Identifying proteins bound to native mitotic ESC chromosomes reveals chromatin repressors are important for compaction

Dounia Djeghloul,Holger Kramer,Jan Löwe,Tanmay A M Bharat,Anne-Céline Kohler,James I Elliott ,Bhavik Anil Patel ,Robert J Klose,Matthias Merkenschlager,Bee Ling Ng ,Amélie Feytout ,Chad Whilding,Abul K Tarafder,Andrew Dimond,Ya Guo,Nicolás Veland ,Miles K Huseyin,Karen E Brown ,Jacky Guy,Amanda G Fisher

doi:10.1038/s41467-020-17823-z

Abstract

Epigenetic information is transmitted from mother to daughter cells through mitosis. Here, to identify factors that might play a role in conveying epigenetic memory through cell division, we report on the isolation of unfixed, native chromosomes from metaphase-arrested cells using flow cytometry and perform LC-MS/MS to identify chromosome-bound proteins. A quantitative proteomic comparison between metaphase-arrested cell lysates and chromosome-sorted samples reveals 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. Deletion of PRC2, Dnmt1/3a/3b or Mecp2 in ESCs leads to an increase in the size of individual mitotic chromosomes, consistent with de-condensation. Similar results were obtained by the experimental cleavage of cohesin. Thus, we identify chromosome-bound factors in pluripotent stem cells during mitosis and reveal that PRC2, DNA methylation and Mecp2 are required to maintain chromosome compaction.

Highlights

Epigenetic information is transmitted from mother to daughter cells through mitosis
Rapidly dividing cultures of mouse ESCs are arrested in metaphase using demecolcine to achieve samples where most (85–90%) cells are in M-phase as judged by propidium iodide (PI) labelling (Fig. 1a, Supplementary Fig. 1a)
The isolation and purification of metaphase chromosomes by flow cytometry offers a different approach for studying chromosome structure and function in mitosis

Summary

Introduction

Epigenetic information is transmitted from mother to daughter cells through mitosis. Here, to identify factors that might play a role in conveying epigenetic memory through cell division, we report on the isolation of unfixed, native chromosomes from metaphase-arrested cells using flow cytometry and perform LC-MS/MS to identify chromosome-bound proteins. A quantitative proteomic comparison between metaphase-arrested cell lysates and chromosome-sorted samples reveals 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. It was originally thought that most DNA sequencespecific transcription factors were actively displaced from chromosomes during mitosis[11], subsequent studies of the Hsp[70] gene promoter[12], or examining the dynamic distribution of Gata[1], FoxaI and Esrrb proteins in cycling cells have revealed that many factors remain bound to mitotic chromosomes, and may occupy a subset of the genomic sites bound during interphase[13,14,15,16]. Our study describes an alternative approach for studying the properties of native mitotic chromosomes and offers a comprehensive catalogue of chromosome-bound proteins in pluripotent mouse ESCs during mitotic division

Methods

Results

Conclusion