Inhibition of rat growth hormone promoter activity by site-specific DNA methylation

The effect of methylation on rat growth hormone (rGH) promoter activity was determined in GH3 cells by measuring rGH-Neo and rGH-CAT fusion gene expression with or without prior in vitro treatment with the site-specific DNA methyltransferases, M-BsuE and M-HhaI. To assay for rGH-promoter-specific effects of DNA methylation, RSV-Neo and RSV-CAT activities with or without M-BsuE, M-HhaI and M-HpaII treatment were measured in parallel cultures of GH3 cells. GH1-Neo and RSV-Neo fusion gene expression was inhibited by in vitro methylation from 44 to 83% as measured by the number of Geneticin-resistant GH3 cell colonies. Methylation of the GH1 promoter by M-BsuE exhibited some selective inhibition of Neo expression as determined by colony numbers, although extensive methylation of non-promoter DNA in GH1-Neo and RSV-Neo by M-HhaI and M-HpaII also inhibited Neo expression. Southern blot analysis of genomic DNA isolated from the Geneticin-resistant GH3 cells indicated that Geneticin-resistance was accompanied by demethylation of the BsuE (ThaI) sites in stably incorporated GH1-Neo DNA but not RSV-Neo DNA. Transient expression of the CAT gene in GH3 cells was selectively inhibited by 60% upon methylation of two BsuE (ThaI) sites in the GH1 promoter of GH1-CAT by M-BsuE. The data demonstrate, for the first time, to our knowledge, a direct effect of DNA methylation on the activity of the rat growth hormone promoter.