Human immunodeficiency virus type 1 (HIV-1) encodes the transactivating protein Tat, which is essential for virus replication and progression of HIV disease. Tat has multiple domains and consequently the molecular mechanisms by which Tat regulates viral and cellular gene expression are complex.P-TEFb, composed of CDK9 and cyclin T, is a global transcription factor for ekaryotic gene transcription as well as a key factor in Hiv Tat transactivation. Brd4 is a mammalian bromodomain protein that binds to acetylated chromatin. In collaboration with Dr. Keiko Ozato, NICHD, we recently reported Brd4 interacts with P-TEFb and plays a major role in P-TEFb regulation. Proteomic analysis revealed that Brd4 interacts with cyclin T1 and Cdk9 that constitutes core positive transcription elongation factor b (P-TEFb). P-TEFb is found in two major pools in the cell. The inactive cytoplasmic fraction contains P-TEFb in association with 7SK/HEXIM1. Brd4 was found associated with active nuclear P-TEFb. 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. These studies provide important insight into P-TEFb regulation and suggest that P-TEFb alternately interacts with Brd4 and the inhibitory subunit to maintain functional equilibrium in the cell. P-TEFb phosphorylates the caboxy-terminal domain (CTD) of the large subunit of RNA polymerase II, leading to a transition from nonprocessive to processive transcription. Brd4 has been shown to recruit P-TEFb to the HIV promoter. However, subsequent events through which Brd4 affects kinase activity of P-TEFb and regulates RNAP II-dependent transcription are not clearly understood. Our results demonstrate a dynamic association of Brd4 with the HIV promoter and regulation of P-TEFb kinase activity by Brd4. Brd4 is released from the HIV transcription complex between +14 and +36. Unexpectedly, our data show that Brd4 inhibits CDK9 kinase activity by inducing phosphorylation of threonine 29, an inhibitory autophosphorylation site in CDK9. Relief of Brd4 inhibition is overcome by the recruitment of a phosphatase to the elongation complex, allowing CDK9 kinase activity to increase transcription from the HIV LTR.
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