CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression [Hi-ChIP]

CTCF is an 11-zinc-finger DNA-binding protein that acts as a transcriptional repressor and insulator, as well as an architectural protein required for 3D genome folding. CTCF mediates long-range chromatin looping and is enriched at the boundaries of topologically associating domains (TADs), which are sub-megabase chromatin structures composed of genomic regions with high contact frequency. Although CTCF is essential for cycling cells and developing embryos, its in vitro removal has only modest effects over gene expression, challenging the generally accepted idea that TADs facilitate enhancer-promoter interactions within gene regulatory landscapes. However, the effects of an altered CTCF-mediated chromatin structure on gene regulation in vivo are poorly understood. Here, we have generated a ctcf knockout mutant in zebrafish that allows us to monitor the effect of CTCF loss of function during embryo patterning and organogenesis. CTCF absence leads to a loss of chromatin structure in zebrafish embryos and affects the expression of thousands of genes, among them many developmental genes. In addition, we show that chromatin accessibility, both at CTCF sites and at developmental cis-regulatory elements (CREs), is severely compromised in ctcf mutants. Probing chromatin interactions from developmental genes at high resolution, we further demonstrate that promoters fail to fully establish long-range contacts with their associated regulatory landscapes, leading to altered gene expression patterns during development. Therefore, our results demonstrate that CTCF and TADs are essential to finetune gene expression during embryonic development, providing the structural basis for the establishment of developmental gene regulatory landscapes. Hi-C assays in wild-type and ctcf zebrafish mutants, at developmental stage 48 hpf