Wang, Andrew P. Hutchins, Jiekai Chen, Hongjie Yao
    Abstract:

    The bromodomain-containing protein BRD4 has been thought to transmit epigenetic information across cell divisions by binding to both mitotic chromosomes and interphase chromatin. UV-released BRD4 mediates the recruitment of active P-TEFb to the promoter, which enhances transcriptional elongation. However, the dynamic associations between BRD4 and P-TEFb and BRD4-mediated gene regulation after UV stress are largely unknown. In this study, we found that BRD4 dissociates from chromatin within 30 min after UV treatment and thereafter recruits chromatin. However, P-TEFb binds tightly to chromatin right after UV treatment, suggesting that no interactions occur between BRD4 and P-TEFb within 30 min after UV stress. BRD4 knockdown changes the distribution of P-TEFb among nuclear soluble and chromatin and downregulates the elongation activity of RNA polymerase II. Inhibition of JNK kinase but not other MAP kinases impedes the interactions between BRD4 and P-TEFb. RNA-seq and ChIP assays indicate that BRD4 both positively and negatively regulates gene transcription in cells treated with UV stress. These results reveal previously unrecognized dynamics of BRD4 and P-TEFb after UV stress and regulation of gene transcription by BRD4 acting as either activator or repressor in a context-dependent manner.

  • BRD4 binds to active enhancers to control cell identity gene induction in adipogenesis and myogenesis.
    Nature communications, 2017
    Co-Authors: Ji-eun Lee, Keiko Ozato, Anup Dey, Young-kwon Park, Sarah Park, Younghoon Jang, Nicholas Waring, Binbin Lai, Weiqun Peng
    Abstract:

    The epigenomic reader BRD4 is an important drug target for cancers. However, its role in cell differentiation and animal development remains largely unclear. Using two conditional knockout mouse strains and derived cells, we demonstrate that BRD4 controls cell identity gene induction and is essential for adipogenesis and myogenesis. BRD4 co-localizes with lineage-determining transcription factors (LDTFs) on active enhancers during differentiation. LDTFs coordinate with H3K4 mono-methyltransferases MLL3/MLL4 (KMT2C/KMT2D) and H3K27 acetyltransferases CBP/p300 to recruit BRD4 to enhancers activated during differentiation. BRD4 deletion prevents the enrichment of Mediator and RNA polymerase II transcription machinery, but not that of LDTFs, MLL3/MLL4-mediated H3K4me1, and CBP/p300-mediated H3K27ac, on enhancers. Consequently, BRD4 deletion prevents enhancer RNA production, cell identity gene induction and cell differentiation. Interestingly, BRD4 is dispensable for maintaining cell identity genes in differentiated cells. These findings identify BRD4 as an enhancer epigenomic reader that links active enhancers with cell identity gene induction in differentiation.

  • The Bromodomain Protein BRD4 Stimulates G1 Gene Transcription and Promotes Progression to S Phase
    The Journal of biological chemistry, 2008
    Co-Authors: Kazuki Mochizuki, Tomohiko Tamura, Hongjie Yao, Anup Dey, Akira Nishiyama, Moon Kyoo Jang, Anu Ghosh, Hiroko Natsume, Keiko Ozato
    Abstract:

    BRD4 is a bromodomain protein that binds to acetylated chromatin. It regulates cell growth, although the underlying mechanism has remained elusive. BRD4 has also been shown to control transcription of viral genes, whereas its role in transcription of cellular genes has not been fully elucidated. Here we addressed the role of BRD4 in cell growth and transcription using a small hairpin (sh) RNA approach. The BRD4 shRNA vector stably knocked down BRD4 protein expression by approximately 90% in NIH3T3 cells and mouse embryonic fibroblasts. BRD4 knockdown cells were growth impaired and grew more slowly than control cells. When synchronized by serum starvation and released, BRD4 knockdown cells were arrested at G(1), whereas control cells progressed to S phase. In microarray analysis, although numerous genes were up-regulated during G(1) in control cells, many of these G(1) genes were not up-regulated in BRD4 knockdown cells. Reintroduction of BRD4 rescued expression of these G(1) genes in BRD4 knockdown cells, allowing cells to progress toward S phase. Chromatin immunoprecipitation analysis showed that BRD4 was recruited to the promoters of these G(1) genes during G(0)-G(1) progression. Furthermore, BRD4 recruitment coincided with increased binding of Cdk9, a component of P-TEFb and RNA polymerase II to these genes. BRD4 recruitment was low to absent at genes not affected by BRD4 shRNA. The results indicate that BRD4 stimulates G(1) gene expression by binding to multiple G(1) gene promoters in a cell cycle-dependent manner.

