title: Visualization of positive transcription elongation factor b (P-TEFb) activation in living cells.
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.
authors.affiliation: From the Departments of Medicine, Microbiology, and Immunology and.
authors.forename: Koh
authors.initials: K
authors.lastname: Fujinaga
authors.affiliation: From the Departments of Medicine, Microbiology, and Immunology and.
authors.forename: Zeping
authors.initials: Z
authors.lastname: Luo
authors.affiliation: the Diabetes and Endocrinology Research Center, University of California, San Francisco, California 94143-0703.
authors.forename: Fred
authors.initials: F
authors.lastname: Schaufele
authors.affiliation: From the Departments of Medicine, Microbiology, and Immunology and matija.peterlin@ucsf.edu.
authors.forename: B Matija
authors.initials: BM
authors.lastname: Peterlin
mesh_terms: D017209:Apoptosis; D001374:Azacitidine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D002474:Cell-Free System; D005816:Genetic Complementation Test; D005990:Glycerol / Q000737:chemistry; D057809:HEK293 Cells; D006367:HeLa Cells; D056572:Histone Deacetylase Inhibitors / Q000737:chemistry; D006801:Humans; D006877:Hydroxamic Acids / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000378:metabolism; D042925:Positive Transcriptional Elongation Factor B / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D013997:Time Factors; D014158:Transcription, Genetic; D000077337:Vorinostat
chemical_list: D001426:Bacterial Proteins; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D008164:Luminescent Proteins; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; C054562:yellow fluorescent protein, Bacteria; D000077337:Vorinostat; D042925:Positive Transcriptional Elongation Factor B; D011493:Protein Kinase C; D001374:Azacitidine; D005990:Glycerol
keywords: 7SK snRNP; drug screening; fluorescence; gene transcription; transcription elongation factor