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. How coordination between these levels is achieved remains relatively unexplored. Here we present Dam&ChIC, a method that experimentally combines scDamID and sortChIC to enable time-resolved and single-cell chromatin profiling at high resolution. Analysis of genome lamina associated domains (LADs) in haploid cells, reveals highly dynamic spatial repositioning of small LADs during interphase and partial inheritance over mitosis. When applied to study random X-inactivation Dam&ChIC reveals that spreading of H3K27me3 on the inactive X chromosome (Xi) overlaps with a remarkable detachment of the same domains from the nuclear lamina. We find that detachment precedes H3K27me3 accumulation on the Xi and occurs after the exit from mitosis. Domains that retain strong genome-lamina interactions are marked by high pre-existing H3K9me3 levels. These findings imply an important role for LADs in regulating H3K27me3 accumulation on the Xi. We anticipate that Dam&ChIC will be instrumental in unravelling interconnectivity and the order of chromatin events that underlie cell-state changes in single cells. Single cell Dam&ChIC was performed for combination of different Dam fusion proteins for DamID (Dam, Dam-LMNB1, Dam-ER, Dam-scFv-K27me3-ER) and antibodies for ChIC (IgG, Lamin B1, H3, H3K4me1, H3K4me3, H3K9me3, H3K27me3, H3K36me3) in haploid Kbm7 cells as well hybrid mESCs undergoing Vitamin C induced differentiation.