Fission yeast Mediator complex and its role in transcriptional regulation

Sammanfattning: Mediator is an evolutionary conserved co-activator complex that regulates transcription of protein encoding genes in eukaryotes. This multiprotein complex was first identified in budding yeast and shown to be indispensable for RNA polymerase II (pol II) dependent transcription. Mediator is a transducer of regulatory signals from gene specific transcription factors to the general transcription machinery. Mediator has both activating and repressive functions, but the regulatory mechanisms are not yet completely understood. In our work, we have used a biochemical approach and identified four proteins, Med12, Med13, Cdk8, and CycC, as subunits of a repressive kinase module present in the Schizosaccharomyces pombe Mediator complex. Taking advantage of a reconstituted in vitro transcription system, we have tried to address the mechanism of transcriptional repression by this subcomplex. We have also addressed the function of the Med15 Mediator subunit, which has been characterized previously in other species. In Paper I we isolated a new form of the S. pombe Mediator complex to near homogeneity and denoted this complex Large (L-) Mediator. The LMediator contains four additional subunit not previously identified in the S. pombe core Mediator complex, but present in many of the large Mediator complexes isolated from higher eukaryotes. In contrast to the core Mediator complex, L-Mediator does not interact with pol II. Based on our biochemistry findings and bioinformatic analysis, our data suggest that Med12, Med13, Cdk8, and CycC form evolutionally conserved kinase module. In Paper II we developed an in vitro transcription system reconstituted with all the general transcription factors purified to near homogeneity, in either native or recombinant form. Fission yeast pol II assisted by TFIIB, TFIIF, TFIIE, TFIIH, and budding yeast TBP could initiate transcription from the S. pombe adh1 promoter in vitro. We found that addition of the core Mediator in complex with pol II could stimulate basal transcription, while L-Mediator suppressed transcription initiation in a dose dependent manner. Based on our findings, we proposed a model explaining the repressive function of L-Mediator. In Paper III we systematically addressed the structural organization of kinase module of L-Mediator. We identified Med13 as a key architectural subunit, anchoring the kinase module to the rest of the Mediator complex. Med13 was sufficient and necessary to occlude pol II from binding to Mediator, whereas deletion of Cdk8 and CycC did not affect Mediator association with pol II. In Paper IV we identified two new S. pombe Mediator components. The evolutionary conserved Med15 subunit associated with the chromatin remodeling protein Hrp1 and formed transient interactions with the S. pombe L-Mediator complex. Genome wide association data demonstrated Med15 association with a distinct subset of Hrp1 bound gene promoters.