Silencing of human methionine adenosyltransferase 1A expression by methylation of the coding region

Abstract

Two genes (MAT1A and MAT2A) encode for the essential enzyme methionine adenosyltransferase (MAT). MAT1A is silenced in hepatocellular carcinoma (HCC), and absence of MAT1A leads to spontaneous development of HCC in mice. Here we investigated the role of methylation in regulating MAT1A expression. There are three MspI/HpaII sites from -1913 to +160 of the human MAT1A gene (numbered relative to the translational start site) at position -977, +10, and +88. Bisulfite treatment and DNA sequencing, and Southern blot analysis showed that methylation at +10 and +88, but not -977, correlated with lack of MAT1A expression. MAT1A promoter construct methylated at -977, +10 or +88 position has 0.7-fold, 3-fold, and 1.6-fold lower promoter activity, respectively. Methylation at -977 and +10 did not inhibit the promoter more than methylation at +10 alone; while methylation at +10 and +88 resulted in a 6-fold reduction of promoter activity. The promoter activity is not affected if these sites are mutated and cannot be methylated. Reactivation of MAT1A correlated with demethylation of +10 and +88. DNase I footprinting analysis using the probe containing nucleotides -346 to +160 and human liver nuclear extract or recombinant TATA binding protein (TBP) showed that HpaII methylation at +10 and +88 prevented TBP binding to the TATA box, but not if the sites were mutated. ChIP analysis confirmed TBP binding to MAT1A only in MAT1A expressing cells. Collectively our data support the novel finding that methylation of the MAT1A coding region can influence TBP binding to the TATA box and shut down gene transcription.