Figure 6. ZEB1 binding site methylation correlates with BRCA1 status and with TAp73 levels in primary ovarian cancers.

(A) Increased methylation of residues within the ZEB binding site in primary tumors from BRCA1 mutation carriers (MUT, N=4) versus non-carriers (WT, N=8). Mean methylated fraction in each tumor is shown for CpG residues (−567, −561 and −542) which exhibit BRCA1-dependent methylation in vitro, assayed by bisulfite sequencing. (B) Reversion to wild-type BRCA1 expression in vivo is associated with hypomethylation of the ZEB1 binding site. Methylation-specific PCR was used to assay methylated (M) and unmethylated (U) resides as in (A), in the primary (BRCA1-deficient) and recurrent “revertant” (BRCA1-expressing) tumor. (C) TAp73 levels correlate with methylation of the ZEB1 binding region in primary ovarian carcinomas. Bisulphite sequencing of the entire ZEB binding region was performed in 25 primary tumors, and the mean fraction of methylated CpG residues was correlated with TAp73 expression assessed by QRT-PCR in the same tumor. P value by Fisher’s Exact Test. (D) Epigenetic regulation of ZEB-1 binding controls p73 expression. Tumors with BRCA1 pathway defects exhibit hypermethylation within p73 intron 1, preventing ZEB1 binding and leading to upregulation of TAp73, which in turn induces pro-apoptotic genes in response to cisplatin-induced DNA damage.