Structure–function relationships explain CTCF zinc finger mutation phenotypes in cancer

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 five cancer-specific 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 that a spectrum of loss-, change- and gain-of-function impacts on CTCF zinc fingers are observed in cell growth regulation and gene regulatory activities. Hence, diverse cellular phenotypes of mutant CTCF are clearly explained by examining structure–function relationships. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00018-021-03946-z.