  • BRD4 associates with mitotic chromosomes throughout early zebrafish embryogenesis.
    Developmental dynamics : an official publication of the American Association of Anatomists, 2008
    Co-Authors: Reiko Toyama, Keiko Ozato, Anup Dey, Martha L. Rebbert, Igor B. Dawid
    Abstract:

    BRD4 is a member of the BET (bromodomains and extraterminal) subfamily of bromodomain proteins that includes chromatin-modifying proteins and transcriptional regulators. BRD4 has a role in cell cycle progression, making it indispensable in mouse embryos and cultured cells. The N-terminal domain of BRD4 participates in a fusion oncogene. BRD4 associates with acetylated histones in chromatin, and this association persists during mitosis implicating BRD4 in epigenetic memory. BRD4 sequence, particularly the bromodomains and ET domain, is conserved in the zebrafish and Xenopus laevis proteins reported here. BRD4 is expressed and localized on mitotic chromosomes in early zebrafish embryos before and after the midblastula transition (MBT), indicating that the BRD4-chromosome association is a conserved property that is maintained even before zygotic transcription. The association of BRD4 with acetylated histones may also be conserved in early embryos as we found that histones H3 and H4 are already acetylated during pre-MBT stages.

  • the bromodomain protein BRD4 is a positive regulatory component of p tefb and stimulates rna polymerase ii dependent transcription
    Molecular Cell, 2005
    Co-Authors: M Jang, Kazuki Mochizuki, Meisheng Zhou, Hosang Jeong, Joh N Ady, Keiko Ozato
    Abstract:

    Summary BRD4 is a mammalian bromodomain protein that binds to acetylated chromatin. Proteomic analysis revealed that BRD4 interacts with cyclinT1 and Cdk9 that constitutes core positive transcription elongation factor b (P-TEFb). BRD4 interacted with P-TEFb in the living nucleus through its bromodomain. About half of P-TEFb was bound to the inhibitory subunit and functionally inactive. BRD4 interacted with P-TEFb that was free of the inhibitory subunit. An increase in BRD4 expression led to increased P-TEFb-dependent phosphorylation of RNA polymerase II (RNAPII) CTD and stimulation of transcription from promoters in vivo. Conversely, a reduction in BRD4 expression by siRNA reduced CTD phosphorylation and transcription, revealing that BRD4 is a positive regulatory component of P-TEFb. In chromatin immunoprecipitation (ChIP) assays, the recruitment of P-TEFb to a promoter was dependent on BRD4 and was enhanced by an increase in chromatin acetylation. Together, P-TEFb alternately interacts with BRD4 and the inhibitory subunit to maintain functional equilibrium in the cell.

Jianxin You - One of the best experts on this subject based on the ideXlab platform.

  • Bromodomain-Containing Protein BRD4 Is Hyperphosphorylated in Mitosis.
    Cancers, 2020
    Co-Authors: Ranran Wang, June F Yang, Erle S. Robertson, Jianxin You
    Abstract:

