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.
Highlights
Positive transcription elongation factor b (P-TEFb) partitions between free positive transcription elongation factor b (P-TEFb) and inactive 7SK small nuclear ribonucleoprotein in cells
Hybrid YC.P-TEFb Proteins Are Incorporated into the 7SK small nuclear ribonucleoprotein (snRNP) and Released by suberoylanilide hydroxamic acid (SAHA)—Originally described by Kerppola [20], Bimolecular fluorescence complementation (BiFC) is based on the formation of active fluorophore by complementary fragments of a fluorescent protein
When YN and YC are brought to close proximity via association between the fusion partners, they form an active fluorophore, allowing protein-protein interactions to be visualized in living cells [20]
Summary
Positive transcription elongation factor b (P-TEFb) partitions between free (active) P-TEFb and inactive 7SK small nuclear ribonucleoprotein (snRNP) in 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. Methods include glycerol gradient centrifugation, co-immunoprecipitation between 7SK snRNP subunits, gel filtration, RNA immunoprecipitation between P-TEFb and 7SK, and differential salt extractions [3, 15,16,17,18,19] We conclude that V-PAC is the first method capable of monitoring P-TEFb activation quantitatively in living cells It can measure the activity of known P-TEFb-releasing agents and be used to screen for yet unknown P-TEFb activators. It may prove invaluable in the development therapeutic approaches for diseases such as AIDS and cancer, in which the activity of P-TEFb plays a major role
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