The mechanism of Sir3-nucleosome interaction and its implications in silent chromatin assembly in Saccharomyces cerevisiae

Silent chromatin domains in the budding yeast S. cerevisiae represent examples of epigenetically heritable heterochromatin, which is a conserved feature of chromosomes in most eukaryotes. The assembly of silent chromatin at telomeres and the silent mating type loci requires the recruitment of the SIR complexes, consisting of the Sir2/Sir4 subcomplex and the Sir3 protein, as well as the proper interactions between SIR complexes and nucleosomes. However, the nature of association of the SIR complex and its subunits with nucleosomes, perhaps the most important step in silent chromatin assembly, is still poorly understood.;In order to understand the mechanism of Sir3-nucleosome association, I set out to identify and define the nucleosomal surfaces that interact with Sir3. My studies showed that the H3 globular domain and the H4 N-terminal tail region are physically and functionally involved in the interaction of nucleosomes with Sir3 in vivo, the recruitment and spreading of Sir3 along chromatin, and silencing of subtelomeric gene expression.;The analysis of Sir3 interaction with reconstituted mononucleosomes allowed us to identify a cooperative mechanism of binding between the Sir3BAH domain and nucleosomes that is sensitive to the mutation of histone H4 lysine 16 to alanine (H4K16A). This suggests that this cooperative binding requires the specific recognition of H4K16 by Sir3BAH. We propose that the cooperative binding is due to a conformational change in the nucleosome that is induced upon binding of one Sir3BAH domain to one side of the nucleosome, which facilitates the binding of another Sir3BAH domain to the other side of the nucleosome.;My studies also showed that the Sir2/Sir4 subcomplex, but not Sir3 protein, efficiently bridges mononucleosomes in vitro, and that the Sir2/Sir4 subcomplex together with Sir3 has a synergistic effect on mononucleosome bridging. This suggested that efficient bridging of nucleosomes, and perhaps the spreading of the SIR complex along the chromatin fiber, require the full SIR complex.;Together, these studies shed light on the mechanism of Sir3-nucleosome interactions and provide insight into the mechanism of association of the SIR complexes with nucleosomes, which form the basis for the formation of functional silent chromatin.