    The epigenetic reader BRD4 binds acetylated histones and plays a central role in controlling cellular gene transcription and proliferation. Dysregulation of BRD4's activity has been implicated in the pathogenesis of a wide variety of cancers. While blocking BRD4 interaction with acetylated histones using BET inhibitors (BETis) has been tested in clinical trials, many cancers have acquired BETi resistance. However, the underlying mechanisms are poorly understood and BETi resistance remains a pressing clinical problem. We previously showed that BRD4 phosphorylation supports stronger chromatin binding and target oncogene expression. In this study, we discovered that BRD4 is hyperphosphorylated by CDK1 during mitosis and determined the major CDK1 phosphorylation sites in BRD4. Using CRISPR/Cas9 gene editing, we replaced endogenous BRD4 with a non-phosphorylatable mutant and demonstrated that CDK1-mediated BRD4 phosphorylation contributes to BETi resistance. CDK1 over-activation frequently observed in cancers has the potential to cause aberrant BRD4 hyperphosphorylation persisting outside of mitosis to strengthen its target gene binding and confer BETi resistance. We found that dual CDK1 and BET inhibition generates a synergistic effect in killing BETi-resistant cancer cells. Our study therefore suggests that CDK1 inhibition can be employed to overcome tumor BETi resistance and improve treatments for BRD4-associated cancers.

  • Uncovering BRD4 hyperphosphorylation associated with cellular transformation in NUT midline carcinoma
    Proceedings of the National Academy of Sciences of the United States of America, 2017
    Co-Authors: Ranran Wang, Cheng Ming Chiang, Benjamin A Garcia, Xing Jun Cao, Katarzyna Kulej, Wei Liu, Margo Macdonald, Jianxin You
    Abstract:

    The epigenetic reader BRD4 plays a vital role in transcriptional regulation, cellular growth control, and cell-cycle progression. Dysregulation of BRD4 function has been implicated in the pathogenesis of a wide range of cancers. However, how BRD4 is regulated to maintain its normal function in healthy cells and how alteration of this process leads to cancer remain poorly understood. In this study, we discovered that BRD4 is hyperphosphorylated in NUT midline carcinoma and identified CDK9 as a potential kinase mediating BRD4 hyperphosphorylation. Disruption of BRD4 hyperphosphorylation using both chemical and molecular inhibitors led to the repression of BRD4 downstream oncogenes and abrogation of cellular transformation. BRD4 hyperphosphorylation is also observed in other cancers displaying enhanced BRD4 oncogenic activity. Our study revealed a mechanism that may regulate BRD4 biological function through phosphorylation, which, when dysregulated, could lead to oncogenesis. Our finding points to strategies to target the aberrant BRD4 signaling specifically for cancer intervention.

  • Mechanistic Analysis of the Role of Bromodomain-containing Protein 4 (BRD4) in BRD4-NUT Oncoprotein-induced Transcriptional Activation
    The Journal of biological chemistry, 2014
    Co-Authors: Ranran Wang, Jianxin You
    Abstract:

    NUT midline carcinoma (NMC) is a rare but highly aggressive cancer typically caused by the translocation t(15;19), which results in the formation of the BRD4-NUT fusion oncoprotein. Previous studies have demonstrated that fusion of the NUT protein with the double bromodomains of BRD4 may significantly alter the cellular gene expression profile to contribute to NMC tumorigenesis. However, the mechanistic details of this BRD4-NUT function remain poorly understood. In this study, we examined the NUT function in transcriptional regulation by targeting it to a LacO transgene array integrated in U2OS 2-6-3 cells, which allow us to visualize how NUT alters the in situ gene transcription dynamic. Using this system, we demonstrated that the NUT protein tethered to the LacO locus recruits p300/CREB-binding protein (CBP), induces histone hyperacetylation, and enriches BRD4 to the transgene array chromatin foci. We also discovered that, in BRD4-NUT expressed in NMC cells, the NUT moiety of the fusion protein anchored to chromatin by the double bromodomains also stimulates histone hyperacetylation, which causes BRD4 to bind tighter to chromatin. Consequently, multiple BRD4-interacting factors are recruited to the NUT-associated chromatin locus to activate in situ transgene expression. This gene transcription function was repressed by either expression of a dominant negative inhibitor of the p300-NUT interaction or treatment with (+)-JQ1, which dissociates BRD4 from the LacO chromatin locus. Our data support a model in which BRD4-NUT-stimulated histone hyperacetylation recruits additional BRD4 and interacting partners to support transcriptional activation, which underlies the BRD4-NUT oncogenic mechanism in NMC.

