In vitro DNA methylation inhibits FMR-1 promoter.

In fragile X syndrome, the FMR-1 gene is changed by a CGG repeat mutation and an abnormal methylation at a CpG-island 5' to the gene. To elicit if methylation itself inactivates the gene, FMR-1 promoter was defined by deletion mapping and primer extension assay and was analyzed by in vitro methylation. Promoter activity was measured by transient expression and chloramphenicol acetyl transferase assay. Although this promoter contains several HpaII sites, it was not affected by methylation with HpaII methylase. However, the promoter was completely repressed by methylation with M. SssI which methylates all cytosines of CpG dinucleotides. This repression could not be overridden by SV-40 enhancer. This study indicates that methylation could be the direct cause of FMR-1 inactivation in fragile X syndrome.