Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Eukaryotic DNA is incorporated into the nucleoprotein structure of chromatin. This structure is essential for the proper storage, maintenance, regulation, and function of the genomes’ constituent genes and genomic sequences. Importantly, cells generate discrete types of chromatin that impart distinct properties on genomic loci; euchromatin is an open and active compartment of the genome, and heterochromatin is a restricted and inactive compartment. Heterochromatin serves many purposes in vivo, from heritably silencing key gene loci during embryonic development, to preventing aberrant DNA repeat recombination. Despite this generally repressive role, the DNA contained within heterochromatin must still be repaired and replicated, creating a need for regulated dynamic access into silent heterochromatin. In this work, we discover and characterize activities that the ATP-dependent chromatin remodeling enzyme SWI/SNF uses to disrupt repressive heterochromatin structure. First, we find two specific physical interactions between the SWI/SNF core subunit Swi2p and the heterochromatin structural protein Sir3p. We find that disrupting these physical interactions results in a SWI/SNF complex that can hydrolyze ATP and slide nucleosomes like normal, but is defective in its ability to evict Sir3p off of heterochromatin. In vivo, we find that this Sir3p eviction activity is required for proper DNA replication, and for establishment of silent chromatin, but not for SWI/SNF’s traditional roles in transcription. These data establish new roles for ATP-dependent chromatin remodeling in regulating heterochromatin. Second, we discover that SWI/SNF can disrupt heterochromatin structures that contain all three Sir proteins: Sir2p, Sir3p and Sir4p. This new disruption activity requires nucleosomal contacts that are essential for silent chromatin formation in vivo. We find that SWI/SNF evicts all three heterochromatin proteins off of chromatin. Surprisingly, we also find that the presence of Sir2p and Sir4p on chromatin stimulates SWI/SNF to evict histone proteins H2A and H2B from nucleosomes. Apart from discovering a new potential mechanism of heterochromatin dynamics, these data also establish a new paradigm of chromatin remodeling enzyme regulation by nonhistone proteins present on the substrate.

DNA double-strand break (DSB) repair is essential for maintenance of genome stability. However, the compaction of the eukaryotic genome into chromatin creates an inherent barrier to any DNA-mediated event, such as during DNA repair. This demands that there be mechanisms to modify the chromatin structure and thus access DNA. Recent work has implicated a host of chromatin regulators in the DNA damage response and several functional roles have been defined. Yet the mechanisms that control their recruitment to DNA lesions, and their relationship with concurrent histone modifications, remain unclear. We find that efficient DSB recruitment of many yeast chromatin regulators is cell-cycle dependent. Furthering this, we find recruitment of the INO80, SWR-C, NuA4, SWI/SNF, and RSC enzymes is inhibited by the non-homologous end joining machinery, and that their recruitment is controlled by early steps of homologous recombination. Strikingly, we find no significant role for H2A.X phosphorylation (γH2AX) in the recruitment of chromatin regulators, but rather that their recruitment coincides with reduced levels of γH2AX. We go on to determine the chromatin remodeling enzyme Fun30 functions in histone dynamics surround a DSB, but does not significantly affect γH2AX dynamics. Additionally, we describe a conserved functional interaction among the chromatin remodeling enzyme, SWI/SNF, the NuA4 and Gcn5 histone acetyltransferases, and phosphorylation of histone H2A.X. Specifically, we find that the NuA4 and Gcn5 enzymes are both required for the robust recruitment of SWI/SNF to a DSB, which in turn promotes the phosphorylation of H2A.X.