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

J Biol Chem. 2015 Jan 16;290(3):1829-36. doi: 10.1074/jbc.M114.605816. Epub 2014 Dec 9.

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: 7SK snRNP; drug screening; fluorescence; gene transcription; transcription elongation factor.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis
  • Azacitidine / chemistry
  • Bacterial Proteins / chemistry
  • Cell-Free System
  • 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
  • RNA-Binding Proteins / metabolism
  • Ribonucleoproteins / metabolism
  • Ribonucleoproteins, Small Nuclear / metabolism*
  • Time Factors
  • Transcription, Genetic
  • Vorinostat

Substances

  • Bacterial Proteins
  • Histone Deacetylase Inhibitors
  • Hydroxamic Acids
  • Luminescent Proteins
  • RNA-Binding Proteins
  • Ribonucleoproteins
  • Ribonucleoproteins, Small Nuclear
  • yellow fluorescent protein, Bacteria
  • Vorinostat
  • Positive Transcriptional Elongation Factor B
  • Protein Kinase C
  • Azacitidine
  • Glycerol