In the past several years we have demonstrated that carcinogenic nickel compounds inactivate g ne expression by enhancing DNA methylation and inhibiting histone acetylation. The long-term goals of this grant proposal are to study the epigenetic mechanisms of nickel carcinogenesis. The short-term goals of the proposal are to study the mechanism by which nickel silences genes through DNA methylation and a newly discovered mechanism involving inhibition of histone H4 acetylation. We believe that these mechanisms involve nickel-binding to histidine at position 18 on the N-terminal tail of histone H4. In the absence of histone acetylation, an acidic region of H2A and H2B interact avidly with the basic 1ysines of the histone H4 tail. The specific aims of the grant proposal are to study the mechanism by which nickel inhibits the acetylation of lysines of H3 and H4 tails with the hypothesis that nickel-binding to histidine 18 on the N-terminal of H4 is responsible for a selective inhibition of H4 acetylation since H3 lacks a metal binding amino acid on its tail. We will study nickel-binding to H4 using the whole protein and model peptides with various analytical tools, such as XAS, in collaboration with inorganic chemists, Drs. Maria Zoroddu and Michael Maroney. We will also invest ate the relationship between histone acetylation and gene silencing in mammalian cells using histone deacetylase inhibitors, such as trichostatin A. We will examine whether trichostatin A can antagonize nickel-induced silencing and can revert nickel-silenced genes both in the G12/G10 mammalian system, as well as the URA3 gene which is silenced by nickel in yeast. We will utilize the chromosome immunoprecipitaton assay to investigate whether nickel effects gene-specific histone H3 or H4 acetylation. This assay involves crosslinking of histories to DNA with formaldehyde after which the DNA is fragmented to 0.5-2 Kb fragments, immunoprecipitated with antibodies against acetylated H3 and H4 and the crosslinked proteins are released and specific DNA sequences associated with acetylated histones are assessed by quantitative PCR. This assay will be applied tooth the G12 nickel-silenced system, as well as the URA3 gene in the nickel- silenced yeast system. Using the Affymetrix GeneChip, we will identify human genes turned-off following exposure to nickel compounds and reactivated by the histone deacetylase inhibitor trichostatin A. These human genes will then be further examined by the chromosome immunoprecipitaton assay and also will be studied in terms of the effects of DNA methylation on their expression. These studies should greatly enhance our body of knowledge concerning the epigenetic mechanisms of nickel carcinogenesis.