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Active DNA demethylation at enhancers during the vertebrate phylotypic period

Nature Genetics volume 48, pages 417–426 (2016)Cite this article

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Abstract

The vertebrate body plan and organs are shaped during a conserved embryonic phase called the phylotypic stage. However, the mechanisms that guide the epigenome through this transition and their evolutionary conservation remain elusive. Here we report widespread DNA demethylation of enhancers during the phylotypic period in zebrafish, Xenopus tropicalis and mouse. These enhancers are linked to developmental genes that display coordinated transcriptional and epigenomic changes in the diverse vertebrates during embryogenesis. Binding of Tet proteins to (hydroxy)methylated DNA and enrichment of 5-hydroxymethylcytosine in these regions implicated active DNA demethylation in this process. Furthermore, loss of function of Tet1, Tet2 and Tet3 in zebrafish reduced chromatin accessibility and increased methylation levels specifically at these enhancers, indicative of DNA methylation being an upstream regulator of phylotypic enhancer function. Overall, our study highlights a regulatory module associated with the most conserved phase of vertebrate embryogenesis and suggests an ancient developmental role for Tet dioxygenases.

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Figure 1: DNA methylome dynamics during vertebrate embryogenesis and the phylotypic stage.
Figure 2: Phylo(−)DMRs are developmentally activated enhancers associated with vertebrate body plan formation.
Figure 3: Active demethylation components bind 5mC and 5hmC during the phylotypic period in vertebrates.
Figure 4: Phylo(−)DMRs are characterized by 5hmC enrichment in vertebrate embryos.
Figure 5: Tet proteins are required for phylo(−)DMR demethylation and body plan formation in zebrafish.

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Acknowledgements

The authors thank D. Secco and A. De Mendoza for critical reading of the manuscript. Spanish and Andalusian government grants BFU2013-41322-P and BIO-396 to J.L.G.-S. supported this work. R.L. was supported by an Australian Research Council Future Fellowship (FT120100862) and a Sylvia and Charles Viertel Senior Medical Research Fellowship, and work in the laboratory of R.L. was funded by the Australian Research Council, National Health and Medical Research Council, and the Raine Medical Research Foundation. O.B. is supported by an Australian Research Council Discovery Early Career Researcher Award (DECRA; DE140101962). The laboratory of M.V. is supported by grants from the Netherlands Organisation for Scientific Research (NWO-VIDI; 864.09.003) and Cancer Genomics Netherlands, a European Research Council starting grant (309384) and the European Union Framework Programme 7 Network of Excellence EpiGeneSys. J.R.E. was supported by the Gordon and Betty Moore Foundation (GBMF3034) and is an Investigator of the Howard Hughes Medical Institute. Work in the laboratory of M.M. is funded by grants from the Ministerio de Economia y Competitividad (BFU2011-23083), Comunidad Autónoma de Madrid (CELLDD-CM), and by the Pro-CNIC Foundation. This work has been supported by a grant from the US National Institutes of Health (National Institute of Child Health and Human Development, grant R01HD069344) to G.J.C.V.

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Authors and Affiliations

  1. Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Perth, Western Australia, Australia

    Ozren Bogdanović, Ethan Ford & Ryan Lister

  2. Centro Andaluz de Biología del Desarrollo, Consejo Superior de Investigaciones Científicas, Universidad Pablo de Olavide, Seville, Spain

    Ozren Bogdanović, Elisa de la Calle Mustienes, Juan J Tena & José Luis Gómez-Skarmeta

  3. Harry Perkins Institute of Medical Research, Perth, Western Australia, Australia

    Ozren Bogdanović, Ethan Ford & Ryan Lister

  4. Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University, Nijmegen, the Netherlands

    Arne H Smits & Michiel Vermeulen

  5. Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    Ruth Williams, Upeka Senanayake & Tatjana Sauka-Spengler

  6. Genomic Analysis Laboratory, Salk Institute for Biological Studies, La Jolla, California, USA

    Matthew D Schultz & Joseph R Ecker

  7. Department of Molecular Developmental Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University, Nijmegen, the Netherlands

    Saartje Hontelez, Ila van Kruijsbergen & Gert Jan C Veenstra

  8. Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain

    Teresa Rayon & Miguel Manzanares

  9. Centre for Integrated Protein Science, Ludwig Maximilians Universität München, Munich, Germany

    Felix Gnerlich & Thomas Carell

  10. Howard Hughes Medical Institute, Salk Institute for Biological Studies, La Jolla, California, USA

    Joseph R Ecker

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Contributions

O.B., M.V., J.L.G.-S. and R.L. designed the study. O.B. and E.F. prepared and sequenced MethylC-seq libraries. The data were analyzed by O.B. with the help of R.L., E.F., M.D.S. and J.R.E. Embryo work was performed by O.B., E.d.l.C.M., J.J.T., T.R., M.M. and J.L.G.-S. The zebrafish sox10 line was prepared by R.W., U.S. and T.S.-S. Xenopus ChIP-seq data were generated by S.H., I.v.K. and G.J.C.V. Quantitative interaction proteomics experiments were performed by A.H.S., F.G., T.C. and M.V. Proteomics data were analyzed by A.H.S. and M.V. The manuscript was written by O.B., A.H.S., M.V., J.L.G.-S. and R.L.

Corresponding authors

Correspondence to Ozren Bogdanović, Michiel Vermeulen, José Luis Gómez-Skarmeta or Ryan Lister.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–18. (PDF 2738 kb)

Supplementary Table 1

Overview of MethylC-seq, TAB-seq and ATAC-seq data used in this study. (XLSX 11 kb)

Supplementary Tables 2–5

Genomic positions and directionality of DMRs. (XLSX 17421 kb)

Supplementary Tables 6–20

Genomic positions of DMRs (FDR = 0.05 and minimum ΔmCG = 0.2). (XLSX 11756 kb)

Supplementary Tables 21 and 22

Conserved gene ontology enrichments. (XLSX 23 kb)

Supplementary Table 23

Genomic positions of VISTA enhancers overlapping phylo(–)DMRs identified in mouse and zebrafish. (XLSX 9 kb)

Supplementary Table 24

Orthologous genes associated with phylo(–)DMRs in zebrafish, Xenopus and mouse. (XLSX 11 kb)

Supplementary Tables 25 and 26

Label-free quantification (LFQ) intensities for zebrafish dome and 24 h.p.f. samples. (XLSX 380 kb)

Supplementary Table 27

Differentially expressed genes in the tet1-tet2-tet3 morphant. (XLSX 173 kb)

Supplementary Table 28

Morpholino and DNA methylation pulldown bait sequences used in this study. (XLSX 9 kb)

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Bogdanović, O., Smits, A., de la Calle Mustienes, E. et al. Active DNA demethylation at enhancers during the vertebrate phylotypic period. Nat Genet 48, 417–426 (2016). https://doi.org/10.1038/ng.3522

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