Transcription activation by targeted recruitment of the RNA polymerase II CTD phosphatase FCP1.

Human FCP1 in association with RNAP II reconstitutes a highly specific CTD phosphatase activity and is required for recycling RNA polymerase II (RNAP II) in vitro. Here we demonstrate that targeted recruitment of FCP1 to promoter templates, through fusion to a DNA-binding domain, stimulates transcription. We demonstrate that a short region at the C-terminus of the FCP1 protein is required and sufficient for activation, indicating that neither the N-terminal phosphatase domain nor the BRCT domains are required for transcription activity of DNA-bound FCP1. In addition, we demonstrate that the C-terminus region of FCP1 suffices for efficient binding in vivo to the RAP74 subunit of TFIIF and is also required for the exclusive nuclear localization of the protein. These findings suggest a role for FCP1 as a positive regulator of RNAP II transcription.

Inhibition of Tat transactivation by the RNA polymerase II CTD-phosphatase FCP1.

OBJECTIVES: To asses the role of the RNAPII carboxy-terminal domain (CTD) phosphatase FCP1 on HIV-1 Tat-mediated transactivation. DESIGN: Construction of expression vectors encoding FCP1 phosphatase and analysis of their functions on Tat activity. METHODS: Basal and Tat-mediated transactivation of HIV-1 long terminal repeat (LTR)-driven transcription was compared, by transient transfections, in the presence of FCP1 phosphatase. Protein interactions were analysed by in vitro binding assays. RESULTS: FCP1 specifically and effectively represses Tat transactivation but not HIV-1 LTR-basal transcription. Protein interaction assays demonstrated that FCP1 specifically and directly binds Tat in vitro. CONCLUSION: The specific and efficient inhibitory function of FCP1 highlights the important role of this CTD-phosphatase in Tat-mediated transactivation, and it suggests that FCP1 might represent a specific target for modulation of Tat activity in infected cells.

The CDK9-associated cyclins T1 and T2 exert opposite effects on HIV-1 Tat activity.

OBJECTIVES: To examine the functional interaction between HIV-1 Tat protein and the cyclin T1 and T2 proteins which, in association with cyclin dependent kinase (CDK)9, are the regulatory subunits of the TAK/P-TEFb cellular complex strictly required for Tat transactivation. DESIGN: HIV-1 long terminal repeat (LTR) reporter plasmid was co-transfected into human and rodent cells with expression vectors encoding Tat and vectors encoding the cyclins T1, T2a and T2b, respectively. METHODS: Tat-mediated transactivation of HIV-1 LTR-driven transcription was compared in the presence or absence of different cyclins T (T1, T2a and T2b), upon co-transfections into human and rodent cell lines. Protein interactions were analysed by in vitro binding assays. RESULTS: It was found that Tat function in rodent cells is enhanced by co-expression of cyclin T1 but not cyclin T2. The N-terminal region (amino acids 1-290) of cyclin T1 is sufficient for this function and for binding to Tat and CDK9. Cyclin T2 binds to CDK9 but not to Tat. Moreover, enforced expression of cyclin T2 inhibits cyclin T1-mediated enhancement of Tat in rodent cells and it represses Tat activity in human cells. CONCLUSION: Efficient Tat transactivation in rodent cells occurs in the presence of human cyclin T1 but not in the presence of cyclin T2; overexpression of cyclin T2 inhibits Tat function in both rodent and human cells.

Transcriptional activity of positive transcription elongation factor b kinase in vivo requires the C-terminal domain of RNA polymerase II.

Phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) is an important step in transcription and the positive transcription elongation factor b (P-TEFb) has been proposed to facilitate elongation at many genes. The P-TEFb contains a catalytic subunit (Cdk9) that, in association with a cyclin subunit (cyclinT1), has the ability to phosphorylate the CTD substrate in vitro. Here, we demonstrate that cyclinT1/Cdk9-mediated transcription requires CTD-containing RNAPII, suggesting that the CTD is the major target of the cyclinT1/Cdk9 complex in vivo. Unlike Cdk7 and Cdk8, two other cyclin-dependent kinases that are capable of phosphorylating the CTD in vitro, we found that only the Cdk9 activates gene expression in a catalysis-dependent manner. Finally, unlike cyclinT1 and T2, we found that the targeted recruitment to promoter DNA of cyclinK (a recently described alternative partner of Cdk9) does not stimulate transcription in vivo. Collectively, our data strongly indicate that the P-TEFb kinase subunits cyclinT/Cdk9 are specifically involved in transcription and the CTD domain of RNAPII is the major functional target of this complex in vivo.

Distinct regions of cyclinT1 are required for binding to CDK9 and for recruitment to the HIV-1 Tat/TAR complex.

Tat-mediated activation of the HIV-1 promoter activity requires Tat-dependent recruitment of the cyclinT1/CDK9 complex (P-TEFb) to the transacting element (TAR) RNA. Tat interaction with the cyclinT1, the regulatory partner of CDK9, results in a specific recruitment of the heterodimer CycT1/CDK9 complex to TAR, whereby it promotes transcription elongation of the HIV-1 LTR-mediated transcription. Using the yeast two-hybrid protein interaction assay we analyzed the binding between cyclinT1 and CDK9. Moreover, using a modified three-hybrid yeast interaction system, we analyzed the recruitment of CycT1 to the Tat/TAR complex. The data presented here demonstrated that distinct domains of cyclinT1 interact with CDK9 and Tat/TAR in vivo. These findings will be instrumental for the designing of proper dominant-negative P-TEFb components capable to interfere with Tat function. J. Cell. Biochem. Suppl. 36: 247-253, 2001.