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

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. Overall design: Capture HiC (CHiC) was performed in the following tissues: blastocyst-derived mouse embryonic stem cells, differentiated XEN cells and midbrain of E11.5 embryos. For ES and XEN cells, 2 individual homozygous embryos were processed and analyzed seperately. WT controls are littermates from isolated embryos. Midbrain samples were pooled from several WT and homozygous mutant embryo.

由CCCTC结合因子（CTCF）基序界定的染色质结构域边界，已被证实能够限制增强子的作用范围。然而，结构域结构的破坏往往仅导致轻微的基因表达变化，而预测边界重排对动物发育的影响仍颇具挑战。本研究靶向包含三个成纤维细胞生长因子（Fibroblast Growth Factor, FGF）基因——Fgf3、Fgf4及Fgf15的结构域内的CTCF基序簇，以检测基因调控与小鼠发育是否对携带多个发育调控因子的染色质结构域破坏格外敏感。删除界定该结构域着丝粒侧边界的四个CTCF基序簇后，FGF基因与跨越删除边界的脑增强子发生了异位相互作用，同时FGF表达被显著诱导，最终导致胚胎围产期致死，并伴随脑膨出与颅面裂表型。杂合性边界缺失足以引发这些完全外显的表型；尤为引人注目的是，仅缺失该簇中朝向脑增强子的单个CTCF基序，即可诱导异位FGF表达并引发脑膨出。不过，这种对结构域结构扰动的表型敏感性，并未延伸至该结构域的所有CTCF介导的边界，也未涉及这三个FGF基因调控的所有发育过程。尽管精确调控FGF4水平对于囊胚发育至关重要，但我们的所有结构扰动均未影响胚胎着床。事实上，删除Fgf3与Fgf4之间的CTCF边界后发现，这些相邻基因在囊胚中实现完全分化的表达模式，是通过不依赖CTCF的机制完成的，且依赖于体内显著的增强子-启动子特异性。本研究揭示了特定结构域边界处的小型序列变异，何以能够产生出人意料的巨大表型效应，并为不同发育环境如何影响染色质结构扰动的表型敏感性提供了重要见解。整体实验设计：对以下组织开展捕获Hi-C（Capture Hi-C, CHiC）实验：囊胚来源的小鼠胚胎干细胞、分化的XEN细胞，以及E11.5胚胎的中脑。对于胚胎干细胞与XEN细胞，分别处理并分析了2个独立的纯合子胚胎；野生型（WT）对照为分离自同窝的胚胎。中脑样本则混合了多只野生型与纯合突变体胚胎。