Assembly and function of heterochromatin

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The objective of this research program is to understand how heterochromatin, a specialized repressive chromosomal domain, assembles from its constituent proteins and to determine how the dynamics of these regions change in response to changing cell cycle position and physiological conditions. Heterochromatin plays important roles in chromosome structure and segregation and is used to regulate the expression of developmental genes and pathways. In the budding yeast, Saccharomyces cerevisiae, heterochromatin contains nucleosomes that are bound by a complex of three proteins, Sir2, Sir3 and Sir4 (Silent information regulator), named the SIR complex. Current models for heterochromatin assembly propose that after recruitment to a DNA element, iterative rounds of Sir2-dependent histone deacetylation and SIR complex recruitment lead to the spreading of heterochromatin. We have identified Asf2, a paralogue of Sir4, that also assembles into core heterochromatic domains. The precise roles of Sir3, Sir4 and Asf2 in the spreading and stability of heterochromatin are poorly understood. This grant proposes the following short-term objectives: 1. Determine the function of Sir4 and Asf2 in heterochromatin dynamics. We have shown the abundance of Sir4 regulates de novo assembly of heterochromatin and that during specific cell cycle stages Sir4 protein levels decline dramatically. We will investigate the mechanism cells used to regulate Sir4 abundance, and if perturbing this pathway alters the the establishment of new domains of heterochromatin. Asf2, in contrast, plays a minor function in transcriptional gene silencing and we will test if instead it regulates the firing of late origins of DNA replication and a specialized recombination event that occurs between heterochromatic loci. We will also use whole genome approaches to explore other novel functions of Asf2-dependent heterochromatin. 2. Determine the mechanism of Sir3 spreading. We have shown that Sir3 can spread along chromatin independently of Sir4/Sir2 and specific histone modifications, suggesting that Sir3 oligomerization drives heterochromatin spreading. We have identified a small loop in the Sir3 N-terminus that may mediate Sir3 oligomerization, and we will test this model using mutants in this loop. 3. Does sub-telomeric heterochromatin generate phenotypic diversity? Sub-telomeric heterochromatin is found in all eukaryotes, but its precise function is poorly understood. Sub-telomeric heterochromatin displays position effect variegation, in which only a subset of telomeres are transcriptionally repressed in a given cell within a population. We will test the model that silencing the expression of different combinations of sub-telomeres is a mechanism cells use to generate phenotypic diversity. By selecting for silencing of specific telomeres, we will test if there is co-regulation of sub-telomeric heterochromatin, and if silencing of specific telomeres is associated with specific phenotypes.