Analysis of methylation in the 5' region of the human alpha-galactosidase A gene containing a binding site for methylated DNA-binding protein/RFX1-4.

We examined methylation in the 5' untranslated region and surrounding sequences of the X-linked human alpha-galactosidase A gene. This region includes a methylation-dependent binding site for the MDBP/RFX1-4 transcription factor family. By bisulfite-based genomic sequencing, we determined the distribution of 5-methylcytosine residues from position +28 to +143 relative to the major transcription start site of the gene in almost 500 molecular clones from different tissues and individuals. The CpG methylation patterns in DNA from females differed from molecule to molecule with no indication of tissue-specificity or age-dependence. In contrast, the CG dinucleotides within this region were completely unmethylated in almost all of the DNA molecules cloned from male tissues. In female eutherian mammals, 50% of the molecules are expected to be methylated in an X-linked gene region in which methylation is coupled to gene silencing. Of the cloned DNA molecules from female tissues, 40% had 4 or more of the 9 CpG's methylated and the MDBP site was methylated in about one-fifth of the female tissue-derived DNA molecules.