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 eukaryotic 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. As pointed out above, the bromodomain protein Brd4 has been shown to interact with the low molecular weight, active P-TEFb complex and recruit P-TEFb to the HIV-1 LTR promoter. The subsequent events through which Brd4 affects CDK9 kinase activity and regulates RNAP II-dependent transcription are not clearly understood. By using a stepwise initiation and elongation complex, our most recent studies provide evidence that Brd4 regulates P-TEFb kinase activity by inducing a negative auto-phosphorylation pathway that regulates transcriptional elongation complexes. Brd4 induces phosphorylation of CDK9 at T29 in the HIV transcription initiation complex. P-TEFb inhibition is transient as Brd4 is released from the transcription complex between +14 and +36. Removal of the phosphate group at T29 by an incoming phosphatase released P-TEFb activity, resulting in increased RNAP II CTD phosphorylation and transcription. Finally, we present evidence by ChIP analysis that T-29 phosphorylated CDK9 is associated with HIV promoter region in the integrated and transcriptionally silent HIV genome. Tat induction of transcription, and release from the latent state, is accompanied by a decrease in T29 phosphorylation. These results suggest that Brd4-induced T29P may play an important step in HIV latency by inhibiting viral transcription.
Showing the most recent 10 out of 14 publications
