A CRISPR Screen Identifies Myc-associated Zinc Finger Protein (MAZ) as an Insulator Functioning at CTCF boundaries in Hox Clusters

The essential CCCTC-binding factor (CTCF) is critical to three-dimensional (3D) genome organization. CTCF binding insulates active and repressed genes within the clusters upon differentiation, but such binding does not participate in boundary formation in all cell types, such as embryonic stem cells. We conducted a genome-wide CRISPR knockout screen to identify genes required for CTCF boundary activity at the cluster, complemented by novel biochemical approaches. This screen identified Myc-associated zinc finger protein (MAZ) as a CTCF insulator co-factor, among other candidates listed herein. MAZ depletion or specific deletion of MAZ motifs within the clusters led to de-repression of posterior genes immediately after CTCF boundaries upon differentiation, which phenocopied deletion of the proximal CTCF motifs. Similar to CTCF, MAZ interacted with the cohesin subunit, RAD21. Upon loss of MAZ, local contacts within topologically associated domains (TADs) were disrupted, as evidenced by HiC analysis. Thus, MAZ is a novel factor sharing insulation properties with CTCF and contributing to the genomic architectural organization.