Functional studies of Mediator in Arabidopsis thaliana and Saccharomyces cerevisiae

Mediator has been shown to be essential for regulation of RNA Polymerase II mediated transcription. Mediator functions as an interface between the general transcriptional machinery and a multitude of DNA binding transcriptional regulators, although the molecular mechanism for the process is elusive. Mediator is a large complex of over twenty subunits, most of which are conserved from yeast to plants to mammals. Many of these subunits are essential for viability in yeast, and mutations in the corresponding genes have global effects on transcription. Mediator was originally identified in Saccharomyces cerevisiae, but has since been described in most eukaryotes. However, until recently the Mediator complex was not identified in plants. This thesis describes the first successful identification and isolation of the Mediator complex from the plant Arabidopsis thaliana. By raising antibodies against candidate A. thaliana Mediator subunits, we were able to purify a multisubunit protein complex. Mass spectrometry and bioinformatics analysis allowed us to identify 21 of these subunits as conserved Mediator components and six as A. thaliana specific subunits. Some of the genes that encode the identified Mediator subunits had earlier been described as components of specific regulatory pathways controlling for example cell proliferation and flowering time. Subsequent genetic analysis confirmed that the A. thaliana Mediator complex is important for several plant signaling pathways, including flowering and stress pathways. This thesis also describes identification of regulators that interact with the A. thaliana Mediator subunit Med25, previously identified as PFT1 (Phytochrome and Flowering Time 1) and implicated in regulation of flowering time in response to light quality. Finally, we describe the function of Mediator in S. cerevisiae using genome-wide approaches. We have carried out a transcriptional switch where half of the genome changes expression and determined Mediator occupancy across the genome before and after such a switch, using ChIP-SEQ on tagged subunits from different Mediator domains. Unexpectedly, we find that Mediator occupancy is limited at most promoters. However, at the highly occupied promoters, we see different modes of changes in occupancy as a result of the transcriptional switch. These highly occupied promoters control genes involved in different stress response pathways. Thus, our results suggest that Mediator function and composition differ considerably between different promoters