Structure of the VP16 transactivator target in the Mediator

Abstract

The human Activator-Recruited Cofactor (ARC)/Mediator co-activator complex interacts with many transcriptional activators and facilitates recruitment of RNA polymerase II to promote target gene transcription. The MED25 (ARC92) subunit is a critical target of the potent Herpes simplex 1 viral transcriptional activator VP16. Here, we determine the solution structure of the MED25 VP16-binding domain (VBD), and define its binding site for the N-terminal portion of the VP16 transactivation domain (TADn). A hydrophobic furrow, formed by a β-barrel and two α-helices in MED25 VBD, interacts tightly with VP16 TADn. Mutations in this furrow prevent binding of VP16 TAD to MED25 VBD and interfere with the ability of over-expressed MED25 VBD to inhibit VP16-dependent transcriptional activation in vivo. This detailed molecular understanding of transactivation by the benchmark activator VP16 could provide important insights into viral and cellular gene activation mechanisms.

Role for Human Mediator Subunit MED25 in Recruitment of Mediator to Promoters by Endoplasmic Reticulum Stress-responsive Transcription Factor ATF6α

“…We present several lines of evidence consistent with the idea that ATF6␣ recruits Mediator to ER stress response genes via direct interactions with its MED25 subunit. First, consistent with our previous observation that the VP16 AD competes with ATF6␣ for binding to Mediator in nuclear extracts (12), we observe that the purified ATF6␣ AD binds stably and specifically in vitro to the purified MED25 VBD, a MED25 domain previously shown to be the binding site on Mediator for the viral VP16 AD (13)(14)(15)(16)(17)(18). Second, in reconstitution experiments, we obtained evidence that Mediator containing a MED25 mutant that lacks the VBD fails to bind efficiently to ATF6␣ but binds to liganded thyroid receptor, which is known to bind to Mediator through LXXLL motifs in its MED1 subunit (33)(34)(35).…”

“…Furthermore, we discovered that the VP16 AD potently competes with ATF6␣ for binding to the Mediator, raising the possibility that the ATF6␣ and VP16 ADs might bind to the same or overlapping surfaces on Mediator. In light of previous studies demonstrating that the VP16 AD binds to the Mediator through its MED25 subunit (13)(14)(15)(16)(17)(18), we initiated experiments to test the possibility that the ATF6␣ AD might also target Mediator through MED25. Below we present our findings, which identify MED25 as a docking site for transcription factor ATF6␣ on the multisubunit human Mediator complex.…”

A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface

“…The function of most tested ADs is conserved among eukaryotes (15,16), even though some key activator targets are not conserved. For example, the herpes virus protein VP16 strongly activates transcription in both yeast and mammalian cells, although human Med25, a critical target of VP16 in humans, is not found in yeast (17,18). Thus, the ability to adapt to different coactivator targets is seemingly a key property of ADs.…”

Functional Characterization of the Serine-Rich Tract of Varicella-Zoster Virus IE62

“…When the IE62 TAD and SRT were moved to the C-terminal end of the IR2P, the IR2P-62TS lost most of its trans-activation activity (Fig. 6C, bar 6 (45). VZV IE62 TAD shows 27% amino acid sequence identity with EHV-1 IEP TAD (Fig.…”

“…6). Both the IE62 TAD and VP16 TAD interact with the VP16-binding domain (VBD; Med25 aa 390 to 553) (17,45) of Mediator 25 which is the same domain with an activator-interacting domain (ACID; Med25 aa 391 to 543) (61). However, the interaction between the IE62 TAD and Mediator 25 was considerably weaker than that involving the VP16 (17,25).…”