Visualization of Positive Transcription Elongation Factor b (P-TEFb) activation in living cells [Cell Biology]

December 9th, 2014 by Fujinaga, K., Luo, Z., Schaufele, F., Peterlin, B. M.

Regulation of transcription elongation by positive transcription elongation factor b (P-TEFb) plays a central role in determining the state of cellular activation, proliferation, and differentiation. In cells, P-TEFb exists in active and inactive forms. Its release from the inactive 7SK small nuclear RiboNucleoProtein (7SK snRNP) complex is a critical step for P-TEFb to activate transcription elongation. However, there exist no good method to analyze this P-TEFb equilibrium in living cells. Only inaccurate and labor-intensive cell-free biochemical assays are currently available. In this study, we present the first experimental system to monitor P-TEFb activation in living cells. We created a Bimolecular Fluorescence Complementation (BiFC) assay to detect interactions between P-TEFb and its substrate, the C-terminal domain (CTD) of RNA polymerase II (RNAPII). When cells were treated with suberoylanilide hydroxamic acid (SAHA), which releases P-TEFb from the 7SK snRNP, cells turned green. Other known P-TEFb-releasing agents including histone deacetylase inhibitors (HDACis), bromodomain and extra-terminal bromodomain inhibitors (BETis) and protein kinase C (PKC) agonists also scored positive in this assay. Finally, we identified 5 azacytidine (AzaC) as a new P-TEFb-releasing agent. This release of P-TEFb correlated directly with the activation of the human immunodeficiency virus (HIV) and HEXamethylene bisacetamide induced protein 1 (HEXIM1) transcription. Thus, our Visualization of P-TEFb Activation by fluorescent Complementation (V-PAC) assay could be used to find new P-TEFb-releasing agents, compare different classes of agents and assess their efficacy singly and/or in combination.