Systematic identification of factors bound to isolated metaphase ESC chromosomes reveals a role for chromatin repressors in compaction
Epigenetic information is transmitted from mother to daughter cells through mitosis. To identify trans-acting factors and cis-acting elements that might be important for conveying epigenetic memory through cell division, we isolated native (unfixed) chromosomes from metaphase-arrested cells using flow cytometry and performed LC-MS/MS to determine the repertoire of chromosome-bound proteins. Quantitative proteomic comparisons between metaphase-arrested cell lysates and chromosome-sorted samples revealed a cohort of proteins that were significantly enriched on mitotic ESC chromosomes. These include pluripotency-associated transcription factors, repressive chromatin-modifiers (such as PRC2 and DNA methyl-transferases) and proteins governing chromosome architecture. We showed that deletion of PRC2, DNMT1/3a/3b or Mecp2 provoked an increase in the size of individual mitotic chromosomes consistent with de-condensation, as did experimental cleavage of cohesin complexes. These data provide a comprehensive inventory of chromosome-bound factors in pluripotent stem cells at mitosis and reveal an unexpected role for chromatin repressor complexes in preserving mitotic chromosome compaction.