The significance of DNA methylation has been an open issue for some time. Many people believe that methylated genes are incapable or less capable of being transcribed than their unmethylated or hypomethylated counterparts. In this application, presented as an R29 application by a member of the Department of Obstetrics and Gynecology, Dr. Phillips proposes to study the significance of methylation in a system involving variably methylated and variably expressing hsp70 genes and selected cell lines. In her preliminary data, Dr. Phillips suggests that, at least for a number of cell lines, the state of DNA methylation for the hsp70 gene is related to the ability of the appropriate transcription factors to bind the promoter region of that gene and to initiate transcription. She proposes to study the role of methylation in hsp70 expression. The studies she proposes to use to explore this question follow 4 specific aims. First, she proposes to measure the relationship between the extent of methylation and the accessibility of the promoter region of the hsp70 genes by DNase I footprinting, and the heat shock-inducible transcriptional activity of the gene as measured by nuclear run-on assays. These studies will be geared to the study of two cell lines in particular, in which one allele of hsp70.1 is fully methylated and the other is only partially methylated. Thus, her experiments here will focus on allelic differences in these cell lines, LK35 and L1210. She will map the methylated and unmethylated DNA for the promoter regions of these genes, for both alleles. These map regions will be correlated with heat-inducible transcriptional activity and accessibility of this region to binding by the appropriate DNA binding proteins.
Specific aim 2 proposes to study the development of hsp70 methylation in newly immortalized mouse cell lines. Dr. Phillips will derive cell lines from BALB/c embryos and proposes to monitor methylation status of the hsp70 genes (70.1 and 70.3) as a function of time in culture by quantitative PCR. She will use this approach to study expression and methylation of hsp70t, which is only expressed only in the testes. In this regard, it is of note that the hsp70 genes are easier to study than many other genes because they have no introns: the entire coding sequence of the gene is contiguous, facilitating interpretation of changes observed. Because hsp70 genes line in the region of chromosome 6 containing the MHC genes, other MHC genes will be studied in this fashion to determine whether this region represents a hot spot for DNA methylation.
Specific aim 3 proposes to study hsp70 gene methylation in Friend MuLV-induced erythroleukemias, as a function of progression of the infected cells to the transformed state. These studies are to be done in collaboration with Yaacov Ben-David, Toronto, Canada, in whose laboratory many such cells have been developed, and DNA extracted at various stages along the path to frank leukemia. Dr. Phillips plans to analyze these stored DNAs for the state of methylation of hsp70 genes and nearby MHC genes, and as well to analyze cells from available lines by nuclear run-on assays to determine the state of transcriptional activity and activatability of the hsp70 and other MHC genes.
Specific aim 4 involves an examination of the effect of demethylation of hsp70 on gene expression. Apparently hsp70 is demethylated when the mouse lymphoma line CH1 is grown as ascites fluid, rather than in culture. Dr. Phillips proposes to collaborate with Robin Anderson, Melbourne, Australia, in studying to what extent demethylation of hsp70 in CH1 changes the ability of these genes to respond to heat shock by transcription.