  • recruitment of BRD4 to the human papillomavirus type 16 dna replication complex is essential for replication of viral dna
    Journal of Virology, 2013
    Co-Authors: Xin Wang, James E. Bradner, Christine M Helfer, Neha J Pancholi, Jianxin You
    Abstract:

    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 associated with acetylated chromatin is important for maintenance of higher order chromatin structure
    Journal of Biological Chemistry, 2012
    Co-Authors: Ranran Wang, Jing Jiao, Christine M Helfer, Jianxin You
    Abstract:

    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.

Alessio Ciulli - One of the best experts on this subject based on the ideXlab platform.

  • SPR-Measured Dissociation Kinetics of PROTAC Ternary Complexes Influence Target Degradation Rate.
    ACS Chemical Biology, 2019
    Co-Authors: Sandra Winkler, Scott J. Hughes, Claire Whitworth, Michael Galant, William Farnaby, Klaus Rumpel, Alessio Ciulli
    Abstract:

    Bifunctional degrader molecules, known as proteolysis-targeting chimeras (PROTACs), function by recruiting a target to an E3 ligase, forming a target/PROTAC/ligase ternary complex. Despite the importance of this key intermediate species, no detailed validation of a method to directly determine binding parameters for ternary complex kinetics has been reported, and it remains to be addressed whether tuning the kinetics of PROTAC ternary complexes may be an effective strategy to improve the efficiency of targeted protein degradation. Here, we develop an SPR-based assay to quantify the stability of PROTAC-induced ternary complexes by measuring for the first time the kinetics of their formation and dissociation in vitro using purified proteins. We benchmark our assay using four PROTACs that target the bromodomains (BDs) of bromodomain and extraterminal domain proteins Brd2, Brd3, and BRD4 to the von Hippel–Lindau E3 ligase (VHL). We reveal marked differences in ternary complex off-rates for different PROTACs t...

  • SPR-measured kinetics of PROTAC ternary complexes influence target degradation rate
    bioRxiv, 2018
    Co-Authors: Sandra Winkler, Scott J. Hughes, Claire Whitworth, Michael Galant, William Farnaby, Klaus Rumpel, Alessio Ciulli
    Abstract:

    Bifunctional degrader molecules, known as proteolysis-targeting chimeras (PROTACs), function by recruiting a target to an E3 ligase, via a target:PROTAC:ligase ternary complex. Despite the importance of this key intermediate species, no method to monitor ternary complex kinetics has been reported, and it remains to be addressed how this might impact on the kinetics of protein degradation. Here, we develop an SPR-based assay to quantify the stability of PROTAC-induced ternary complexes by measuring for the first time the kinetics of their formation and dissociation. We benchmark our assay using four PROTACs that target the bromodomains (BDs) of BET proteins Brd2, Brd3 and BRD4 to the E3 ligase VHL. We reveal marked differences in ternary complex off-rates for different PROTACs that form either cooperative or non-cooperative complexes. The positively cooperative degrader MZ1 forms comparatively stable and long-lived ternary complexes with either BRD4BD2 or Brd2BD2 and VHL. Equivalent complexes with Brd3BD2 are destabilized due to a single amino acid difference (Glu/Gly swap) present in the bromodomain. We observe that this difference in ternary complex dissociative half-life correlates to a greater initial rate of intracellular degradation of Brd2 and BRD4 relative to Brd3. These findings establish a novel assay to measure the kinetics of PROTAC ternary complexes and elucidate the important kinetic parameters that drive effective target degradation.

