Genome-wide analysis of the Mediator transcription complex

Abstract

Transcription regulation is an essential process that enables living organisms to develop, to respond to extra-cellular signals and to environmental changes. In S. cerevisiae more than 300 proteins are required for accurate transcription regulation. This thesis focuses on one of the more central transcription regulatory complexes, known as Mediator. Mediator is highly conserved from yeast to human and is required for the expression of nearly all protein-coding genes. Initially, Mediator was suggested to form a molecular bridge between gene-specific transcription factors and RNA Polymerase II. The presences of negatively and positively acting subunits as well as enzymatic activity does not agree with this simple model and suggest that Mediator may function as a signal processor that integrates signalling pathways and the regulation of transcription. The objective of the work described in this thesis was to investigate the role of Mediator subunits by systematically analysing the effects of deleting individual subunits by microarray expression profiling. Previous microarray experiments showed that the expression of nearly all transcripts is affected upon disrupting essential subunits of Mediator or RNA Polymerase II. To detect such global expression changes, normalization of microarray experiments requires a set of transcripts that do not vary between the different samples. In chapter 2 we describe the development and use of external RNA controls for normalization of microarray expression profiling experiments. In control experiments, we show that external RNA controls can accurately report global mRNA changes. Furthermore, we demonstrate that conditions such as heat-shock or starvation can affect global transcript levels. This illustrates that global changes occur more frequently than is currently assumed in microarray studies. Finally, a yeast stationary phase experiment exemplified the importance and power of external RNA controls when large global changes occur, especially when global transcript changes are unbalanced. In chapter 3 we applied DNA microarrays to examine the effects of deleting individual Mediator subunits. The results reveal that the microarray expression profiles correlate well with the structural organization of individual subunits leading to the proposal that expression-profiling is useful for structure-function analyses. In addition, different modules within Mediator were found to have antagonistic effects on the transcription of the same set of genes. Epistasis analyses, with microarray expression profiles as transcriptional phenotypes, demonstrate that MED2 and MED18 are epistatic to CDK8. The functional consequences of one of these pathways is mapped further and reveals that Cdk8-mediated phosphorylation of Med2 affects the transcription of a single transcription factor regulon. Together, this shows that intra-complex phosphorylation mediated by Cdk8 can regulate specificity of transcription regulation. This demonstrates that microarray expression-profiling can uncover new signal transduction pathways, delineate their components and precisely determine the consequences of single amino acid modifications. In chapter 4 we further investigated the regulation of Mediator activity by Cdk8. We show that Med15, another subunit from the Tail, is a potential target of Cdk8. Med15 migrates as a higher mobility, non-diffuse band on a protein gel when Cdk8 is absent. This illustrates that Med15 is phosphorylated dependent on Cdk8. In addition we show that Cdk8 can phosphorylate Med15 in vitro. Precise mapping of the Cdk8-dependent phosphorylated residues by mass-spectrometry is currently underway. Upcoming experiments should illustrate whether Med15 phosphorylation results in similar regulatory events as with Med2. Chapter 5 is a general discussion of the results that are described in this thesis and discusses possible mechanisms through which Mediator can integrate signal transduction pathways and regulation of transcription.