Michela Venturi:
The Schizosaccharomyces pombe Mediator. Roles in heterochromatinization and analysis of additional subunits Med9/Med11

Date: 28-03-2013    Supervisor: Steen Holmberg

In the past several years great attention has been dedicated to the characterization of the Mediator complex in a different range of model organisms. Mediator is a conserved co-activator complex involved in transcriptional regulation and it conveys signals from regulatory transcription factors to the basal transcription machinery. Mediator was initially isolated from Saccharomyces cerevisiae based on its ability to render a RNA polymerase II in vitro transcription system responsive to activators. Additionally, structural studies have revealed striking structural similarities between S. cerevisiae, Schizosaccharomyces pombe and mammalian Mediator.

In our study, we have taken the S. pombe Mediator into consideration and characterized genetically and biochemically two subunits already know in S. cerevisiae, Med9 and Med11, but still not identified in the S. pombe Mediator. Genetic analysis has shown that med9+ is a nonessential gene, while deletion of med11+ resulted in unviable cells. These results are in line with those obtained in S. cerevisiae. Isolation of S. pombe Mediator by the tandem affinity purification method and Co-IP experiments lead to the conclusion that Med9 and Med11 might not belong to the Mediator complex, but our results did not exclude it completely either. Our attempts to demonstrate the presence of these two subunits in the Mediator complex remain inconclusive primarily due to the lack of proper expression of the tagged versions of the proteins. However, we have paved a way to further experiments that might shed light on the structure of the S. pombe Mediator.

S. pombe is a well known and used model organism for heterochromatin pathway studies. S. pombe has large and complex centromeres comprising a central core surrounded by inner and outer repetitive sequences. Silencing and heterochromatinization of the repeats depend on the RNA interference (RNAi) pathway. RNAi relies on transcription of the centromeric repeats by RNA Pol II. Centromeric transcripts are processed into siRNA by the RNAi machinery, leading to the recruitment and accumulation at centromeres of several interacting protein complexes and histonemodifying enzymes. Consistent with transcription being performed by RNA Pol II, centromeric transcripts are poly-adenylated and specific mutations in RNA Pol II subunits impair heterochromatin formation. The involvement of RNA Pol II in heterochromatin assembly indicates that the Mediator complex may also play a role in heterochromatin biology.

This feature allowed us to carry out several studies, taking three Mediator mutants into consideration: Δmed9, Δmed12, and Δmed18. We analyzed these Mediator deletion mutants in relation to the transcriptional regulation of centromeric repeats and thus in heterochromatin formation, centromere function and chromosome segregation. The mitotic loss of a non-essential mini-chromosome was affected in a Δmed18 strain. Also, our data demonstrated that Med18 regulates transcriptional repression of the centromeric repeats thereby affecting the functionality of the centromeres. The same results were obtained by Thorsen et al. (Epigenetics.Chromatin., 5, 19, 2012) using a different strain background. Moreover, we also observed that Med18 and Med9 were involved in telomere length maintenance. In summary, our data have demonstrated and confirmed the importance of Mediator function in the heterochromatic pathway, both at centromere and telomere levels.