  • selective small molecule induced degradation of the bet bromodomain protein BRD4
    ACS Chemical Biology, 2015
    Co-Authors: Michael Zengerle, Kwokho Chan, Alessio Ciulli
    Abstract:

    The Bromo- and Extra-Terminal (BET) proteins BRD2, BRD3, and BRD4 play important roles in transcriptional regulation, epigenetics, and cancer and are the targets of pan-BET selective bromodomain inhibitor JQ1. However, the lack of intra-BET selectivity limits the scope of current inhibitors as probes for target validation and could lead to unwanted side effects or toxicity in a therapeutic setting. We designed Proteolysis Targeted Chimeras (PROTACs) that tether JQ1 to a ligand for the E3 ubiquitin ligase VHL, aimed at triggering the intracellular destruction of BET proteins. Compound MZ1 potently and rapidly induces reversible, long-lasting, and unexpectedly selective removal of BRD4 over BRD2 and BRD3. The activity of MZ1 is dependent on binding to VHL but is achieved at a sufficiently low concentration not to induce stabilization of HIF-1α. Gene expression profiles of selected cancer-related genes responsive to JQ1 reveal distinct and more limited transcriptional responses induced by MZ1, consistent wit...

James E. Bradner - One of the best experts on this subject based on the ideXlab platform.

  • Signal-Dependent Recruitment of BRD4 to Cardiomyocyte Super-Enhancers Is Suppressed by a MicroRNA
    Cell reports, 2016
    Co-Authors: Matthew S. Stratton, James E. Bradner, Charles Y. Lin, Priti Anand, Philip D. Tatman, Bradley S. Ferguson, Sean T. Wickers, Amrut V. Ambardekar, Carmen C. Sucharov, Saptarsi M. Haldar
    Abstract:

    Summary BRD4 governs pathological cardiac gene expression by binding acetylated chromatin, resulting in enhanced RNA polymerase II (Pol II) phosphorylation and transcription elongation. Here, we describe a signal-dependent mechanism for the regulation of BRD4 in cardiomyocytes. BRD4 expression is suppressed by microRNA-9 (miR-9), which targets the 3′ UTR of the BRD4 transcript. In response to stress stimuli, miR-9 is downregulated, leading to derepression of BRD4 and enrichment of BRD4 at long-range super-enhancers (SEs) associated with pathological cardiac genes. A miR-9 mimic represses stimulus-dependent targeting of BRD4 to SEs and blunts Pol II phosphorylation at proximal transcription start sites, without affecting BRD4 binding to SEs that control constitutively expressed cardiac genes. These findings suggest that dynamic enrichment of BRD4 at SEs genome-wide serves a crucial role in the control of stress-induced cardiac gene expression and define a miR-dependent signaling mechanism for the regulation of chromatin state and Pol II phosphorylation.

  • the bromodomain inhibitor jq1 and the histone deacetylase inhibitor panobinostat synergistically reduce n myc expression and induce anticancer effects
    Clinical Cancer Research, 2016
    Co-Authors: Jeyran Shahbazi, James E. Bradner, Pei Yan Liu, Bernard Atmadibrata, Glenn M Marshall, Richard B Lock, Tao Liu
    Abstract:

    Purpose: Patients with neuroblastoma associated with MYCN oncogene amplification experience a very poor prognosis. BET bromodomain inhibitors are among the most promising novel anticancer agents as they block BRD3 and BRD4 from activating oncogene transcription. However, treatment with BET bromodomain inhibitors alone does not result in cancer remission in many murine models. Experimental Design: MYCN -amplified neuroblastoma cells were treated with vehicle control, the BET bromodomain inhibitor JQ1, the histone deacetylase inhibitor panobinostat, or the combination of JQ1 and panobinostat. Genes modulated by JQ1, panobinostat, or the combination therapy were identified by Affymetrix microarray, and cell proliferation and apoptosis were examined by Alamar blue assays and flow cytometry analysis. Modulation of LIN28B promoter activity by BRD3 and BRD4 was examined by chromatin immunoprecipitation and luciferase assays. In addition, neuroblastoma-bearing mice were treated with vehicle control, JQ1, and/or panobinostat. Results: LIN28B was one of the top genes synergistically reduced by JQ1 and panobinostat. BRD3 and BRD4 directly bound to the LIN28B gene promoter and activated LIN28B gene transcription, and knocking down LIN28B reduced the expression of N-Myc protein, but not N-Myc mRNA. JQ1 and panobinostat synergistically reduced LIN28B gene and N-Myc protein expression, and synergistically induced growth inhibition and apoptosis in neuroblastoma cells, but not normal nonmalignant cells in vitro . In neuroblastoma-bearing mice, JQ1 and panobinostat synergistically and considerably reduced N-Myc protein expression in tumor tissues and blocked tumor progression. Conclusions: Our findings have identified a novel strategy to reduce the N-Myc oncoprotein expression and a novel therapeutic approach for the treatment of aggressive neuroblastoma. Clin Cancer Res; 22(10); 2534–44. ©2016 AACR .

