4D Chromatin Dynamics Resolved During Early Random X Chromosome Inactivation

X chromosome inactivation 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 reorganization across the entire chromosome. To investigate the four-dimensional chromatin dynamics during early inactivation stages, we applied the multi-omics sequencing technique HiRES (Hi-C and RNA-seq employed simultaneously), which simultaneously detects the three-dimensional genome and transcriptome in single cells, in a mouse embryonic stem cell line with induced random inactivation. This three-dimensional genome and transcriptome dual-omics data allowed us to identify random inactivation trajectories at single-cell resolution. We characterized multiple layers of X-chromosome reorganization and discovered a transient structural state shared by both X chromosomes, associated with biallelic X-inactive specific transcript (Xist) expression. By constructing single-cell inactivation trajectories, we found that most chromatin remodeling either accompanied or followed gene silencing. Further analysis of interaction decay kinetics revealed that topologically associating domain (TAD) attenuation began from loss of interactions on TAD anchors. This study thus provides a detailed depiction of fine-scale chromatin reorganization during the initiation of random X chromosome inactivation.