Differential regulation of the alcohol dehydrogenase 1B (ADH1B) and ADH1C genes by DNA methylation and histone deacetylation

The human class I alcohol dehydrogenase (ADH) genes (ADH1A, ADH1B, and ADH1C) differ in expression during development and in various tissues. They are repressed in the HepG2 human hepatoma cell line. We hypothesized that epigenetic modifications play a role in this repression and that class I ADH gene expression would be enhanced upon global inhibition of DNA methylation and histone deacetylation. Southern blotting was used to assess the methylation status of each class I ADH gene. HepG2 and HeLa cells were treated with either the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), the histone deacetylase inhibitor Trichostatin A (TSA), or both in combination, and class I ADH gene expression was analyzed. Chromatin immunoprecipitation assays were performed to analyze histone H3 acetylation. Transient transfections and gel mobility shift assays were used to analyze the role that methylation plays in inhibiting transcription factor binding and promoter function. We show that the upstream regions of ADH1A, ADH1B, and ADH1C are methylated in HepG2 cells. 5-Aza-2'-deoxycytidine treatment enhanced expression of both ADH1B and ADH1C. Trichostatin A treatment elevated expression of ADH1C. ADH1A expression was not stimulated by either 5-aza-dC or TSA. H3 histones associated with a methylated upstream region of ADH1B were hyperacetylated in TSA-treated, but not in 5-aza-dC-treated, HepG2 cells. A methylated upstream region of ADH1C achieved histone H3 hyperacetylation upon either 5-aza-dC or TSA treatment. Methylation of the ADH1B proximal promoter in vitro decreased its activity to 54% and inhibited the binding of the upstream stimulatory factor. These findings suggest that the class I ADH genes are regulated by epigenetic mechanisms in human hepatoma cells. The temporal and tissue-specific expression of these genes may in part result from differences in epigenetic modifications and the availability of key transcription factors.