Mediator co‐activator function is controlled by activator‐induced structural shifts

The human Mediator complex is a general factor that helps regulate expression of nearly all protein‐coding genes and thus lies at the heart of transcriptional regulation. Activator binding triggers major structural shifts within Mediator, suggesting a straightforward means to spatially and temporally regulate Mediator activity. 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 reveal a non‐overlapping set of co‐regulatory 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 structural shifts regulate Mediator function in gene‐specific ways.