Disruption of a -35 kb Enhancer Impairs CTCF Binding and <i>MLH1</i> Expression in Colorectal Cells.

Abstract

<b>Purpose:</b><i>MLH1</i> is a major tumor suppressor gene involved in the pathogenesis of Lynch syndrome and various sporadic cancers. Despite their potential pathogenic importance, genomic regions capable of regulating <i>MLH1</i> expression over long distances have yet to be identified.<b>Experimental Design:</b> Here, we use chromosome conformation capture (3C) to screen a 650-kb region flanking the <i>MLH1</i> locus to identify interactions between the <i>MLH1</i> promoter and distal regions in <i>MLH1</i>-expressing and nonexpressing cells. Putative enhancers were functionally validated using luciferase reporter assays, chromatin immunoprecipitation, and CRISPR-Cas9-mediated deletion of endogenous regions. To evaluate whether germline variants in the enhancer might contribute to impaired <i>MLH1</i> expression in patients with suspected Lynch syndrome, we also screened germline DNA from a cohort of 74 patients with no known coding mutations or epimutations at the <i>MLH1</i> promoter.<b>Results:</b> A 1.8-kb DNA fragment, 35 kb upstream of the <i>MLH1</i> transcription start site enhances <i>MLH1</i> gene expression in colorectal cells. The enhancer was bound by CTCF and CRISPR-Cas9-mediated deletion of a core binding region impairs endogenous <i>MLH1</i> expression. A total of 5.4% of suspected Lynch syndrome patients have a rare single-nucleotide variant (G > A; rs143969848; 2.5% in gnomAD European, non-Finnish) within a highly conserved CTCF-binding motif, which disrupts enhancer activity in SW620 colorectal carcinoma cells.<b>Conclusions:</b> A CTCF-bound region within the <i>MLH1</i>-35 enhancer regulates <i>MLH1</i> expression in colorectal cells and is worthy of scrutiny in future genetic screening strategies for suspected Lynch syndrome associated with loss of MLH1 expression. <i>Clin Cancer Res; 24(18); 4602-11. ©2018 AACR</i>.