Author/Editor		Fujinaga, Koh; Luo, Zeping; Schaufele, Fred; Peterlin, Matija Boris
Title		Visualization of positive transcription elongation factor b (P-TEFb) activation in living cells
Type		članek
Vol. and No.		Letnik 290, št. 3
Publication year		2015
Volume		str. 1829-1836
ISSN		0021-9258 - The Journal of biological chemistry
Language		eng
Abstract		Regulation of transcription elongation by positive transcription elongation factor b (P-TEFb) plays a central role in determining the state of cell activation, proliferation, and differentiation. In cells, P-TEFb exists in active and inactive forms. Its release from the inactive 7SK small nuclear ribonucleoprotein complex is a critical step for P-TEFb to activate transcription elongation. However, no good method exists 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 assay to detect interactions between P-TEFb and its substrate, the C-terminal domain of RNA polymerase II. When cells were treated with suberoylanilide hydroxamic acid, which releases P-TEFb from the 7SK small nuclear ribonucleoprotein, they turned green. Other known P-TEFb-releasing agents, including histone deacetylase inhibitors, bromodomain and extraterminal bromodomain inhibitors, and protein kinase C agonists, also scored positive in this assay. Finally, we identified 5%-azacytidine as a new P-TEFb-releasing agent. This release of P-TEFb correlated directly with activation of human HIV and HEXIM1 transcription. Thus, our visualization of P-TEFb activation by fluorescent complementation 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.
Keywords		transcription living cells cell activation transkripcija žive celice aktivacija celic