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 gene appear to be causal events in the majority of vestibular schwannomas, involvement of another potentially important mechanism, transcriptional inactivation, 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 NF2 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 mobility shift assay indicated recognition by nuclear protein of the unusually long ( approximately 66 bp) sequences in this region. Recognition was inhibited by either mutation of the binding core sequence or by methylation of three CpG sites. Point mutations at these CpG sites significantly decreased promoter activity, suggesting the importance of these sites. In 14 of 23 vestibular schwannomas, these three CpG sites were methylated in a site-specific manner and the methylation status was consistent with the expression of NF2 mRNA.


CONCLUSIONS
Suppressed expression by aberrant methylation or mutation of the promoter elements could be an alternative mechanism for inactivation of the NF2 gene.
Kino
T
T
Department of Neurosurgery, Kumamoto University School of Medicine, Kumamoto 860-8556, Japan.
Takeshima
H
H
Nakao
M
M
Nishi
T
T
Yamamoto
K
K
Kimura
T
T
Saito
Y
Y
Kochi
M
M
Kuratsu
J
J
Saya
H
H
Ushio
Y
Y
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D001932:Brain Neoplasms / Q000235:genetics*; D003001:Cloning, Molecular; D018899:CpG Islands / Q000235:genetics / Q000502:physiology*; D019175:DNA Methylation; D020868:Gene Silencing; D016515:Genes, Neurofibromatosis 2; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009442:Neurilemmoma / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D014162:Transfection