Visualization of Positive Transcription Elongation Factor b (P-TEFb) Activation in Living Cells

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 ri...

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Bibliographic Details
Published in:The Journal of biological chemistry 2015-01, Vol.290 (3), p.1829-1836
Main Authors: Fujinaga, Koh, Luo, Zeping, Schaufele, Fred, Peterlin, B. Matija
Format: Article
Language:English
Subjects:
7SK snRNP
Apoptosis
Azacitidine - chemistry
Bacterial Proteins - chemistry
Cell-Free System
drug screening
fluorescence
Gene Regulation
gene transcription
Genetic Complementation Test
Glycerol - chemistry
HEK293 Cells
HeLa Cells
Histone Deacetylase Inhibitors - chemistry
Humans
Hydroxamic Acids - chemistry
Luminescent Proteins - chemistry
Microscopy, Fluorescence
Plasmids - metabolism
Positive Transcriptional Elongation Factor B - metabolism
Protein Kinase C - metabolism
Protein Structure, Tertiary
Ribonucleoproteins - metabolism
Ribonucleoproteins, Small Nuclear - metabolism
RNA-Binding Proteins - metabolism
Time Factors
transcription elongation factor
Transcription, Genetic
Vorinostat
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Summary: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.Positive transcription elongation factor b (P-TEFb) partitions between free (active) P-TEFb and inactive 7SK small nuclear ribonucleoprotein (snRNP) in cells. Bimolecular fluorescence complementation (BiFC) detects interactions between active P-TEFb and its C-terminal domain substrate in vivo. BiFC follows the release of P-TEFb from 7SK snRNP in living cells. This system is the first to monitor P-TEFb activation in living cells.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M114.605816