The VP16 activation domain establishes an active mediator lacking CDK8 in vivo.
VP16 has been widely used to unravel the mechanisms underlying gene transcription. Much of the previous work has been conducted in reconstituted in vitro systems. Here we study the formation of transcription complexes at stable reporters under the control of an inducible Tet-VP16 activator in living cells. In this simplified model for gene activation VP16 recruits the general factors and the cofactors Mediator, GCN5, CBP, and PC4, within minutes to the promoter region. Activation is accompanied by only minor changes in histone acetylation and H3K4 methylation but induces a marked promoter-specific increase in H3K79 methylation. Mediated through contacts with VP16 several subunits of the cleavage and polyadenylation factor (CPSF/CstF) are concentrated at the promoter region. We provide in vitro and in vivo evidence that VP16 activates transcription through a specific MED25-associated Mediator, which is deficient in CDK8.
Uhlmann
Thomas
T
Gene Expression, National Research Center for Environment and Health, Marchionini-Strasse 25, D-81377 Munich, Germany.
Boeing
Stefan
S
Lehmbacher
Michael
M
Meisterernst
Michael
M
D001665:Binding Sites; D056850:Cyclin-Dependent Kinase 8; D018844:Cyclin-Dependent Kinases / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D056892:Mediator Complex; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
D017405:Herpes Simplex Virus Protein Vmw65; C483045:MED25 protein, human; D056892:Mediator Complex; D015534:Trans-Activators; C095686:CDK8 protein, human; D056850:Cyclin-Dependent Kinase 8; D018844:Cyclin-Dependent Kinases