Abstract
The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. In addition to its canonical role in transcriptional activation, recent studies have demonstrated that S. cerevisiae Mediator can interact directly with nucleosomes, and their histone tails. Mutations in Mediator subunits have shown that Mediator and certain chromatin structures mutually impact each other structurally and functionally in vivo. We have taken a UV photo cross-linking approach to further delineate the molecular basis of Mediator chromatin interactions and help determine whether the impact of certain Mediator mutants on chromatin is direct. Specifically, by using histone tail peptides substituted with an amino acid analog that is a UV activatible crosslinker, we have identified specific subunits within Mediator that participate in histone tail interactions. Using Mediator purified from mutant yeast strains we have evaluated the impact of these subunits on histone tail binding. This analysis has identified the Med5 subunit of Mediator as a target for histone tail interactions and suggests that the previously observed effect of med5 mutations on telomeric heterochromatin and silencing is direct.
Highlights
The eukaryotic Mediator complex is a transcriptional coactivator for a wide variety of DNA-bound transcription factors and serves additional intricate roles in the regulation of transcription [1]
Since deacetylated H4 K16 chromatin is a hallmark of silenced heterochromatin [27,28], these findings suggested that direct interactions between Mediator and a specialized chromatin structure at telomeres could lead to the targeting of Mediator to heterochromatin and its effect on silencing at these loci
We adopted Bpa(benzoyl-phenylalanine)-mediated UV crosslinking as a method to identify the Mediator subunits that are in close proximity to the H4 tail peptide when it is bound to Mediator
Summary
The eukaryotic Mediator complex is a transcriptional coactivator for a wide variety of DNA-bound transcription factors and serves additional intricate roles in the regulation of transcription [1]. A separate subset of proteins termed the Cdk module is variably associated with the core Mediator subunits [7,8]. Transcriptional profiling in vivo demonstrated that other Mediator subunits are essential for transcription of virtually all genes in S. cerevisiae [13], suggesting the complex was a general transcription factor. A number of genetic screens and experiments in S. cerevisiae have established an important role for some Mediator subunits in transcriptional repression and silencing [14,15,16,17,18,19,20]. Our recent work on telomeric silencing [21] and Mediator-chromatin [22] interactions suggests that the mechanism used by Mediator to facilitate repression involves an effect on chromatin
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