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DNA methylation is dispensable for changes in global chromatin architecture but required for chromocentre formation in early stem cell differentiation

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Abstract

Epiblast stem cells (EpiSCs), which are pluripotent cells isolated from early post-implantation mouse embryos (E5.5), show both similarities and differences compared to mouse embryonic stem cells (mESCs), isolated earlier from the inner cell mass (ICM) of the E3.5 embryo. Previously, we have observed that while chromatin is very dispersed in E3.5 ICM, compact chromatin domains and chromocentres appear in E5.5 epiblasts after embryo implantation. Given that the observed chromatin re-organization in E5.5 epiblasts coincides with an increase in DNA methylation, in this study, we aimed to examine the role of DNA methylation in chromatin re-organization during the in vitro conversion of ESCs to EpiSCs. The requirement for DNA methylation was determined by converting both wild-type and DNA methylation-deficient ESCs to EpiSCs, followed by structural analysis with electron spectroscopic imaging (ESI). We show that the chromatin re-organization which occurs in vivo can be re-capitulated in vitro during the ESC to EpiSC conversion. Indeed, after 7 days in EpiSC media, compact chromatin domains begin to appear throughout the nuclear volume, creating a chromatin organization similar to E5 epiblasts and embryo-derived EpiSCs. Our data demonstrate that DNA methylation is dispensable for this global chromatin re-organization but required for the compaction of pericentromeric chromatin into chromocentres.

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Acknowledgements

We thank Ren Li for assistance in preparing the sections for correlative fluorescence and electron spectroscopic imaging. The research was supported by the Canadian Institutes of Health Research. DPB-J holds the Canada Research Chair in Molecular and Cellular Imaging.

Author contribution

VH-Z helped design, carried out the experiments, helped to analyse the data, and wrote the paper. PR-G developed the method to convert ESCs to EpiSCs, provided data on converted EpiSCs, and helped to write the paper. DPB-J helped design the experiments, helped to analyse the data, and helped to write the paper.

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Authors and Affiliations

  1. Department of Cell and Molecular Biology, Faculty of Biology, College of Science, University of Tehran, Tehran, Iran

    Vahideh Hassan-Zadeh

  2. Epigenetics Programme, The Babraham Institute, Babraham Research Campus, Cambridge, UK

    Peter Rugg-Gunn

  3. Programme in Genetics and Genome Biology, The Hospital for Sick Children, 686 Bay Street, Toronto, ON, M5G 0A4, Canada

    David P. Bazett-Jones

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  1. Vahideh Hassan-Zadeh
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Correspondence to David P. Bazett-Jones.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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The authors declare that they have no conflict of interest.

Funding

The funding received was the Operating Grant number 229881 from the Canadian Institutes of Health Research.

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The datasets supporting the conclusions of this article are included within the article.

Electronic supplementary material

Supplementary Table S1

Primer sequences used for gene expression analysis (DOCX 15 kb)

Fig. S1

The average cluster sizes in WT and DNA methylation-deficient ESC and ESC-derived EpiSCs. The average cluster size in WT, DKO and TKO ESC-derived EpiSCs are 8,702, 9,421 and 10,009 nm2 respectively. These values are five to six times that observed in WT and DNA methylation-deficient ESCs. Using a t-test, the statistical significance of the greater than 5-fold increase in the cluster size distribution of the EpiSCs compared to the corresponding ESC lines is represented by a p-value of 0.0001 (DOCX 46 kb)

Fig. S2

The number of characteristic chromocentres is significantly reduced in TKO ESC-derived EpiSCs. The number of H3K9me3-enriched foci with characteristic chromocentre morphology (red bars) and dispersed chromatin (blue bars) in WT and TKO ESC-derived EpiSCs is displayed. The chi-square test shows that the difference in chromocentre morphology between WT and TKO ESC-derived EpiSCs is highly significant (χ2 =30.59, df=1, p-value<0.005) (DOCX 17 kb)

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Hassan-Zadeh, V., Rugg-Gunn, P. & Bazett-Jones, D.P. DNA methylation is dispensable for changes in global chromatin architecture but required for chromocentre formation in early stem cell differentiation. Chromosoma 126, 605–614 (2017). https://doi.org/10.1007/s00412-017-0625-x

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