Functional Characterisation of C2H2-type Zinc Finger Proteins CTCF and ZNF512B

Abstract

The C2H2-zinc finger (ZF)-type family of proteins is the most abundant group of transcription factors involved in gene regulation. We showed this family is poorly-characterised as approximately 50% of members have little or no biological function known. A quarter of C2H2-ZF proteins are intolerant to genetic variation with over 100 inherited genetic diseases with causal links to this family of proteins. CCCTC-binding factor (CTCF) and ZNF512B are well- and poorly-characterised C2H2-ZF proteins, respectively. We examined their gene regulatory and protein interactions in the context of normal biology and disease. For CTCF, we examined five cancer-specific somatic mutations in ZFs and showed they reduced the affinity of binding to its core consensus sequence, as well as abrogate insulator function and transcriptional activity. Using pulldown-mass spectrometry, we examined the impact of additional non-DNA-contacting CTCF ZF mutations on protein-protein interactions (PPIs) and identified binding partners which were both gained and lost. To detect normal CTCF PPIs often missed by traditional proteomic approaches, we applied the first known biotin-dependent proximity labeling screen (BioID) for CTCF. We identified known as well as novel interactors which were further validated using co-immunoprecipitation (CoIP) assays. ZNF512B was a highly-enriched binding partner and was confirmed by CoIP, but little was known of its normal biological function. Biochemical analysis of ZNF512B revealed specific interactions with the nucleosome remodeling and deacetylation (NuRD) complex, specifically the RBBP4 subunit via an ‘RRK’ motif. Reduced ZNF512B expression levels fully inhibited the growth and clonogenicity of neuronal precursor cells (NPCs) and led to significant downregulation of neuronal-related genes. Also, we showed that ZNF512B expression significantly increases upon differentiation of NPCs into neurons. These findings have provided new insights into C2H2-ZF biology.