The polymorphic variant rs1800734 influences methylation acquisition and allele-specific TFAP4 binding in the MLH1 promoter leading to differential mRNA expression

Abstract

Expression of the mismatch repair gene MutL homolog 1 (MLH1) is silenced in a clinically important subgroup of sporadic colorectal cancers. These cancers exhibit hypermutability with microsatellite instability (MSI) and differ from microsatellite-stable (MSS) colorectal cancers in both prognosis and response to therapies. Loss of MLH1 is usually due to epigenetic silencing with associated promoter methylation; coding somatic mutations rarely occur. Here we use the presence of a colorectal cancer (CRC) risk variant (rs1800734) within the MLH1 promoter to investigate the poorly understood mechanisms of MLH1 promoter methylation and loss of expression. We confirm the association of rs1800734 with MSI+ but not MSS cancer risk in our own data and by meta-analysis. Using sensitive allele-specific detection methods, we demonstrate that MLH1 is the target gene for rs1800734 mediated cancer risk. In normal colon tissue, small allele-specific differences exist only in MLH1 promoter methylation, but not gene expression. In contrast, allele-specific differences in both MLH1 methylation and expression are present in MSI+ cancers. We show that MLH1 transcriptional repression is dependent on DNA methylation and can be reversed by a methylation inhibitor. The rs1800734 allele influences the rate of methylation loss and amount of re-expression. The transcription factor TFAP4 binds to the rs1800734 region but with much weaker binding to the risk than the protective allele. TFAP4 binding is absent on both alleles when promoter methylation is present. Thus we propose that TFAP4 binding shields the protective rs1800734 allele of the MLH1 promoter from BRAF induced DNA methylation more effectively than the risk allele.

Transcription Factor AP4 Mediates Cell Fate Decisions: To Divide, Age, or Die

“…Interestingly, the rs1800734 G > A mutation disrupts the AP4 binding sequence, CAGCTG, located at the promoter of MLH1 . The loss of AP4 binding leads to an increase in DNA methylation at the promoter region and the epigenetic silencing of MLH1 [ 21 ]. In another study, Liu and colleagues found that the rs1800734 G > A mutation in several colorectal cancer cell lines enhances the long-range interactions between rs1800734 and the promoter and 3′ UTR region of the doublecortin-like kinase 3 gene ( DCLK3 ), resulting in an increase in the expression of this potential oncogene [ 22 ].…”

The MLH1 polymorphism rs1800734 and risk of endometrial cancer with microsatellite instability

“…We carried out a meta-analysis and found no evidence of MSI EC risk association for rs1800734 (OR = 1.06 CI 0.85-1.33 p = 0.60) or any other SNPs in the MLH1 region (supplementary table 2), after correction for multiple testing. While the sample set is relatively small and the findings will need replicating, a similarly sized MSI CRC sample set gave a strong rs1800734 risk association (CRC MSI cases n = 170, controls n = 2686, OR = 1.95, 95% CI 1.50-2.55, p = 8.04 × 10 −7 , [16]). We estimate that we had 99% power to detect an OR of this magnitude for MSI EC.…”

“…In CRC tumour tissue stratified by genotype (but not in normal tissue), we previously found that rs1800734 acted as an MLH1 expression quantitative trait locus (eQTL) and a methylation quantitative trait locus meQTL (meQTL), with the risk allele associated with higher methylation and lower mRNA expression [16]. We were therefore interested to see whether this was true for EC tumours given that there was no association observed between rs1800734 and EC risk.…”

“…This is predominantly due to somatic silencing by promoter hypermethylation in both types of cancer, and less frequently caused by germline pathogenic variants [12]. In CRC a promoter polymorphism, rs1800734 in the 5′untranslated region of MLH1 is strongly associated with an increased risk of MSI cancer, as well as hypermethylation and reduced MLH1 transcription [13][14][15][16][17]. This polymorphism has no association with microsatellite stable (MSS) CRC and shows a much weaker association in data sets unstratified by MSI status.…”

“…This polymorphism has no association with microsatellite stable (MSS) CRC and shows a much weaker association in data sets unstratified by MSI status. Using artificially de-methylated MSI CRC cell lines heterozygous for rs1800734, we have previously shown that methylation accumulation occurs more quickly on the risk (A) allele than the protective (G) allele and that this is accompanied by an allelic bias in MLH1 transcription, with more expression from the protective allele [ 16 ]. We have suggested that the risk allele is more prone to methylation accumulation due to disruption of the binding site of transcription factor TFAP4, which binds strongly to the protective allele only [ 16 , 18 , 19 ].…”