Investigation of the events underlying gene silencing in association with aberrant CpG island hypermethylation in cancer

My thesis work, performed under the advisement of Dr. Stephen Baylin, was designed to characterize the relationship between CpG methylation density and histone deacetylase activity in order to better understand the processes involved in the establishment and maintenance of CpG island transcriptional silencing in mammalian cancer cells.;In my studies, I primarily focused on two genes containing a promoter region CpG island -p15 (CDKN2B) and MLH1. For each gene the CpG island is normally maintained free of cytosine methylation, but it can become hypermethylated and transcriptionally silenced in human cancer. p15 encodes a cyclin dependent kinase inhibitor and is methylated and silenced specifically in primary acute leukemia (Hannon, et al., 1994; Stone, et al., 1995; Herman, et al., 1996; Drexler, et al., 1998). MLH1 encodes a mismatch repair protein and is methylated and transcriptionally silenced in sporadic colon, endometrial, and gastric carcinomas (Lengauer, et al., 1998; Herman, et al., 1998; Kane, et al., 1997; Esteller, et al., 1999; Fleisher, et al., 1999).;In order to study the relationship between methylation density and transcriptional silencing, I first characterized the methylation patterning of the p15 CpG island in normal lymphocytes and found the methylation-free region to be very small with borders that varied between individuals. I then characterized in detail the aberrant methylation of p15 found in primary acute leukemia. Transcriptional silencing of p15 in association with CpG island methylation in primary acute leukemia was found to be largely a function of CpG methylation density and not site-specific methylation.;After determining the importance of CpG methylation-associated transcriptional silencing, I investigated the relationship between CpG methylation density and higher order chromatin. Using the histone deacetylase inhibitor Trichostatin A (TSA), I showed that histone deacetylase activity is not necessary for the maintenance of transcriptional silencing at endogenous, methylated CpG island promoters, but that histone deacetylase activity plays a role in the silencing of promoters that have been slightly demethylated using the demethylating agent 5-aza-2'deoxycytidine (5Aza-dC). Inhibition of histone deacetylase activity using TSA increased the transcriptional activity of four important genes in cancer (MLH1, p15, TIMP3 , and p16) following 5Aza-dC treatment. This increased transcription was not the result of further demethylation, but was instead the result of an increased number of expressing cells. Thus, although DNA methylation and histone deacetylation appear to act as synergistic layers for the silencing of genes in cancer, dense CpG island methylation is dominant for the stable maintenance of a silent state at these loci.