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

Abstract

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 modi®ca-tions 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-speci®c 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 ®rst evidence suggesting a role for DNA methylation in the transcriptional inactivation of the BRCA1 in human breast cancer.

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

“…Our laboratory (Mancini et al, 1998) and others have previously shown site-speci®c e ects of DNA methylation within the promoter regions of a variety of tumour suppressor genes. However, similar information concerning the NF1 gene has been lacking.…”

“…For example, methylation of exonic CpGs is considered to be a major contributor to point mutations due to the hypermutability of 5-methylcytosine. In fact, up to 30% of germ-line mutations leading to human genetic disease occur as C to T transitions at CpG dinucleotides which frequently result in protein truncating mutations (Mancini et al, , 1998Rodenhiser et al, 1997;Cooper and Krawczak, 1989). DNA methylation within gene regulatory regions, on the other hand, may mediate gene inactivation either by causing general alterations in chromatin con®guration or by interfering with the binding of transcription factors.…”

“…Several putative transcription factor binding motifs are present in NF1, including a CRE (cAMP response) element and an SRE (serum response) element located 16 and 14 bases respectively from the transcription start site (Hajra et al, 1994). We have shown previously that methylation abolishes CREB binding in the BRCA1 gene (Mancini et al, 1998). Therefore, we sought to identify putative regulatory motifs in the NF1 promoter where methylation might exert its e ect, by using gel mobility shift assays to determine the binding potential of proteins from neuroblastoma nuclear extracts to methylated and unmethylated oligonucleotides corresponding to the CpG-containing CRE element.…”

“…Di erential DNA methylation at CpG islands that contain gene regulatory regions is known to contribute to the complex regulatory mechanisms governing eukaryotic gene expression (Bird, 1987;Laird and Jaenisch, 1994;Graessman and Graessman, 1993;Razin and Cedar, 1991;Barlow, 1993). Variant methylation patterns at speci®c transcription-factor binding motifs within regulatory regions may result in temporal and tissuespeci®c patterns of gene expression (Issa et al, 1996a,b;Iguchi-Ariga and Scha ner, 1989;Comb and Goodman, 1990;Clark et al, 1997;Mancini et al, 1998). Particularly relevant in the context of cancer syndromes have been reports of methylation within promoter regions of tumour suppressor genes contributing to the multistage process of tumour progression (Laird and Jaenisch, 1994;Issa et al, 1996a,b;Mancini et al, 1998;Greger et al, 1989;Ohtani-Fujita et al, 1993).…”

“…Variant methylation patterns at speci®c transcription-factor binding motifs within regulatory regions may result in temporal and tissuespeci®c patterns of gene expression (Issa et al, 1996a,b;Iguchi-Ariga and Scha ner, 1989;Comb and Goodman, 1990;Clark et al, 1997;Mancini et al, 1998). Particularly relevant in the context of cancer syndromes have been reports of methylation within promoter regions of tumour suppressor genes contributing to the multistage process of tumour progression (Laird and Jaenisch, 1994;Issa et al, 1996a,b;Mancini et al, 1998;Greger et al, 1989;Ohtani-Fujita et al, 1993). Our laboratory has previously shown that in the BRCA1 gene, site-speci®c methylation at a putative CRE binding motif interferes with CREB binding, suggesting that aberrant DNA methylation at BRCA1 regulatory sequences may play a role in the transcriptional inactivation of this gene (Mancini et al, 1998).…”