Disruption of a 35 KB enhancer impairs CTCF binding and MLH1 expression in colorectal cells
| Autor: | Liu, Q, Thoms, JAI, Nunez, AC, Huang, Y, Knezevic, K, Packham, D, Poulos, RC, Williams, R, Beck, D, Hawkins, NJ, Ward, RL, Wong, JWH, Hesson, LB, Sloane, MA, Pimanda, JE |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
Adult
Aged 80 and over Male congenital hereditary and neonatal diseases and abnormalities CCCTC-Binding Factor nutritional and metabolic diseases DNA Methylation Middle Aged Colorectal Neoplasms Hereditary Nonpolyposis DNA Mismatch Repair digestive system diseases Gene Expression Regulation Neoplastic Enhancer Elements Genetic Cell Line Tumor Humans Female Oncology & Carcinogenesis CRISPR-Cas Systems Colorectal Neoplasms Promoter Regions Genetic MutL Protein Homolog 1 neoplasms Germ-Line Mutation Aged |
| Popis: | © 2018 American Association for Cancer Research. Purpose: MLH1 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 MLH1 expression over long distances have yet to be identified. Experimental Design: Here, we use chromosome conformation capture (3C) to screen a 650-kb region flanking the MLH1 locus to identify interactions between the MLH1 promoter and distal regions in MLH1-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 MLH1 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 MLH1 promoter. Results: A 1.8-kb DNA fragment, 35 kb upstream of the MLH1 transcription start site enhances MLH1 gene expression in colorectal cells. The enhancer was bound by CTCF and CRISPR-Cas9–mediated deletion of a core binding region impairs endogenous MLH1 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. Conclusions: A CTCF-bound region within the MLH1-35 enhancer regulates MLH1 expression in colorectal cells and is worthy of scrutiny in future genetic screening strategies for suspected Lynch syndrome associated with loss of MLH1 expression. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|