In mammalian female cells, random X chromosome inactivation (rXCI) serves as a mechanism to balance X-linked gene dosage, achieved by silencing one X chromosome in a random fashion. This inactivation leads to extensive reorganization of the chromosome's three-dimensional (3D) structure. To understand the dynamic chromatin changes occurring in the early phases of this process, we employed HiRES, a multi-omics sequencing approach that captures both 3D genome architecture and transcriptomic information at the single-cell level. This combined genome-transcriptome dataset enabled us to trace XCI lineage development with single-cell precision, revealing multiple structural changes within the X chromosome during inactivation. Importantly, we observed a unique transient inactivation state affecting both X chromosomes, induced by the biallelic expression of Xist. By constructing single-cell inactivation trajectories, we found that chromatin remodeling largely paralleled or followed the onset of gene silencing. A closer look at interaction decay kinetics showed that TAD weakening initiated through loss of interactions at TAD anchor points. Altogether, this study provides a highly detailed model of chromatin reorganization during the early steps of rXCI.

X染色体失活,三维基因组,染色质重组,单细胞多组学,基因沉默,TAD,Xist