An evaluation of the effects of CRISPR/cas9-mediated editing of the Dxz4 locus on regulation of the mouse inactive X chromosome in Patski cells [RNA-seq]

The mammalian inactive X chromosome (Xi) condenses into a bipartite structure with two superdomains of frequent long-range contacts separated by a boundary or hinge region. Using in situ DNase Hi-C in mouse cells with deletions or inversions within the hinge, we show that the conserved repeat locus Dxz4 alone is sufficient to maintain the bipartite structure and that Dxz4 orientation controls the distribution of long-range contacts on the Xi. Frequent long-range contacts between Dxz4 and the telomeric superdomain are either lost after its deletion or shifted to the centromeric superdomain after its inversion. This massive reversal in contact distribution is consistent with the reversal of CTCF motif orientation at Dxz4. Decondensation of the Xi after Dxz4 deletion is associated with partial restoration of TADs normally attenuated on the Xi, and with an increase in chromatin accessibility and CTCF binding, but few changes in gene expression, in accordance with multiple epigenetic mechanisms ensuring X silencing. We propose that Dxz4 represents a structural platform for frequent long-range contacts with multiple loci in a direction dictated by the orientation of a bank of CTCF motifs at Dxz4, which may work as a ratchet to form the distinctive bipartite structure of the condensed Xi. RNA-seq experiment on F1 hybrid wild-type and CRISPR/cas9-modified Patski cells.

哺乳动物失活X染色体（inactive X chromosome, Xi）会凝聚为二分结构，由两个以高频长程相互作用为特征的超级结构域构成，二者由边界区域或铰链区域相互分隔。通过对铰链区域存在缺失或倒位的小鼠细胞开展原位DNase Hi-C实验，我们证实：仅保守重复位点Dxz4即可维持该二分结构，且Dxz4的取向可调控Xi上长程相互作用的分布。Dxz4与端粒超级结构域之间的高频长程相互作用，在Dxz4缺失后会完全消失，而在其发生倒位后则会转移至着丝粒超级结构域。这种相互作用分布的大规模反转，与Dxz4位点处CCCTC结合因子（CTCF）基序取向的反转完全一致。Dxz4缺失后Xi的去凝聚现象，与Xi上通常被抑制的拓扑关联结构域（topologically associating domains, TADs）的部分恢复相关，同时伴随着染色质开放性与CTCF结合水平的提升，但基因表达仅发生极少变化，这契合多种维持X染色体沉默的表观遗传调控机制。我们提出，Dxz4可作为一类结构平台，介导同多个基因座间的高频长程相互作用，其作用方向由Dxz4位点处一簇CTCF基序的取向所决定；该结构或可作为棘轮机制，促成凝聚态Xi独特的二分结构。本研究针对F1杂交野生型细胞与经CRISPR/Cas9编辑的Patski细胞开展了RNA测序（RNA-seq）实验。