| Unlike in somatic cells, cancer cells adopt an aberrant pattern of methylation as well as histone acetylation, and therefore distort the chromatin structure. Chapters 2--4 of this thesis look at mechanisms carried out by the recently cloned DNA demethylase, how its demethylation activity is closely linked with the semblance of acetylation of chromatin, and how this relationship can be skewed in cancer. The three intriguing mechanisms described provide attractive models by which to explain general genome wide demethylation, site specific demethylation of genes upon their activation, and the relationship between aberrant methylation and histone acetylation in cancer. The thesis begins by characterizing the mechanism of demethylation carried out by the bona fida DNA demethylase---an enzyme identified and cloned in our laboratory found to demethylate both hemi and double-stranded DNA in vitro. This enzyme manifests the removal of methyl groups from DNA without damaging the DNA and is therefore a candidate protein responsible for hypomethylation seen during development as well as in transformed cells. One essential property of an enzyme that removes methylation from wide regions of the genome could be processivity. Southern blot analysis and sodium bisulfite mapping experiments determine that purified demethylase demethylates DNA in a processive manner in vitro. Experiments in Chapter 3 demonstrate how an active demethylase enzyme is involved in shaping patterns of methylation relative to the state of histone acetylation. We present evidence suggesting demethylase activity is directed by the state of histone acetylation, therefore contrasting the accepted dogma, and suggesting that the local histone acetylation state determines the resulting methylation pattern. Aberrant DNA methylation and histone deacetylation are frequently associated with silencing of tumor suppressor genes in cancer and yet cannot simply be explained by the level of methyltransferase(s) enzyme(s) present in the cells. Chapter 4 therefore focuses on a new candidate family of proteins found to inhibit the activity of histone acetyltransferases called INHATs, that might explain this phenomenon. This novel class of proteins, not belonging to the family of methyltransferase proteins, is shown to modify DNA methylation patterns by preventing histone acetylation of ectopically methylated DNA resulting in hypermethylation. (Abstract shortened by UMI.)... |
