Resolving the 4D chromatin dynamics during early random X chromosome inactivation

X chromosome inactivation (XCI) is a process that compensates X-linked gene dosage in mammalian female cells. The silencing of a randomly selected chromosome is accompanied by dramatic three-dimensional (3D) reorganization across the entire chromosome. To investigate the 4D chromatin dynamics during early inactivation stages, we applied the multi-omics sequencing technique HiRES, which simultaneously detects 3D genome and transcriptome in single cells, in a mouse embryonic stem cell line with induced random XCI. The 3D genome and transcriptome dual-omics data allowed us to identify XCI lineages at single-cell resolution. We characterized multiple layers of chromosome reorganization during random XCI and discovered a transient structural inactivation state for both X chromosomes resulted from biallelic Xist expression. By constructing single-cell inactivation trajectories, we found that a vast majority of chromatin remodeling either accompanied or followed gene silencing. Further analysis of interaction decay kinetics revealed that TAD attenuation began from loss of interactions on TAD anchors. This study thus drew a detailed depiction of fine-scale chromatin reorganization during the initiation of random XCI. To initiate random X-chromosome inactivation (rXCI), we induced the differentiation of female hybrid mouse embryonic stem cells (mESCs) obtained from a cross between the C57B6/J and CAST/EiJ mouse strains by removing 2i/Lif. Subsequently, we harvested cells at multiple time points during the differentiation process, spanning from day 0 to day 4. We then utilized HiRES technology to simultaneously obtain 3D genome and transcriptome data at the single-cell level.