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. Our most recent studies provide evidence that Brd4 regulates P-TEFb kinase activity and that P-TEFb activity is regulated in the transcription elongation complex. Briefly, 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 and activates transcription. Finally, we present evidence by ChIP analysis that promoter bound CDK9 is phosphorylated at T29 in the latent state. 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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC005753-15
Application #
7592552
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
2007
Total Cost
$324,896
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Bonome, Tomas; Levine, Douglas A; Shih, Joanna et al. (2008) A gene signature predicting for survival in suboptimally debulked patients with ovarian cancer. Cancer Res 68:5478-86
Gegonne, Anne; Weissman, Jocelyn D; Lu, Hanxin et al. (2008) TFIID component TAF7 functionally interacts with both TFIIH and P-TEFb. Proc Natl Acad Sci U S A 105:5367-72
Cho, Won-Kyung; Zhou, Meisheng; Jang, Moon Kyoo et al. (2007) Modulation of the Brd4/P-TEFb interaction by the human T-lymphotropic virus type 1 tax protein. J Virol 81:11179-86
Zhou, Meisheng; Lu, Hanxin; Park, Hyeon et al. (2006) Tax interacts with P-TEFb in a novel manner to stimulate human T-lymphotropic virus type 1 transcription. J Virol 80:4781-91
Gegonne, Anne; Weissman, Jocelyn D; Zhou, Meisheng et al. (2006) TAF7: a possible transcription initiation check-point regulator. Proc Natl Acad Sci U S A 103:602-7
Jang, Moon Kyoo; Mochizuki, Kazuki; Zhou, Meisheng et al. (2005) The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription. Mol Cell 19:523-34
Ammosova, Tatyana; Washington, Kareem; Debebe, Zufan et al. (2005) Dephosphorylation of CDK9 by protein phosphatase 2A and protein phosphatase-1 in Tat-activated HIV-1 transcription. Retrovirology 2:47
Watanabe, Takahiro; Sugaya, Makoto; Atkins, April M et al. (2003) Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen prolongs the life span of primary human umbilical vein endothelial cells. J Virol 77:6188-96
Zhou, Meisheng; Deng, Longwen; Kashanchi, Fatah et al. (2003) The Tat/TAR-dependent phosphorylation of RNA polymerase II C-terminal domain stimulates cotranscriptional capping of HIV-1 mRNA. Proc Natl Acad Sci U S A 100:12666-71
Zhou, M; Kashanchi, F; Jiang, H et al. (2000) Phosphorylation of the RAP74 subunit of TFIIF correlates with Tat-activated transcription of the HIV-1 long terminal repeat. Virology 268:452-60

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