A systematic survey of the Cys2His2 zinc finger DNA-binding landscape

Abstract

Cys2His2 zinc fingers (C2H2-ZFs) comprise the largest class of metazoan DNA-binding domains. Despite this domain's well-defined DNA-recognition interface, and its successful use in the design of chimeric proteins capable of targeting genomic regions of interest, much remains unknown about its DNA-binding landscape. To help bridge this gap in fundamental knowledge and to provide a resource for design-oriented applications, we screened large synthetic protein libraries to select binding C2H2-ZF domains for each possible three base pair target. The resulting data consist of >160 000 unique domain–DNA interactions and comprise the most comprehensive investigation of C2H2-ZF DNA-binding interactions to date. An integrated analysis of these independent screens yielded DNA-binding profiles for tens of thousands of domains and led to the successful design and prediction of C2H2-ZF DNA-binding specificities. Computational analyses uncovered important aspects of C2H2-ZF domain–DNA interactions, including the roles of within-finger context and domain position on base recognition. We observed the existence of numerous distinct binding strategies for each possible three base pair target and an apparent balance between affinity and specificity of binding. In sum, our comprehensive data help elucidate the complex binding landscape of C2H2-ZF domains and provide a foundation for efforts to determine, predict and engineer their DNA-binding specificities.

Transcription factor–DNA binding: beyond binding site motifs

“…For example, large-scale B1H assays and structural analyses have shown that in C2H2-zf proteins, residues at the finger–finger interface and the order of fingers within an array frequently influence specificity [48,49]. In particular, adjacent fingers can constrain mutual finger orientation and influence the positioning of specificity-determining residues [46,50].…”

Toward clinical and molecular understanding of pathogenic variants in the ZBTB18 gene

“…By homology modeling, we illustrated a possible alteration in function caused by missense variants. ZBTB18 encodes for a protein with four C2H2 Zinc fingers, structures found in nearly half of DNA‐binding factors and known to be involved in numerous biological processes (Persikov et al., ). Our homology model shows how the recurrent p.Arg464His substituting variant in the C2H2 structure of ZNF 3 probably affects DNA‐binding properties.…”

Enabling functional genomics with genome engineering

“…However, other results suggest widespread genomic interactions with these proteins that can lead to off-target transcriptional dysregulation (Falahi et al 2013;Grimmer et al 2014). Surveys of ZF nuclease specificities demonstrate that off-target effects can be prevalent (Cornu et al 2008;Pattanayak et al 2011), although this off-target activity may be mitigated by optimized design tools that minimize confounding factors such as context-dependent ZF domain effects (Isalan et al 2001;Maeder et al 2008;Sander et al 2011;Gupta et al 2012;Persikov et al 2015).…”