Activator-Mediator binding regulates Mediator-cofactor interactions.

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.