Replication dynamics identifies the folding principles of the inactive X chromosome [4C]

Chromosome-wide late replication is an enigmatic hallmark of the inactive X chromosome (Xi). How it is established and what it represents remains obscure. By single-cell DNA replication sequencing, here we show that the entire Xi is reorganized to replicate rapidly and uniformly in late S-phase upon X-chromosome inactivation (XCI), reflecting its relatively uniform structure revealed by 4C-seq. Despite this uniformity, only a subset of the Xi became earlier replicating in SmcHD1-mutant cells. In the mutant, these domains protruded out of the Xi core, contacted each other, and became transcriptionally reactivated. 4C-seq suggested that they constituted the outermost layer of the Xi even before XCI and were rich in escape genes. We propose that this default positioning forms the basis for their inherent heterochromatin instability in cells lacking the Xi-binding protein SmcHD1 or exhibiting XCI escape. These observations underscore the importance of 3D genome organization on heterochromatin stability and gene regulation. Overall design: BrdUIP Repli-seq, RNA-seq, and 4C-seq experiments were performed in wild-type and SmcHD1 mutant female JB4/EI7HZ2 mouse embryonic stem cells, day-9 differentiated cells, and neural stem cells (NSCs). Single-cell Repli-seq (scRepli-seq) was performed in wild-type and SmcHD1 mutant NSCs. We only deposited single-cell data sets which passed our filtering steps.