GENETIC ASPECTS OF DNA METHYLATION

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In many organism, including humans, DNA is modified by methylation of certain cytosines. DNA methylation is essential in mammals, and is used in X-chromosome inactivation and in the regulation of genes, such as those subject to genomic imprinting. Over-expression of the mouse DNA methyltransferase (MTase) gene can induce tumorigenic transformation of cultured cells, engineered reduction of MTase activity can slow tumor progression, and considerable circumstantial evidence suggests that aberrant methylation of tumor suppressor genes can cause cancer. In addition, there is evidence that methylation serves in "genome defense" systems in fungi, plants and animals. The long-term goal of the research proposed here is to elucidate the function and control of DNA methylations in eukaryotics by taking advantage of an outstanding model system the fungus Neurospora crassa. Methylation is dispensable in Neurospora which facilitates genetic analyses of this process and the tools and materials required to dissect methylation are at hand. Specific aims are: 1. To identify the cis-acting signals that control establishment and maintenance of DNA methylation. Sequence requirements will be further defined and new models will be tested. 2. To isolate proteins that bind DNA sequences that signal DNA methylation and/or are methylated, and to determine their function by isolation and disruption of the corresponding genes. 3. To identify other trans-acting components of the methylation machinery. Methylation genes that we have already identified and additional genes identified by insertional mutagenesis will be isolated and characterized. 4. To elucidate the role of histone acetylation in the control of DNA methylation. These studies are based on our finding that Trichostatin A (TSA), a histone deacetylase inhibitor, causes regional loss of DNA methylation. Alternate models will be tested by: assessing the acetylation of histones associated with unmethylated, methylated but TSA-insensitive, and TSA-sensitive sequences; testing whether TSA-induced loss of methylation depends on transcription; testing the function of candidate cis-acting elements; testing the effect of mutations in histones H3 and H4 on methylation; testing whether DNA methylation affects histone acetylation. 5. To elucidate the relationship between replication timing and DNA methylation. Methylation mutants and methods that we have developed to study replication in Neurospora will be used to investigate whether either DNA methylation or histone hypoacetylation cause late replication of DNA.