  • selective target protein degradation via phthalimide conjugation
    Science, 2015
    Co-Authors: Georg E Winter, Amanda L Souza, Joshiawa Paulk, Justin M Roberts, Sirano Dhepaganon, Dennis L Buckley, James E. Bradner
    Abstract:

    Small-molecule antagonists disable discrete biochemical properties of protein targets. For multi-domain protein targets, the pharmacologic consequence of drug action is limited by selective disruption of one domain-specific activity. More broadly, target inhibition is kinetically limited by the durability and degree of target engagement. These features of traditional drug molecules are challenging to the development of inhibitors targeting transcription factors and chromatin-associated epigenetic proteins, which function as multi-domain biomolecular scaffolds and generally feature rapid association and dissociation kinetics. We therefore devised a chemical strategy to prompt ligand-dependent target protein degradation, via chemical conjugation with derivatized phthalimides that hijack the function of the Cereblon E3 ubiquitin ligase complex. Using this approach, we converted an acetyl-lysine competitive antagonist that displaces BET bromodomains from chromatin (JQ1) to a phthalimide-conjugated ligand that prompts immediate Cereblon-dependent BET protein degradation (dBET1). Expression proteomics confirms high specificity for BET family members BRD2, BRD3 and BRD4 among 7429 proteins detected. Degradation of BET bromodomains is associated with a more rapid and robust apoptotic response compared to bromodomain inhibition in primary human leukemic blasts, and dBET1 exhibits in vivo efficacy in a human leukemia xenograft. The reach of this approach is illustrated by a second series of probes that degrade the cytosolic signaling protein, FKBP12. Together, these findings identify a facile and general new strategy to control target protein stability, with implications for approaching previously intractable protein targets.

  • recruitment of BRD4 to the human papillomavirus type 16 dna replication complex is essential for replication of viral dna
    Journal of Virology, 2013
    Co-Authors: Xin Wang, James E. Bradner, Christine M Helfer, Neha J Pancholi, Jianxin You
    Abstract:

    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.

  • Targeting STAT5 in Leukemia Through Inhibition of Bromodomain Proteins
    Blood, 2012
    Co-Authors: Suhu Liu, James E. Bradner, Sarah R. Walker, Erik Nelson, Robert Cirulli, Michael Xiang, David A. Frank
    Abstract:

