Bromodomain Protein Brd4 Regulates Human Immunodeficiency Virus Transcription through Phosphorylation of CDK9 at Threonine 29

Abstract

Positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 (CDK9) and cyclin T, is a global transcription factor for eukaryotic gene expression, as well as a key factor for human immunodeficiency virus (HIV) transcription elongation. P-TEFb phosphorylates the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II), facilitating the transition from nonprocessive to processive transcription elongation. Recently, 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 type 1 long terminal repeat (LTR) promoter. However, the subsequent events through which Brd4 regulates CDK9 kinase activity and RNAP II-dependent transcription are not clearly understood. Here we provide evidence that Brd4 regulates P-TEFb kinase activity by inducing a negative pathway. Moreover, by analyzing stepwise initiation and elongation complexes, we demonstrate that P-TEFb activity is regulated in the transcription complex. Brd4 induces phosphorylation of CDK9 at threonine 29 (T29) in the HIV transcription initiation complex, inhibiting CDK9 kinase activity. P-TEFb inhibition is transient, as Brd4 is released from the transcription complex between positions ؉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 chromatin immunoprecipitation studies showing that CDK9 with phosphorylated T29 is associated with the HIV promoter region in the integrated and transcriptionally silent HIV genome.The largest subunit of eukaryotic RNA polymerase II (RNAP II) has a carboxyl-terminal domain (CTD) consisting of tandem repeats of the consensus heptad peptide Tyr-SerPro-Thr-Ser-Pro-Ser, which is conserved among most eukaryotes (3,17,30). The CTD is phosphorylated at Ser 2 (Ser 2P) and Ser 5 (Ser 5P) as RNAP II progresses through the transcription initiation and elongation. At least three cyclindependent kinases are capable of phosphorylating the RNAP II CTD in the regulation of different stages of mRNA synthesis. The first kinase, cyclin-dependent kinase 7 (CDK7), along with MAT1 and cyclin H, forms a complex known as CDKactivating kinase which is part of the general transcription factor TFIIH (9, 23, 35, 45). As analyzed both in vitro and in vivo, TFIIH facilitates promoter clearance by preferentially phosphorylating Ser 5 of the RNAP II CTD within the promoter region (18,36,40). The second kinase, CDK9, is a cdc2-like serine/threonine kinase that was initially isolated by screening a human cDNA library by using oligonucleotide probes to identify CDK-related proteins (12). Consistent with the process of selection, the 43-kDa CDK9 protein shares a high degree of homology with cdc2, cdk2, cdk3, and cdk5. Understanding the function of CDK9 was facilitated by the discovery that CDK9 was the kinase subunit of positive transcription elongation factor b (P-TEFb), which supports transcri...

Cross-talk Among RNA Polymerase II Kinases Modulates C-terminal Domain Phosphorylation

“…186 phosphorylation and negatively by Thr-29 phosphorylation. It has been shown previously that during early elongation, BRD4 dissociates from the elongation complex, and the protein phosphatase PP2A specifically dephosphorylates the Thr-29 sites on CDK9 (16). However, CDK9 Thr-186 phosphorylation is only dephosphorylated by PPM1A/B (17), suggesting that Thr-186 phosphorylated by BRD4 persists through elongation.…”

“…Phosphorylation of Thr-29 inactivates its kinase activity, whereas phosphorylation of CDK9 at Thr-186 is crucial for its activity (15,16). The kinase(s) directly responsible for these phosphorylation events has not been identified although the presence of BRD4 is known to be a requirement (16,17). We therefore asked whether either of these sites is phosphorylated directly by BRD4.…”

Nucleophosmin Contributes to the Transcriptional Activation Function of the Epstein-Barr Virus EBNA1 Protein

“…The amino acid 61-to-83 region mediates an interaction with Brd4, an acetylated-histone-binding protein known to contribute to the transcriptional activation of both cellular and viral genes (7)(8)(9)(10). The central Gly-Arg region contributes to interactions with the histone chaperones NAP1 and TAF-I (11,12).…”

Obtenção das quinases dependentes de ciclinas CDK9 e CDK11 humanas utilizando um sistema bacteriano de expressão (E. coli)

“…2009 *A fosforilação da CDK9 em Thr29 é observada como resultado da associação do PTEFb com Brd4 (ZHOU et al, 2009).…”

“…Uma mudança crítica na composição dos complexos de transcrição é observada no início da fase de elongação -equivalente ao intervalo entre as posições +14 e +36 para o HIV-1 -que inclui a liberação de TFIIH e Brd4, os quais mantêm a CDK9 inativa por impedir a fosforilação da Thr186 e desfosforilação da Thr29, respectivamente. Para a desfosforilação da Thr29 é necessária ainda a associação da fosfatase PP2A ao complexo de transcrição, que ocorre neste mesmo intervalo ZHOU et al, 2009). Com a dispersão dos fatores inibitórios, ocorre a ativação de novo da CDK9 no interior do complexo de elongação, evitando desta maneira que o P-TEFb fosforile seus alvos prematuramente durante a iniciação.…”