Differential nucleosome organization in human interphase and metaphase chromosomes [ChemMap]

Mitosis induces profound changes in chromosome structure, impacting nucleosome organization. Here, we employed chemical mapping to achieve single-base-pair resolution of nucleosome positioning in human interphase and metaphase chromosomes, unveiling distinct organizing principles between the two states. During interphase, we observe greater variability in internucleosome spacing, with a higher abundance of shorter linkers compared to metaphase, reflecting the genome's adaptability in accommodating nucleosome arrays for gene expression. Local analyses further uncover differential nucleosome landscapes at key euchromatin landmarks, including promoters, enhancers, and insulators, in each state. Moreover, we analyzed the relationship between DNA cyclizability and dyad positioning during mitosis. Our results indicate that in metaphase, nucleosomes exhibit significantly higher cyclizability around the dyad, whereas during interphase, nucleosomes more frequently position DNA with higher cyclizability in the nucleosome shoulder near regulatory genomic regions. Together, this study offers novel insights into the intricate interplay between DNA mechanics and nucleosome dynamics during mitosis. Overall design: Through PiggyBac transgenic expression of an shRNA directed against a shared DNA sequence present in all human H4 genes and RNAi-resistant H4S47C cDNA, we generated H4S47C-substituted HeLa S3 stable cell lines suitable for efficient chemical cleavage of nucleosomes. Using these cells, we performed chemical nucleosome mapping experiments in both interphase and synchronized mitotic cells, with two replicates for each condition.