Identification of the cis-acting region in the NF2 gene promoter as a potential target for mutation and methylation-dependent silencing in schwannoma

Background: Although mutational inactivation and allelic loss in the NF2 ge ne appear to be causal events in the majority of vestibular schwannomas, in volvement of another potentially important mechanism, transcriptional inact ivation, has not been investigated. Results: We cloned and functionally characterized the 5'-flanking region of the human NF2 gene and identified the molecular mechanisms that regulate N F2 expression. Luciferase assay and site-directed mutagenesis demonstrated that a 70-base pair (bp) region (-591 to -522 bp from the translation start site) was essential for the basic expression of the NF2 gene. A gel mobili ty shift assay indicated recognition by nuclear protein of the unusually lo ng (approximate to 66 bp) sequences in this region. Recognition was inhibit ed by either mutation of the binding core sequence or by methylation of thr ee CpG sites. Point mutations at these CpG sites significantly decreased pr omoter activity, suggesting the importance of these sites. In 14 of 23 vest ibular schwannomas, these three CpG sites were methylated in a site-specifi c manner and the methylation status was consistent with the expression of N F2 mRNA. Conclusions: Suppressed expression by aberrant methylation or mutation of t he promoter elements could be an alternative mechanism for inactivation of the NF2 gene.