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 now identify 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. A recent report that BRD4 activates transcription of a subset of genes independent of PTEFb supports our conclusions. 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 thus far. 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 three primary CTD kinases engage in crosstalk directly, as well as indirectly through 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 speculative 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. Elongation is dependent on the stimulation of TOP1activity, which relieves the torsional stress on the DNA duplex imposed by RNA Pol II 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.
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