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 for methylation defects that can result in aberrant expression and mutagenesis. We have established a methylation map of the promoter region of the neurofibromatosis (NF1) gene and demonstrated functional sensitivity for methylation at specific sites for the SP1 and CRE binding (CREB) proteins in the NF1 regulatory region. We evaluated the methylation status of CpG dinucleotides within five promoter subregions in the human and mouse homologues of the neurofibromatosis (NF1) genes. Three 5' subregions were found to be consistently methylated in all the tissues analysed. In contrast, DNA methylation was absent in the vicinity of the transcription start site bounded by SP1 recognition sequences. Gelshift assays showed that methylation specifically 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-specific manner with the maintenance of a methylation-free promoter region bounded by SP1 binding sites that allow an accessible promoter to be retained. When these SP1 boundaries are breached, methylation can sweep in, rendering the promoter inaccessible for specific methylation-sensitive transcription factors and leading to a loss of functional integrity of the methylation-free CpG island.

CpG methylation within the 5' regulatory region of the BRCA1 gene is tumor specific and includes a putative CREB binding site.

Breast cancer is a genetic disease arising from a series of germ-line and/or somatic DNA changes in a variety of genes, including BRCA1 and BRCA2. DNA modifications have been shown to occur by a number of mechanisms that include DNA methylation. In some cases, the aberrant methylation of CpGs within 5' regulatory regions has led to suppression of gene activity. In this report we describe a variation in the pattern of DNA methylation within the regulatory region of the BRCA1 gene. We found no evidence of methylation at CpGs within the BRCA1 promoter in a variety of normal human tissues. However, screening of a series of randomly sampled breast carcinomas revealed the presence of CpG methylation adjacent to the BRCA1 transcription start site. One such methylated CpG occurs at a putative CREB (cAMP-responsive element binding) transcription factor binding site in the BRCA1 promoter. Gelshift assays with methylated and unmethylated BRCA1/CREB binding site oligonucleotides demonstrate that this site is sensitive to site-specific CpG methylation. These data suggest that aberrant DNA methylation at regulatory sequences in the BRCA1 locus may play a role in the transcriptional inactivation of the BRCA1 gene within subclones of breast tumors. This study represents the first evidence suggesting a role for DNA methylation in the transcriptional inactivation of the BRCA1 in human breast cancer.

Homonucleotide tracts, short repeats and CpG/CpNpG motifs are frequent sites for heterogeneous mutations in the neurofibromatosis type 1 (NF1) tumour-suppressor gene.

Neurofibromatosis type 1 (NF1) is among the most common human genetic disorders, having a constellation of cutaneous and skeletal manifestations, intellectual impairment, and an increased risk for a variety of malignancies. The NF1 gene has a high spontaneous mutation rate and is also associated with a variety of sporadic cancers in the general population. While a number of laboratories are involved in a coordinated effort to identify NF1 mutations, an important gap in our knowledge is an understanding of the mechanisms responsible for NF1 mutagenesis. In this present paper we describe our analysis of the sequence environment in the NF1 gene at those sites where small deletions, insertions and nucleotide substitution mutations have been reported. Our objective was to determine whether specific nucleotide sequences commonly occur at these mutation sites within the NF1 gene. We assessed how frequently independent NF1 mutations occur at the site of short direct repeats, single nucleotide repeats (homonucleotides) and at CpG and CpNpG motifs. We have established that homonucleotide and short direct repeats are commonly involved in the majority of small deletions and insertions analysed. Substitution mutations are frequently associated with homonucleotide repeats and methylatable CpG dinucleotides and CpNpG trinucleotides. We suggest that NF1 mutations are acquired and retained by cells through an intricate balancing of repair and replication mechanisms. Such mutations may provide a proliferative advantage for that cell and its progeny.

Site and sequence specific DNA methylation in the neurofibromatosis (NF1) gene includes C5839T: the site of the recurrent substitution mutation in exon 31.

CpG dinucleotides provide hotspots for transitional mutations in a variety of genes, some leading to genetic diseases in humans. Although this phenomenon is attributed to cytosine methylation at such sites, direct and specific observations of CpG methylation at the sites of recurrent mutations are lacking. We have used a bisulfite genomic sequencing method to analyze DNA methylation within three representative exons from the neurofibromatosis type 1 (NF1) gene, well recognized for its high frequency of spontaneous mutations. We observed that the cytosine methylation within NF1 exons 28, 29, and 31 is restricted to CpG dinucleotides, including the CpG dinucleotide present at the site of the recurrent NF1 mutation (C5839T; also referred to as R1947X). At several sites, clone-specific methylation differences were also observed. Our results provide experimental evidence for the hypothesis that methylatable CpGs in the NF1 gene contribute to spontaneous germline mutations associated with this gene, by showing that DNA methylation does occur at all CpGs contained within these representative NF1 exons. As well, the DNA methylation seen at the common mutation site in exon 31 may explain why this site is frequently mutated. Methylation-dependent mutagenesis may also provide a basis for some somatic (second hit) mutations which disable the normal allele and result in the development of NF1 associated symptoms.