Structure and Dynamics of Chromosomal Domains

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The objective of this research program is to understand how specialized chromosomal domains are assembled from their constituent proteins, and to determine how the dynamics of these regions change in response to changing physiological conditions. Specific regions of chromosomes are assembled into specialized structures composed of DNA packaged into chromatin and coated by specific chromosomal proteins. Although these structures are often viewed as static elements they are responsive to changing cellular conditions, their boundaries can change and they can move to new locations on chromosomes. This program is currently investigating the structure and dynamics of one model chromosomal domain, silent chromatin, in the budding yeast, Saccharomyces cerevisiae. Heterochromatic domains play a central role in the structure and transmission of chromosomes, and in the regulation of cell identity and proliferation. In budding yeast heterochromatin contains nucleosomes that are hypoacetylated and demethylated and are bound by a complex of three proteins, Sir2, Sir3 and Sir4 (Silent information regulator), named the SIR complex.  Sir2 is a conserved NAD-dependent protein deacetylase and creates the hypoacetylated domains of nucleosomes within heterochromatin.  Sir3 and Sir4 are histone-binding proteins that are believed to spread on chromatin and form a repressive structure.  Current models for SIR complex assembly of heterochromatin propose that after recruitment to a DNA element, iterative rounds of histone deacetylation and SIR complex binding lead to spreading of heterochromatin.  Although nucleation of heterochromatin is well understood, the precise role of the Sir proteins in the spreading and stability of heterochromatin is poorly understood. We are focusing on two aspects of Sir3 and Sir4 function:1. To determine the mechanism of Sir3 spreading.  Although mutations in all three SIR complex proteins abolish heterochromatin formation, it has been difficult to determine if all three proteins are required for both nucleation and spreading. We have created and characterized mutants of Sir3 that disrupt its ability to bind Sir4, and have shown that mutant Sir3 can spread and form silent chromatin with near wild type efficiency.  Our current model proposes that Sir3 spreading is driven by Sir3 oligomerization, and not changes in histone modifications. We have identified a small loop in the Sir3 N-terminus that may mediate Sir3 oligomerization, and our characterization of mutations in this region will allow us to rigorously test our model.2. To determine how cell cycle dependent phosphorylation of Sir3 and Sir4 regulate the stability of silent chromatin.  Silent chromatin is significantly more stable in G1 than in mitosis, but the mechanism that controls this stability is unknown.  We will examine cell cycle changes in the composition and modification of the SIR complex and test if these changes modulate the stability of silent chromatin using simple transcriptional readouts, as well as FRAP and biochemical assays.  Our initial focus will be on phosphorylation of Sir3 and Sir4, which are phosphorylated at different stages of the cell cycle, and Asf2, a novel core component of silent chromatin.Significance.  Work on budding yeast has pioneered the understanding of heterochromatin, and this proposal addresses a number of fundamental questions in chromatin biology: 1) How important are histone modifications for the spreading of chromatin domains? 2) Heterochromatin is thought to be formed from polymers of heterochromatin proteins, but little is known about how these proteins polymerize and if as polymers they bypass the need for specific histone marks, 3) How do cells regulate chromatin structures during the cell cycle?