Activator-Mediator binding regulates Mediator-cofactor interactions

Abstract

The 26-subunit, 1.2 MDa human Mediator complex is essential for expression of perhaps all protein-coding genes. Activator binding triggers major structural shifts within Mediator, suggesting a straightforward means to spatially and temporally regulate Mediator activity. By using mass spectrometry (MudPIT) and other techniques, we have compared the subunit composition of Mediator in three different structural states: bound to the activator SREBP-1a, VP16, or an activator-free state. As expected, consensus Mediator subunits were similarly represented in each sample. However, we identify a set of cofactors that interact specifically with activator-bound but not activator-free Mediator, suggesting activator binding triggers new Mediator-cofactor interactions. Furthermore, MudPIT combined with biochemical assays reveals a nonoverlapping set of coregulatory factors associated with SREBP-Mediator vs. VP16-Mediator. These data define an expanded role for activators in regulating gene expression in humans and suggest that distinct, activator-induced structural shifts regulate Mediator function in gene-specific ways.

Fig. 1.

Activator binding triggers new Mediator-cofactor interactions. (A) Orthogonal purification scheme for confirmation of Mediator-associated cofactors. (B) Silver-stained acrylamide gels representing various stages in the orthogonal purification: IP input, A/G-beads only negative control, and CDK8 or MED1 IP elutions. Subunit identities are listed at the right. (C) Western blots for various cofactors identified from the MudPIT analysis. Note most, but not all, cofactors were confirmed as Mediator-associated in this assay. LRP130, HADHA, SKIV2L2, and SnoN did not track with Mediator through the orthogonal purification, suggesting these factors likely interact directly with the SREBP activation domain and not Mediator.

Fig. 2.

Distinct cofactors associate with SREBP-Mediator vs. VP16-Mediator. (A) Silver-stained acrylamide gels showing glycerol gradient fractions from the purification protocols shown in B. Mediator-containing fractions are denoted by the red boxes. (C) Quantitative Western blots confirm MudPIT data. Mediator-associated factors probed in immunoblotting experiments are shown at left. Factors shown in black font were observed to be present in both the SREBP-Mediator and VP16-Mediator samples, whereas factors shown in blue font were observed only in the SREBP-Mediator sample by MudPIT.

Fig. 3.

A model that summarizes the results and implications of this study. Mediator-cofactor interactions do not occur in the activator-free state; rather, activator binding signals a shift in Mediator structure only when engaged at the promoter, providing a straightforward means by which Mediator activity can be controlled in a spatial and temporal fashion. Note that activator binding not only enables Mediator-cofactor interactions, but different activators, which induce different structural shifts within Mediator, trigger interaction with distinct sets of coregulatory factors, providing a mechanism by which Mediator can adopt activator-specific functionality.