Site-specific DNA methylation in the neurofibromatosis (NF1) promoter interferes with binding of CREB and SP1 transcription factors

Tumour suppressor genes and growth regulatory genes are frequent targets fo r methylation defects that can result in aberrant expression and mutagenesi s, We have established a methylation map of the promoter region of the neur ofibromatosis (NF1) gene and demonstrated functional sensitivity for methyl ation at specific sites for the SP1 and CRE binding (CREB) proteins in the NF1 regulatory region. We evaluated the methylation status of CpG dinucleot ides within five promoter subregions in the human and mouse homologues of t he neurofibromatosis (NF1) genes, Three 5' subregions vc ere found to be co nsistently methylated in all the tissues analysed. In contrast, DNA methyla tion was absent in the vicinity of the transcription start site bounded by SP1 recognition sequences. Gelshift assays showed that methylation specific ally inhibits the CREB transcription factor from binding to its recognition site at the NF1 transcription start site. Furthermore, SP1 elements within the NF1 promoter are methylation sensitive, particularly when methylation is present on the antisense strand. We propose that for NF1 as with several other tumour suppressor genes, CpG methylation occurs in a complex, site-s pecific manner with the maintenance of a methylation-free promoter region b ounded by SP1 binding sites that allow an accessible promoter to be retaine d. When these SP1 boundaries are breached, methylation can sweep in, render ing the promoter inaccessible for specific methylation-sensitive transcript ion factors and leading to a loss of functional integrity of the methylatio n-free CpG island.