Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development [Cut&Run]

Chromatin domain boundaries delimited by CTCF motifs have been shown to restrict the range of enhancers. However, disruption of domain structure often results in mild gene expression changes, and predicting the impact of boundary rearrangements on animal development remains challenging. By targeting clusters of CTCF motifs in a domain with three FGF genes­—Fgf3, Fgf4, and Fgf15­—we tested whether gene regulation and mouse development may be particularly sensitive to disruption of chromatin domains with multiple developmental regulators. Deletion of a cluster of four CTCF motifs that defines the centromeric boundary of this domain­, resulted in ectopic interactions of the FGF genes with brain enhancers located across the deleted boundary, and a strong induction of FGF expression that led to perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary loss was enough to cause these fully penetrant phenotypes, and strikingly, loss of the single CTCF motif within the cluster that is oriented towards the brain enhancers, was sufficient to induce ectopic FGF expression and cause encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF-mediated boundaries of this domain nor to all developmental processes controlled by these three FGF genes. Although precise control of FGF4 levels is essential to regulate blastocyst development, none of our structural perturbations affected implantation. In fact, deletion of a CTCF boundary between Fgf3 and Fgf4, showed that the ability of these neighboring genes to have fully divergent expression patterns in blastocysts is achieved through CTCF-independent mechanisms and relies on remarkable enhancer-promoter specificity in vivo. Our work highlights how small sequence variants at certain domain boundaries can have a surprisingly outsized phenotypic effect and provides important insight into how different developmental contexts affect phenotypic sensitivity to perturbation of chromatin structure.