Retrospective and multifactorial single-cell profiling reveals sequential chromatin reorganization during X inactivation

The regulation of gene expression is governed at multiple levels of chromatin organization. However, how gene regulation is coordinated remains relatively unexplored. Here we develop Dam&ChIC, a method that enables retrospective and multifactorial chromatin profiling in single cells. Dam&ChIC employs in vivo chromatin labeling with m6A to acquire a past chromatin state, coupled with an antibody-mediated readout to capture the present chromatin state. Analyses of diverse chromatin combinations highlight its versatility and superior resolution. By tracking LAD inheritance over the cell cycle, we showcase that Dam&ChIC provides retrospective single-cell chromatin data. Importantly, by applying Dam&ChIC in random X chromosome inactivation, we disentangle the temporal order of chromatin remodeling events. Our data reveals that upon mitotic exit, the inactive X chromosome undergoes extensive genome-lamina detachment, preceding H3K27me3 spreading. We anticipate that Dam&ChIC will be instrumental in unraveling the interconnectivity and order of gene-regulatory events underlying cell-state changes during development. Overall design: Single cell Dam&ChIC was performed for combination of a Dam fusion protein for DamID (Dam-LMNB1) and antibody for ChIC (H2AK119Ub) in haploid Kbm7 cells as well hybrid mESCs undergoing Vitamin C induced differentiation. Also, scDam&T-seq was performed on the same differentiation system with a Dam fusion protein for DamID (Dam-LMNB1).