The VP16 Activation Domain Establishes an Active Mediator Lacking CDK8 in Vivo

Abstract

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.The herpes simplex virus-encoded protein VP16 is a potent activator that controls the transcription of immediate early viral genes through the interaction with host cell factors (1). VP16 harbors a strong activation domain that functions well in both yeast and human cells when tethered to independent DNA-binding domains (2-4). Based on these properties, fusion proteins of VP16 with GAL4 or DNA-binding domains of other transactivators have been widely used as models to investigate the basic principles of transcription activation.In conjunction with molecular interactions involving the general transcription factors TFIIA, 3 TBP, TFIIB, and the TBPassociated factors (5-9), VP16 increases the number of functional pre-initiation complexes in vitro. Furthermore, VP16 stimulates open complex formation, which relates to interactions with the p62 subunit of TFIIH and the general cofactor PC4 (10).Comparative binding studies and functional experiments pointed to a key role for the cofactors CBP, STAGA/TFTC, and Mediator (11-16) in activation by VP16 (17). CBP interacts specifically with the C-terminal (H2) region of VP16, whereas Mediator binds tightly to both the N-terminal (H1) and C-terminal (H2) regions of the activation domain (18, 19). The histone acetyltransferase activity of CBP (p300) is critical for activation by VP16 in chromatin (20, 21). The other major histone acetyltransferase, GCN5, which is part of the SAGA-STAGA/ TFTC complex, is also recognized by VP16 (17,22,23). Further related to chromatin accessibility, the remodeling Swi/Snf complex interacts with VP16 (24).VP16 binds Mediator tightly and specifically through the MED17 and the MED25 subunits (19,25). Overexpression of the MED25 Mediator-binding domain has a dominant negative effect on VP16 activation, which substantiates the critical influence of a MED25-associated Mediator (19). Furthermore, Mediator apparently functions synergistically with other target proteins such as the histone acetyltransferases, CBP and GCN5, that are also necessary for activation by VP16 in living cells (17).Here, we use an inducible Tet-VP16 in combination...

Polycomb Repressive Complex 1 (PRC1) Disassembles RNA Polymerase II Preinitiation Complexes

“…In the case of VP16, Mediator interacts via MED25 (46,47) and TFIID via TAF9 (48). VP16 is known to recruit both complexes to DNA in vitro and in vivo (49).…”

RNA Polymerase II C-terminal Heptarepeat Domain Ser-7 Phosphorylation Is Established in a Mediator-dependent Fashion

“…Jurkat nuclear extracts were prepared as described previously (39). Jurkat nuclear extracts were immunodepleted using anti-RNAPII (1C7), MED15 (1H7), and MED25 (VC1) rat monoclonal antibodies, anti-TBP, TFIIH p62, CDK7, and CDK9 rabbit polyclonal antibodies (Santa Cruz sc-273, sc-292, sc-529, and sc-8338, respectively), and anti-TFIIB (IIB8) mouse monoclonal antibodies (Santa Cruz sc-23875) or anti-CDK8 goat polyclonal antibodies (Santa Cruz sc-1521).…”

“…The salt concentration was adjusted to a final concentration of 60 mM KCl. The substrate used in the kinase assay, GST-CTD, was expressed in Escherichia coli and affinity-purified on glutathione-Sepharose as described previously (39). Approximately 0.1 g of GST-CTD was included in a single 50-l reaction.…”

“…Here we used an active nuclear extract from human Jurkat T-cells as a source for general factors, RNAPII and CTD kinases. The DNA template comprised five GAL4 binding sites upstream of the adenovirus ML core promoter (39). Templates were then washed briefly, and proteins bound to them were eluted and analyzed in Western blots.…”

“…More importantly, its presence closely correlated to the formation of active transcription complexes (39,46). We, therefore, asked whether Mediator-associated RNAPII is phosphorylated at Ser-7.…”