CTCF zinc finger mutations produce cellular phenotypes explained by structure-function relationships

Abstract CCCTC-binding factor (CTCF) plays fundamental roles in transcriptional regulation and chromatin architecture maintenance. CTCF is also a tumour suppressor frequently mutated in cancer, however the structural and functional impact of mutations have not been examined. We performed molecular and structural characterisation of 5 CTCF missense zinc finger (ZF) mutations occurring within key intra- and inter-ZF residues. Functional characterisation of CTCF ZF mutations revealed a complete (L309P, R339W, R377H) or intermediate (R339Q) abrogation as well as an enhancement (G420D) of the anti-proliferative effects of CTCF. DNA binding at select sites was disrupted and transcriptional regulatory activities abrogated. Molecular docking and molecular dynamics confirmed that mutations in residues specifically contacting DNA bases or backbone exhibited loss of DNA binding. However, R339Q and G420D were stabilised by the formation of new primary DNA bonds, contributing to gain-of-function. Our data confirm a spectrum of loss-, change- and gain-of-function impacts in CTCF zinc fingers are observed in cell growth regulation and gene regulatory activities. We have established that these diverse cellular phenotypes in CTCF are explained by examining structure-function relationships.