MAPPING PATTERNS OF CPG ISLAND METHYLATION IN NORMAL AND NEOPLASTIC-CELLS IMPLICATES BOTH UPSTREAM AND DOWNSTREAM REGIONS IN DE-NOVO METHYLATION

Promoter region CpG island methylation is associated with tumor suppre ssor gene silencing in neoplasia, Gen-Bank sequence analyses revealed that a number of CpG islands are juxtaposed to multiple Alu repeats, w hich have been proposed as ''de novo methylation centers.'' These isla nds 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 norma l tissue, these islands were embedded between densely methylated flank ing regions containing multiple Alu repeats, These methylated flanks w ere segregated from the unmethylated, island CpG sites by Sp1-rich bou ndary regions. Finally, in human fibroblasts overexpressing DNA methyl transferase, de novo methylation of the Ecad CpG island initially invo lved sequences at both ends of the island and the adjacent, flanking r egions and progressed with time to encompass the entire CpG island reg ion, Together, these data suggest that boundaries exist at both ends o f 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.