Abstract
CCCTC-binding factor (CTCF) is critical to three-dimensional genome organization. Upon differentiation, CTCF insulates active and repressed genes within Hox gene clusters. We conducted a genome-wide CRISPR knockout (KO) screen to identify genes required for CTCF-boundary activity at the HoxA cluster, complemented by biochemical approaches. Among the candidates, we identified Myc-associated zinc-finger protein (MAZ) as a cofactor in CTCF insulation. MAZ colocalizes with CTCF at chromatin borders and, similar to CTCF, interacts with the cohesin subunit RAD21. MAZ KO disrupts gene expression and local contacts within topologically associating domains. Similar to CTCF motif deletions, MAZ motif deletions lead to derepression of posterior Hox genes immediately after CTCF boundaries upon differentiation, giving rise to homeotic transformations in mouse. Thus, MAZ is a factor contributing to appropriate insulation, gene expression and genomic architecture during development.
Highlights
CCCTC-binding factor (CTCF) is critical to three-dimensional genome organization
We focused on the CTCF boundary that forms upon embryonic stem cells (ESCs) differentiation into cervical MNs16,17
In this study, we demonstrated that an unbiased genome-wide CRISPR screen coupled with biochemical approaches enabled the identification of factors that function to and in conjunction with CTCF
Summary
CCCTC-binding factor (CTCF) is critical to three-dimensional genome organization. Upon differentiation, CTCF insulates active and repressed genes within Hox gene clusters. CTCF functions as a boundary between active and repressed chromatin domains, decorated by Trithorax and Polycomb, respectively, at Hox clusters upon differentiation of mouse embryonic stem cells (ESCs) into cervical motor neurons (MNs)[16,17]. This dynamic compartmentalization of Hox clusters into antagonistic domains allows CTCF-mediated looping to reshape regulatory interactions. During differentiation, additional regulatory factors appear to be necessary to foster CTCF-mediated insulation properties To identify such putative factors affecting CTCF-boundary activity, we devised an unbiased genome-wide loss-of-function genetic screen involving a functional CTCF boundary within the. Through a series of functional assays performed in vitro and in vivo during development, we demonstrate that MAZ is integral to appropriate gene expression and architectural genome organization in the context of CTCF and cohesin
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