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On the mechanisms of transcriptional repression in the yeast Saccharomyces cerevisiae


Ehrenhofer-Murray,  Ann Elizabeth
Independent Junior Research Groups (OWL), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Ehrenhofer-Murray, A. E. (2003). On the mechanisms of transcriptional repression in the yeast Saccharomyces cerevisiae. Habilitation Thesis, Humboldt-Universität, Berlin.

Summary Gene silencing is a mechanism widely used by eukaryotic organisms both to ensure proper gene expression during development and to prevent detrimental effects on chromatin. Missing or inappropriate silencing can cause misexpression of genes, ultimately disrupting cellular programming. Therefore, learning about the mechanisms that lead to gene inactivation has broad medical relevance for understanding how defects in epigenetic processes can disturb cellular programs and thus can cause disease. This work reviews my contributions to the mechanistic understanding of gene silencing in the model organism Saccharomyces cerevisiae. I have probed the relationship between replication and silencing in several ways: i)Together with my research group, we have discovered a novel mechanism for the reestablishment of epigenetic imprints after DNA replication. Specifically, we have identified a physical interaction between the histone acetyltransferase (HAT) complex SAS-I and chromatin assembly factors, suggesting that this HAT complex is recruited to the replication fork in order to restore histone acetylation marks on the freshly assembled chromatin. Since chromatin maturation also includes other modifications, this suggests that chromatin assembly factors serve as platforms for chromatin modifying activities to reestablish epigenetic patterns of histone modifications on newly replicated chromatin. ii)I have investigated the involvement of replication proteins in silencing and have found a novel role for the DNA clamp and replication processivity factor PCNA, the clamp loader RF-C, the DNA polymerase e, and the licensing factor Cdc45 in silencing. Interestingly, these proteins are functionally tightly connected to each other, and they are involved in the recognition of replicated DNA by the chromatin assembly complexes. This suggests that their role in silencing is to recruit chromatin assembly complexes, and thus chromatin modifying activities, to the replication fork. iii)I have studied the relationship between replication initiation, the initiator complex ORC and silencing and have found that the initiation and silencing functions of ORC can be separated. Thus, ORC has an initiation-independent silencing function, which likely is to interact with the silencing establishment factor Sir1 in order to recruit other silencing complexes to repressed genomic regions. Furthermore, we have investigated silencing forms in S. cerevisiae that are alternative to the classical forms of silencing at the silent mating-type loci HMR/ HML and at telomeres. i) We found that the rDNA silencing factor Net1, which is a component of the RENT complex, also can function in HMR silencing by interacting with silencer binding proteins, suggesting that RENT is anchored to rDNA sequences by a similar mechanism. ii)We have furthermore addressed the question whether other silenced regions exist in the yeast genome. We have identified a novel yeast silencer, the origin of replication of the endogenous 2µ plasmid. Silencing by this silencer required the known silencing proteins. Significantly, it also required the histone deacetylase Hst3 and thus constituted a novel class of silencing. This is the first demonstration of a singular cellular function for Hst3. Origins of replication are binding sites for ORC. However, not every ORC binding site is a silencer. This finding gives us the opportunity to determine what converts an origin into a silencer and what distinguishes regular origins from silencers. In summary, this work contributes to the understanding of multiple aspects of the role of replication in gene silencing. These findings are discussed in the light of other recent developments to present a unified model for repressed chromatin in yeast and larger eukaryotes.