Systematic identification of factors bound to isolated metaphase ESC chromosomes reveals a role for chromatin repressors in compaction
Djeghloul D., Kohler A-C., Patel B., Kramer H., Veland N., Whilding C., Dimond A., Elliott J., Feytout A., Bharat TAM., Tarafder AK., Löwe J., Ng BL., Guo Y., Brown K., Guy J., Merkenschlager M., Fisher AG.
<jats:title>Abstract</jats:title><jats:p>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.</jats:p>