Analysis of factors bound to native metaphase ESC chromosomes following purification by flow cytometry

Stem cells are able to self-renew and also generate differentiated progeny. How gene expression patterns are transmitted through mitosis remains unresolved. Conventional studies have relied upon rigorous cell-cycle-stage synchronisation to find factors in mitotic lysates that could ‘bookmark’ the genome. Here we use an alternative approach, purifying native-unfixed mitotic chromosomes from different cell types using flow cytometry and directly identifying chromosome-bound proteins by LC-MS/MS. This revealed a rich profile of pluripotency- and lineage-specific transcription factors that remained bound to mitotic chromosomes in pluripotent ESCs and lymphocytes, as well as factors implicated in chromosome condensation, architecture and gene silencing. Removal of DNA methylation, PRC2 activity, or in situ cleavage of cohesin, each resulted in the reduced compaction of mitotic chromosomes. These data provide a comprehensive catalogue of bookmarking factors in pluripotent versus lineage-restricted cells and demonstrate an expected duality of function by candidates in regulating both gene expression and mitotic chromosome structure.