Mapping Patterns of CpG Island Methylation in Normal and Neoplastic Cells Implicates Both Upstream and Downstream Regions inde Novo Methylation

Abstract

Promoter region CpG island methylation is associated with tumor suppressor gene silencing in neoplasia. GenBank sequence analyses revealed that a number of CpG islands are juxtaposed to multiple Alu repeats, which have been proposed as "de novo methylation centers." These islands also contain multiple Sp1 elements located upstream and downstream of transcription start, which have been shown to protect CpG islands from methylation. We mapped the methylation patterns of the E-cadherin (E-cad) and von Hippel-Lindau (VHL) tumor suppressor gene CpG island regions in normal and neoplastic cells. Although unmethylated in normal tissue, these islands were embedded between densely methylated flanking regions containing multiple Alu repeats. These methylated flanks were segregated from the unmethylated, island CpG sites by Sp1-rich boundary regions. Finally, in human fibroblasts overexpressing DNA methyltransferase, de novo methylation of the Ecad CpG island initially involved sequences at both ends of the island and the adjacent, flanking regions and progressed with time to encompass the entire CpG island region. Together, these data suggest that boundaries exist at both ends of a CpG island to maintain the unmethylated state in normal tissue and that these boundaries may be progressively overridden, eliciting the de novo methylation associated with tumor suppressor gene silencing in neoplasia.

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

“…However, methylation within non-CpG island regions is thought to contribute to the maintenance of a heterochromatinized state which precludes binding of transcription factors (Tate and Bird, 1993;Antequera et al, 1990;Nan et al, 1996). Since it has been shown that CpG dinucleotides in bulk chromatin outside CpG-islands may become hypomethylated in some tumours, altered methylation within this NF1 region could compromise the gene's structural integrity and alter NF1 expression (Gra et al, 1997).…”

“…Several studies in the literature point to a potential role for SP1 binding sites in maintaining the CpG island in an unmethylated and transcriptionally active state (Gra et al, 1997;Clark et al, 1997;Macleod et al, 1994;Brandeis et al, 1994). In contrast to many other transcription factors, SP1 has until recently been shown to bind to its recognition sequence regardless of its methylation status.…”

“…Previous studies have shown that the SP1 transcription factor is critical for both transcription (Macleod et al, 1994;Brandeis et al, 1994;Holler et al, 1988;Worrad and Schultz, 1997) as well as for the maintenance of methylation-free status within CpGrich regions (Clark et al, 1997;Gra et al, 1997). Analysis of the NF1 promoter using TFSEARCH localized several putative SP1 motifs.…”

“…Recently, Gra et al (1997) have described how multiple SP1 elements both upstream and downstream of transcription start sites are common features of many CpG islands. This group has generated detailed maps of methylation patterns anking the E-cad and VHL tumour suppressor genes and suggested that methylation boundaries established around CpG islands may be overridden by de novo methylation, contributing to tumour suppressor gene silencing in neoplasia.…”

Quantification of Epigenetic and Genetic 2nd Hits in CDH1 During Hereditary Diffuse Gastric Cancer Syndrome Progression

“…Moreover, this CpG island has been described as a hallmark of promoter methylation in human cancers. 24,25 CDH1 promoter methylation analysis was performed in all tumor DNA samples. The EpiTect Bisulfite Kit (Qiagen, Valencia, Calif) was used to treat 200 ng of DNA.…”