Fig. 2

TFAP4 binding occurs in EC cells but does not result in rs1800734 allelic bias of MLH1 expression or promoter methylation. a TFAP1 binds to MSS but not MLH1 methylated MSI EC cells. The line graph shows relative TFAP4 enrichment at UCSC coordinates proximal to the MLH1 promoter in HEC1A (MSS) cells and NOU1 (MSI) cells untreated and 0 days, 4 days and 11 days post 48 h 5′-azacytidine. Relative TFAP4 enrichment was determined after normalization with input DNA using the ΔΔCT method. Error bars show the standard error of the mean (n = 3). HEC1A cells show TFAP4 binding but AzaC treatment did not reactivate TFAP4 expression in Nou1 cells. b MLH1 expression in EC cells shows no rs1800734 allelic bias. RNA was extracted from HEC1A cells and NOU1 cells, untreated, 0 days, 4 days and 11 days post 48 h 5′-azacytidine treatment. The bar chart shows relative mRNA expression levels with error bars showing the standard error of the mean (n = 3). Percentages represent the proportion of G or A reads out of the total rs1800734 sequences for each cell line/time point. NOU1 MLH1 expression is activated by treatment with 5-azacytidine, with the highest expression 0 days post-treatment. Re-repression occurs at 4 days and 11 days post-treatment. There is no significant allelic bias at any stage. c 5′-Azacytidine treatment of NOU1 cells removes MLH1 promoter methylation but no allelic bias is seen in the control or at any post-treatment time point as methylation is re-acquired