    Abstract 399 Introduction: The transcription factor STAT5 is constitutively activated in many forms of hematologic malignancies, including chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). STAT5 can be activated by constitutively activated tyrosine kinases or autocrine and paracrine secretion of cytokines signaling through Jak kinases. STAT5 is essential for the pathogenesis of neoplasms induced by BCR-ABL1 and Jak2V617F, as well as for leukemia stem cell self-renewal. Development of tyrosine kinases inhibitors (TKIs), such as imatinib, has greatly improved the outcome of patients with leukemias harboring aberrantly activated oncogenic tyrosine kinases. However, TKIs used as a single agent only achieve significant success in CML, with very limited benefit in the more aggressive ALL. Moreover, patients with CML who initially respond well may acquire resistance to TKIs with the progression of their disease. In fact, increased activity of STAT5 is often associated with CML progression and may underlie resistance to TKIs. Importantly, leukemia cells that are resistant to TKIs remain sensitive to STAT5 inhibition, and dual inhibition of both tyrosine kinases and STAT5 leads to more efficient reduction of leukemia cell viability. Thus targeting STAT5 alone or in combination is a promising therapeutic strategy for many hematological malignancies. While many strategies directly inhibit STAT5, we considered the possibility that STAT5 association with co-regulatory proteins is essential for STAT5 function and therefore targeting this association may be a suitable therapeutic strategy. Methods and Results: Given the importance of BET bromodomain proteins in chromatin remodeling necessary for transcription, we tested the activity of the BET bromodomain inhibitor JQ1 on STAT5-dependent transcriptional activity. Using both heterologous reporter systems and endogenous STAT5 target genes, we found that JQ1, but not its inactive enantiomer, potently and specifically inhibited STAT5-dependent gene expression. Inhibition of STAT5 dependent gene regulation was also replicated by another BET bromodomian inhibitor, iBET, further demonstrating that BET inhibition inhibits STAT5. Since JQ1 inhibits BET family members Brd2, Brd3, BRD4, and BrdT, we asked which BET family member is specifically associated with STAT5 transcriptional function. To do this, we utilized shRNA to knock-down each bromodomain protein and determined the effect on STAT5 activity. We found that knocking-down Brd2, but not Brd3 or BRD4, reduces STAT5 target gene expression, indicating that Brd2 is specifically involved in regulating STAT5 transcriptional function. JQ1 can reduce STAT5 transcriptional activity without inhibiting STAT5 phosphorylation or STAT5 binding to its genomic binding sites. Similarly, knocking-down Brd2 can reduce STAT5 target gene expression without influencing STAT5 phosphorylation. We hypothesize that Brd2 regulates STAT5 transcriptional function by acting as a co-activator for STAT5. Thus through blocking Brd2, JQ1 can inhibit STAT5 transcriptional function without directly targeting STAT5 itself. In a group of aggressive T cell acute lymphoblastic leukemia (T-ALL) cell lines, where constitutively activated STAT5 contributes to leukemia cell survival, knocking-down Brd2 renders leukemia cells more sensitive to TKI induced apoptosis. In addition, combined treatment with TKIs and JQ1 showed strong synergy in inducing T-ALL leukemia cells apoptosis and reducing viability. Overexpressing a constitutively active form of STAT5 rescues these leukemia cells from death induced by TKIs and JQ1, indicating an important role of STAT5 as a target for TKI and JQ1 induced cell death in T-ALL cells. Conclusion: We found that the BET bromodomain inhibitor JQ1 can reduce STAT5 transcriptional function by blocking Brd2 without reducing STAT5 phosphorylation or STAT5 DNA binding. In addition, the combination of TKIs and JQ1 induces T-ALL leukemia cell apoptosis and reduces survival in a synergistic manner, and represents a rational drug combination for treating this sub-group of highly aggressive leukemias. Disclosures: Bradner: Tensha Therapeutics: Consultancy, Equity Ownership, Scientific founder Other.

Peter M Howley - One of the best experts on this subject based on the ideXlab platform.

  • the BRD4 extraterminal domain confers transcription activation independent of ptefb by recruiting multiple proteins including nsd3
    Molecular and Cellular Biology, 2011
    Co-Authors: Shaila Rahman, Mathew E Sowa, Matthias Ottinger, Jennifer A Smith, Yang Shi, Wade J Harper, Peter M Howley
    Abstract:

    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.

  • kaposi s sarcoma associated herpesvirus latency associated nuclear antigen interacts with bromodomain protein BRD4 on host mitotic chromosomes
    Journal of Virology, 2006
    Co-Authors: Jianxin You, Gerald V Denis, Viswanathan Srinivasan, William J Harrington, Mary E Ballestas, Kenneth M Kaye, Peter M Howley
    Abstract:

    The latency-associated nuclear antigen (LANA) of Kaposi's 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.

  • interaction of the bovine papillomavirus e2 protein with BRD4 tethers the viral dna to host mitotic chromosomes
    Cell, 2004
    Co-Authors: Jianxin You, Keiko Ozato, Jennie L Croyle, Akiko Nishimura, Peter M Howley
    Abstract:

    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.