Studies of the chromatin form of yeast Mediator and the function of its tail module

Abstract: Mediator is a large, multifunctional complex that is conserved in eukaryotes. It was first discovered in Saccharomyces cerevisiaeas required for transcriptional activators to function in a reconstituted in vitro system. Mediator is also important for stimulation of basal, unregulated transcription, and transcriptional repression. In yeast, Mediator consists of 25 subunits divided into head, middle, and tail modules, and is intermittently associated with a Cdk8 kinase module (CKM). The head and middle bind to the RNA polymerase II (Pol II) while the tail is responsible for binding to gene-specific transcriptional regulators. Most head and middle subunits are essential, whereas all tail module subunits are encoded by non-essential genes. CKM is mostly involved in transcriptional repression by binding Mediator in a way that sterically blocks the binding of Pol II to Mediator. Mediator is traditionally purified from the ‘non-chromatin’ fraction of whole-cell extracts. Since most Mediator functions occur in a chromatin context, we set out to purify Mediator from the chromatin fraction of cell extracts. We performed affinity-purification using strains expressing epitope-tagged Mediator subunits, combined with mass spectrometry to reveal the composition of chromatin-bound Mediator. We found that Mediator in chromatin interacts with several protein complexes involved in different aspects of gene expression. Several of them, such as CPF, CF IA, and TFIIB have been shown to be involved in gene looping. Using Chromatin immunoprecipitation (ChIP)-seq experiments, we localized Mediator occupancy genome-wide. As expected, we found enrichment of Mediator at gene promoters, but also at Chromatin Interaction Domain boundaries (CIDBs), which are important for chromatin organization and transcriptional regulation. We also investigated the yeast Tail module function in detail. Individually, Tail subunit mutants are non-essential, but med15/med16 or med5/med15 strain are lethal. We used the N-degron system to conditionally deplete Med15/Med16 or Med5/Med15 and studied their effects on global gene expression using MicroArray assays. Several meiosis and sporulation genes were upregulated in the med5/med15 and med15/med16. In support of this, Rck1 which is a repressor of meiosis and sporulation rate in diploid cells, was downregulated in both strains. All strains where Med15 expression was depleted showed downregulation of several target genes for the Ace2 transcription factor which is important for cell cycle progression through the G1 cell cycle phase. Accordingly, all our med15-degron strains showed a G1-phase arrest in flow cytometry assays. Finally, we investigated the tail module subunit Med2 which has a phosphorylation site at position S208. We found that point mutations of S208A led to downregulation of several genes that are usually expressed during anaerobic growth. We also found that a med2 strain was unable to grow under anaerobic conditions.