A key unsolved question in chromosome biology is how epigenetic gene silencing is restricted to a specific chromosomal domain. Boundary elements are insufficient to explain how silent chromatin is contained as their deletion often results in only a mild spread of silencing. We have identified an anti-silencing mechanism that operates in euchromatin independently of boundary elements. Specifically, we have shown that the conserved histone variant H2A.Z is a euchromatin-specific protein that restricts the ectopic spread of silencing mediated by the Sir2/3/4 complex in S. cerevisiae. As such, H2A.Z represents the first intrinsic component of euchromatin identified whose specific function is to antagonize heterochromatin formation. We and others have also defined a protein complex, the Swr1 complex, that is required for the deposition of H2A.Z in vivo. In order to understand how euchromatin is formed and regulated, we wish to determine how the Swr1 deposition complex is directed to correct chromosomal locations in euchromatin, how H2A.Z deposition and activity are modulated by regulatory factors, and how H2A.Z functions to specify euchromatin. To accomplish these goals we will 1) Define DMA signals that specify H2A.Z. deposition, 2) Identify recognition proteins that specify the chromosomal sites H2A.Z deposition, 3) Determine whether H2A.Z-mediated anti-silencing is regulated by phosphorylation, an 4) Analyze the mechanism of anti-silencing by H2A.Z. This work is highly relevant to human oncogenesis. In human cancer, tumor suppressor genes are inactivated by two mechanisms: mutation and gene silencing. Silencing is potentially reversible and therefore the inactivation of silencing offers great therapeutic promise for the treatment of human malignancies. This proposal focuses entirely on how gene silencing is reversed in cells, and therefore is directly relevant to the goal of activating epigenetically silenced tumor suppressor genes in human tumors to arrest their growth and/or induce apoptosis. Indeed, chemical inhibitors of histone deacetylases, which are required for epigenetic gene silencing in all systems characterized from yeast to humans, are in clinical trials as anticancer drugs. ? ?
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