Umeå University's logo

Mediator is a multi-unit molecular complex that plays a key role in transferring signals from transcriptional regulators to RNA polymerase II in eukaryotes. We have combined biochemical purification of the Sac-charomyces cerevisiae Mediator from chromatin with chromatin immunoprecipitation in order to reveal Mediator occupancy on DNA genome-wide, and to identify proteins interacting specifically with Mediator on the chromatin template. Tandem mass spectrometry of proteins in immunoprecipitates of mediator complexes revealed specific interactions between Mediator and the RSC, Arp2/Arp3, CPF, CF 1A and Lsm complexes in chromatin. These factors are primarily involved in chromatin remodeling, actin assembly, mRNA 3'-end processing, gene looping and mRNA decay, but they have also been shown to enter the nucleus and participate in Pol II transcription. Moreover, we have found that Mediator, in addition to binding Pol II promoters, occupies chromosomal interacting domain (CID) boundaries and that Mediator in chromatin associates with proteins that have been shown to interact with CID boundaries, such as Sth1, Ssu72 and histone H4. This suggests that Mediator plays a significant role in higher-order genome organization.

Yeast cell subjected to many different stresses elicit an acute transcriptional stress response mediated by the Msn2 transcription factor, which alters expression of both a stress specific-cohort of genes as well as a common cohort of genes that changes expression in a stereotypic fashion upon exposure to any of a wide variety of stresses. We have shown by dynamic single cell analysis that stresses regulate Msn2 activity through cytoplasm to nuclear relocalization but do so in an unusual way: stresses induce increased frequency of bursts of short-lived, recurrent periods of Msn2 nuclear localization with different stresses eliciting different patterns of bursts. Moreover, genetically identical cells subject to an identical stress can behave quite differently, with some cells mounting a robust nuclear occupancy of Msn2 while others show no nuclear localization at all. We have proposed that this idiosyncratic behavior allows populations of cells to “hedge their bet” as to what will be the optimum strategy for surviving the ensuing stress. We have used computational modeling and single cell analysis to determine that bursting is a consequence of noise in the stress signaling pathways amplified by the small number of Msn2 molecules in the cell. Moreover, we have applied genome wide chromatin immunoprecipitation and nucleosome profiling to address how different stresses determine where Msn2 binds under a particular stressful conditions, and thus what genes are regulated by that stress, and how that binding affects, and is affected by, nucleosome positioning and other transcription factor binding. These results provide in vivo validation of Widon's model of indirect cooperativity of transcription factor binding, mediated by partial unwinding of nucleosomes by one transcription factor to allow access for a second transcription factor to a previously occluded binding site. Finally, we have addressed the “bet hedging” hypothesis by showing that persistence of the Msn2-mediated stress response yields cell growth arrest and have identified the targets responsible for that growth arrest. We have applied experimental evolution paradigms to address the relative fitness of cells exhibiting stochastic stress responses versus those with a uniform response. In short, our results indicate that the stress response is complex and that complexity is critical for cell survival.

The transcription factor Msn2 mediates a significant proportion of the environmental stress response, in which a common cohort of genes changes expression in a stereotypic fashion upon exposure to any of a wide variety of stresses. We have applied genome-wide chromatin immunoprecipitation and nucleosome profiling to determine where Msn2 binds under stressful conditions and how that binding affects, and is affected by, nucleosome positioning. We concurrently determined the effect of Msn2 activity on gene expression following stress and demonstrated that Msn2 stimulates both activation and repression. We found that some genes responded to both intermittent and continuous Msn2 nuclear occupancy while others responded only to continuous occupancy. Finally, these studies document a dynamic interplay between nucleosomes and Msn2 such that nucleosomes can restrict access of Msn2 to its canonical binding sites while Msn2 can promote reposition, expulsion and recruitment of nucleosomes to alter gene expression. This interplay may allow the cell to discriminate between different types of stress signaling.

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.

Development in plants is controlled by abiotic environmental cues such as day length, light quality, temperature, drought, and salinity. These signals are sensed by a variety of systems and transmitted by different signal transduction pathways. Ultimately, these pathways are integrated to control expression of specific target genes, which encode proteins that regulate development and differentiation. The molecular mechanisms for such integration have remained elusive. We here show that a linear 130-amino-acids-long sequence in the Med25 subunit of the Arabidopsis thaliana Mediator is a common target for the drought response element binding protein 2A, zinc finger homeodomain 1, and Myb-like transcription factors which are involved in different stress response pathways. In addition, our results show that Med25 together with drought response element binding protein 2A also function in repression of PhyB-mediated light signaling and thus integrate signals from different regulatory pathways.