BRD4 is emerging as an important player in cancer: in the etiology of cancer, metastasis, and epithelial-mesenchymal transition. Indeed, inhibition of BRD4 dramatically reduces aggressive AML, promotes regression of NUT midline carcinoma, and is immuno-modulatory, making it an attractive therapeutic target. Additionally, BRD4 regulates cell cycle progression, transcription elongation and viral infection. The wide range of BRD4 activities makes understanding its molecular mechanism(s) of action of interest to a broad community. Despite the wide-ranging interest in BRD4, there is relatively little understanding of its function. By binding to acetylated chromatin through its bromodomains, BRD4 recruits a variety of transcription factors to regions of active chromatin, including super-enhancers and typical enhancers and thus plays a passive role in transcriptional regulation. The goals of our studies is to determine whether BRD4 plays an active role in regulating transcription. Indeed, we have discovered that BRD4 is an atypical kinase that directly regulates eukaryotic transcription, providing new insight(s) into BRD4 activity and function. The goal of our studies is to further characterize the mechanisms by which BRD4 regulates transcription. BRD4, a bromodomain protein that contributes to the regulation of transcription initiation and elongation, has been implicated in regulation of both primary and metastatic tumor growth. However, the molecular mechanism(s) of BRD4 function remains unknown. Since BRD4 binds acetylated histones in chromatin, it has been presumed that it functions to recruit a critical transcriptional regulator (PTEFb) to promoters. We have identified BRD4 as a Pol II CTD Ser2 kinase that phosphorylates the CTD of Pol II independent of other CTD kinases, both in vitro and in vivo, providing a novel function for this tumor regulatory protein, which fundamentally alters our understanding of its mechanism of action. Although PTEFb, which is recruited by BRD4, was thought to be the predominant CTD Ser2 kinase thus far, we have found that BRD4 is equally capable of phosphorylating the CTD Ser2. As we have shown, CTD Ser2 is phosphorylated both under conditions where PTEFb is excluded from the preinitiation complex and in stem cell lines deficient in PTEFb. Our findings provide a mechanistic basis for several functional studies which demonstrated that loss of BRD4 causes transcription termination and embryonic lethality. Based on our results, we have suggested a new model of transcription initiation in which the initial phosphorylation of the Pol II CTD Ser2 is mediated by BRD4 during the transition from transcription initiation to elongation, and only subsequently by PTEFb during elongation. More recently, we have examined the regulation of BRD4 and the other CTD kinases, CDK7 and CDK9 in their phosphorylation of the Pol II CTD. Until now, characterization of Pol II CTD phosphorylation patterns has been focused primarily on the sequential timing of CTD phosphorylation patterns laid by respective CTD kinases. However, little has been known about the mechanisms responsible for the regulation of CTD kinases which results in the shifting patterns of CTD phosphorylations. We have discovered that the three primary CTD kinases engage in crosstalk directly, as well as indirectly through the transcription factor, TAF7, to actively modulate their ability to phosphorylate the Pol II CTD. We have identified two distinct layers that contribute to the modulation of CTD kinase activity: 1) direct regulatory mechanisms mediated by reciprocal phosphorylations of BRD4, PTEFb and TFIIH kinases and 2) indirect mechanisms mediated by TAF7 interactions. These findings lead to a model in which CTD kinase interactions serve to ensure that CTD phosphorylation events occur in an orderly and sequential fashion. BRD4 kinase activity also plays a critical role in transcription elongation. Transcription elongation is associated with the accumulation of torsional stress which impedes further elongation. Topoisomerase I (Top1) functions to remove torsional stress by transiently breaking one strand, allowing axial rotation of the DNA. Besides the relaxation of the excess torsional stress generated during transcription elongation, Top1 has been also linked to early events during transcription initiation in vitro. However, the mechanisms coordinating Top1 activity with RNA synthesis throughout the different stages of the transcription cycle remain obscure. We have shown that a positive feedback loop develops between Top1 and RNAPII during pause-release. As the carboxyl-terminal domain (CTD) of RNAPII is progressively phosphorylated, Top1 is stimulated above its intrinsic relaxation rate. This activation is strongly dependent on the kinase activity of BRD4 which phosphorylates serine 2 of CTD and participates in the regulation of the early events of transcription elongation. Our results demonstrate that phosphorylated RNAPII is required for maximal Top1 activity and activated Top1 is needed for efficient elongation. Top I stimulation is strongly dependent on the kinase activity of BRD4 and its phosphorylation of Ser2-CTD. Through its activation of Top 1, BRD4 regulates RNAPII pause-release. The coupling between the generation and removal of transcriptionally generated supercoils allows tuning of DNA topology to spur elongation while preserving the negative torsional stress at start sites that assists promoter melting. Considering the importance of Top1 inhibitors as anti-neoplastic agents and the compelling interest in the anti-cancer activity of BRD4/BET-inhibitors, these finding provide a new rationale for drug discovery and new approaches for combinatorial treatments. Most of the steps in, and many of the factors contributing to, glucocorticoid receptor (GR) regulated gene induction are currently unknown. The roles of BRD4 and NELF-E, a negative regulator of transcription, were examined using a novel competition assay, based on a validated chemical kinetic model of steroid hormone action. This assay was used to define their sites and mechanisms of action. BRD4 is a kinase involved in numerous initial steps of gene induction. Consistent with its complicated biochemistry, BRD4 was found to alter both the maximal activity (Amax) and the steroid concentration required for half-maximal induction (EC50) of GR-mediated gene expression by acting at a minimum of three different kinetically-defined steps. The action at two of these steps is dependent on BRD4 concentration while the third step requires the association of BRD4 with P-TEFb. BRD4 was also found to bind to NELF-E, a component of the NELF-complex. Unexpectedly, NELF-E modifies GR induction in a manner that is independent of the NELF complex. Several of the kinetically-defined steps of BRD4 were related to its known biochemical actions. However, novel actions of BRD4 and of NELF-E in GR controlled gene induction have been uncovered. The model-based competition assay is also unique in being able to order, for the first time, the sites of action of the various reaction components, which is GR - Cdk9 - BRD4 -induced gene - NELF-E. This ability to order factor actions will assist efforts to reduce the side-effects of steroid treatments.
| Devaiah, Ballachanda N; Gegonne, Anne; Singer, Dinah S (2016) Bromodomain 4: a cellular Swiss army knife. J Leukoc Biol 100:679-686 |
| Baranello, Laura; Wojtowicz, Damian; Cui, Kairong et al. (2016) RNA Polymerase II Regulates Topoisomerase 1 Activity to Favor Efficient Transcription. Cell 165:357-71 |
| Pradhan, Madhumita A; Blackford Jr, John A; Devaiah, Ballachanda N et al. (2016) Kinetically Defined Mechanisms and Positions of Action of Two New Modulators of Glucocorticoid Receptor-regulated Gene Induction. J Biol Chem 291:342-54 |
| Devaiah, Ballachanda N; Singer, Dinah S (2013) Two faces of brd4: mitotic bookmark and transcriptional lynchpin. Transcription 4:13-7 |
| Devaiah, Ballachanda N; Lewis, Brian A; Cherman, Natasha et al. (2012) BRD4 is an atypical kinase that phosphorylates serine2 of the RNA polymerase II carboxy-terminal domain. Proc Natl Acad Sci U S A 109:6927-32 |
| Devaiah, Ballachanda N; Singer, Dinah S (2012) Cross-talk among RNA polymerase II kinases modulates C-terminal domain phosphorylation. J Biol Chem 287:38755-66 |
