Cooperative Binding of Sir Proteins to Nucleosomes and Its Implications for Silent Chromatin Assembly in Saccharomyces Cerevisiae
CitationLu, Chenning. 2015. Cooperative Binding of Sir Proteins to Nucleosomes and Its Implications for Silent Chromatin Assembly in Saccharomyces Cerevisiae. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractSilent chromatin, or heterochromatin, refers to regions of the genome in which genes are constitutively repressed. These regions are important for regulating developmental genes and for maintaining genome stability, and are epigenetically inherited. In Saccharomyces cerevisiae, subtelomeres and silent mating type loci are assembled into silent chromatin by the Silent Information Regulator (SIR) complex, composed of Sir2, Sir3 and Sir4, which deacetylates histones and spreads along chromatin. Many questions remain regarding the mechanism of Sir protein spreading along chromatin and the mechanism of epigenetic inheritance of silent chromatin domains. It has been hypothesized that the lateral Sir-Sir protein interactions together with Sir-nucleosome interactions cooperatively recruit Sir proteins to spread along chromatin.
In my thesis project, I set out to test this cooperativity hypothesis by examining the interaction of Sir proteins with well-defined in vitro reconstituted mono- and di-nucleosomes. Using electrophoretic mobility shift assay (EMSA), I find that Sir3, the main nucleosome-binding component of the SIR complex, associates with nucleosomes cooperatively, involving the dimerization of Sir3 bound to neighboring nucleosomes. I demonstrate that this inter-nucleosomal cooperativity is mediated by the Sir3 C-terminal winged helix (wH) dimerization domain and is further stabilized by the Sir4 coiled-coil (CC) domain, which mediates both Sir4 homodimerization and Sir3-Sir4 interactions. There is functional redundancy from the two domains in mediating binding cooperativity, as suggested by the measurement of cooperativity free energy. Surprisingly, my binding measurements suggest that there are no Sir-Sir protein interactions on the same nucleosome. Moreover, by using an in vitro bridging assay, I show that Sir3 effectively bridges free nucleosomes in solution and that its wH domain is required for its bridging activity. My in vitro results are corroborated by in vivo ChIP-seq results showing that either the Sir3 wH domain or the Sir4 CC domain alone could mediate weak spreading of Sir3 protein, away from recruitment sites, under Sir3 overexpression conditions. However, mutations in both domains abolish the spreading completely.
Both histone H4 lysine 16 (H4K16) acetylation and histone H3 lysine 79 (H3K79) methylation are hallmarks of euchromatin in S. cerevisiae. I quantify the effect of either modification alone and both modifications in combination on Sir3-nucleosome binding affinity. This shows that either modification alone decreases Sir3 binding affinity towards nucleosomes by 3-4 fold, and that the two modifications work together to reduce the binding affinity even further. Statistical mechanical modeling of the nucleosome binding results indicate that the combined effect of H4K16 acetylation and H3K79 methylation can account for partitioning of Sir3 between silent and active chromatin regions in vivo.
Our findings and their quantitative analysis suggest that SIR complexes spread along chromatin discontinuously, arguing against the stepwise polymerization model for silent chromatin assembly.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:17467201
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