Anti-CDK8 monoclonal antibody, clone 6I5

Background: Processive elongation of the integrated HIV-1 provirus is dependent on recruitment of P-TEFb by the viral Tat protein to the viral TAR RNA element. P-TEFb kinase activity requires phosphorylation of Thr186 in the T-loop of the CDK9 subunit. In resting CD4(+)T cells, low levels of T-loop phosphorylated CDK9 are found, which increase significantly upon activation. This suggests that the phosphorylation status of the T-loop is actively regulated through the concerted actions of cellular proteins such as Ser/Thr phosphatases. We investigated the role of phosphatase PPM1A in regulating CDK9 T-loop phosphorylation and its effect on HIV-1 proviral transcription. Results: We found that overexpression of PPM1A inhibits HIV-1 gene expression during viral infection and this required PPM1A catalytic function. Using an artificial CDK tethering system, we further found that PPM1A inhibits CDK9, but not CDK8 mediated activation of the HIV-1 LTR. SiRNA depletion of PPM1A in resting CD4(+)T cells increased the level of CDK9 T-loop phosphorylation and enhanced HIV-1 gene expression. We also observed that PPM1A protein levels are relatively high in resting CD4+T cells and are not up-regulated upon T cell activation. Conclusions: Our results establish a functional link between HIV-1 replication and modulation of CDK9 T-loop phosphorylation by PPM1A. PPM1A represses HIV-1 gene expression by inhibiting CDK9 T-loop phosphorylation, thus reducing the amount of active P-TEFb available for recruitment to the viral LTR. We also infer that PPM1A enzymatic activity in resting and activated CD4(+)T cells are likely regulated by as yet undefined factors.

Transcription corepressors are general regulators controlling the expression of genes involved in multiple signaling pathways and developmental programs. Repression is mediated through mechanisms including the stabilization of a repressive chromatin structure over control regions and regulation of Mediator function inhibiting RNA polymerase 11 activity. Using whole-genome arrays we show that the Arabidopsis thaliana corepressor LEUNIG, a member of the GroTLE transcription corepressor family, regulates the expression of multiple targets in vivo. LEUNIG has a role in the regulation of genes involved in a number of different physiological processes including disease resistance, DNA damage response, and cell signaling. We demonstrate that repression of in vivo LEUNIG targets is achieved through histone deacetylase (HDAC)-dependent and -independent mechanisms. HDAC-dependent mechanisms involve direct interaction with HDA19, a class 1 HDAC, whereas an HDAC-independent repression activity involves interactions with the putative Arabidopsis Mediator components AtMED14/SWP and AtCDK8/HEN3. We suggest that changes in chromatin structure coupled with regulation of Mediator function are likely to be utilized by LEUNIG in the repression of gene transcription.