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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Bird, A. DNA methylation patterns and epigenetic memory. Genes Dev. 16, 6–21 (2002).
Santos, F., Hendrich, B., Reik, W. & Dean, W. Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev. Biol. 241, 172–182 (2002).
Borgel, J. et al. Targets and dynamics of promoter DNA methylation during early mouse development. Nat. Genet. 42, 1093–1100 (2010).
Andersen, I.S., Reiner, A.H., Aanes, H., Aleström, P. & Collas, P. Developmental features of DNA methylation during activation of the embryonic zebrafish genome. Genome Biol. 13, R65 (2012).
Smith, Z.D. et al. A unique regulatory phase of DNA methylation in the early mammalian embryo. Nature 484, 339–344 (2012).
Jiang, L. et al. Sperm, but not oocyte, DNA methylome is inherited by zebrafish early embryos. Cell 153, 773–784 (2013).
Potok, M.E., Nix, D.A., Parnell, T.J. & Cairns, B.R. Reprogramming the maternal zebrafish genome after fertilization to match the paternal methylation pattern. Cell 153, 759–772 (2013).
Wang, L. et al. Programming and inheritance of parental DNA methylomes in mammals. Cell 157, 979–991 (2014).
Seisenberger, S. et al. Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers. Phil. Trans. R. Soc. Lond. B 368, 20110330 (2013).
Lee, M.T., Bonneau, A.R. & Giraldez, A.J. Zygotic genome activation during the maternal-to-zygotic transition. Annu. Rev. Cell Dev. Biol. 30, 581–613 (2014).
Domazet-Lošo, T. & Tautz, D. A phylogenetically based transcriptome age index mirrors ontogenetic divergence patterns. Nature 468, 815–818 (2010).
Irie, N. & Kuratani, S. Comparative transcriptome analysis reveals vertebrate phylotypic period during organogenesis. Nat. Commun. 2, 248 (2011).
Kalinka, A.T. et al. Gene expression divergence recapitulates the developmental hourglass model. Nature 468, 811–814 (2010).
Cokus, S.J. et al. Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215–219 (2008).
Lister, R. et al. Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133, 523–536 (2008).
Yu, M. et al. Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell 149, 1368–1380 (2012).
Spruijt, C.G. et al. Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell 152, 1146–1159 (2013).
Oswald, J. et al. Active demethylation of the paternal genome in the mouse zygote. Curr. Biol. 10, 475–478 (2000).
Smith, Z.D. et al. DNA methylation dynamics of the human preimplantation embryo. Nature 511, 611–615 (2014).
Guo, H. et al. The DNA methylation landscape of human early embryos. Nature 511, 606–610 (2014).
Lister, R. et al. Global epigenomic reconfiguration during mammalian brain development. Science 341, 1237905 (2013).
McLean, C.Y. et al. GREAT improves functional interpretation of cis-regulatory regions. Nat. Biotechnol. 28, 495–501 (2010).
Dutton, K.A. et al. Zebrafish colourless encodes sox10 and specifies non-ectomesenchymal neural crest fates. Development 128, 4113–4125 (2001).
Stadler, M.B. et al. DNA-binding factors shape the mouse methylome at distal regulatory regions. Nature 480, 490–495 (2011).
Hon, G.C. et al. Epigenetic memory at embryonic enhancers identified in DNA methylation maps from adult mouse tissues. Nat. Genet. 45, 1198–1206 (2013).
Bogdanovic, O. et al. Dynamics of enhancer chromatin signatures mark the transition from pluripotency to cell specification during embryogenesis. Genome Res. 22, 2043–2053 (2012).
Shen, Y. et al. A map of the cis-regulatory sequences in the mouse genome. Nature 488, 116–120 (2012).
Hontelez, S. et al. Embryonic transcription is controlled by maternally defined chromatin state. Nat. Commun. 6, 10148 (2015).
Creyghton, M.P. et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl. Acad. Sci. USA 107, 21931–21936 (2010).
Rada-Iglesias, A. et al. A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470, 279–283 (2011).
Heintzman, N.D. et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat. Genet. 39, 311–318 (2007).
Illingworth, R.S. et al. Orphan CpG islands identify numerous conserved promoters in the mammalian genome. PLoS Genet. 6, e1001134 (2010).
Long, H.K. et al. Epigenetic conservation at gene regulatory elements revealed by non-methylated DNA profiling in seven vertebrates. eLife 2, e00348 (2013).
Cohen, N.M., Kenigsberg, E. & Tanay, A. Primate CpG islands are maintained by heterogeneous evolutionary regimes involving minimal selection. Cell 145, 773–786 (2011).
Krebs, A.R., Dessus-Babus, S., Burger, L. & Schübeler, D. High-throughput engineering of a mammalian genome reveals building principles of methylation states at CG rich regions. eLife 3, e04094 (2014).
Wachter, E. et al. Synthetic CpG islands reveal DNA sequence determinants of chromatin structure. eLife 3, e03397 (2014).
Visel, A., Minovitsky, S., Dubchak, I. & Pennacchio, L.A. VISTA Enhancer Browser—a database of tissue-specific human enhancers. Nucleic Acids Res. 35, D88–D92 (2007).
Visel, A. et al. ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457, 854–858 (2009).
Huang, W., Sherman, B.T. & Lempicki, R.A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009).
Huang, W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).
Pauli, A. et al. Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis. Genome Res. 22, 577–591 (2012).
Paranjpe, S.S., Jacobi, U.G., van Heeringen, S.J. & Veenstra, G.J. A genome-wide survey of maternal and embryonic transcripts during Xenopus tropicalis development. BMC Genomics 14, 762 (2013).
Auclair, G., Guibert, S., Bender, A. & Weber, M. Ontogeny of CpG island methylation and specificity of DNMT3 methyltransferases during embryonic development in the mouse. Genome Biol. 15, 545 (2014).
Shen, L. et al. Tet3 and DNA replication mediate demethylation of both the maternal and paternal genomes in mouse zygotes. Cell Stem Cell 15, 459–470 (2014).
Siepel, A. et al. Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 15, 1034–1050 (2005).
Piasecka, B., Lichocki, P., Moretti, S., Bergmann, S. & Robinson-Rechavi, M. The hourglass and the early conservation models—co-existing patterns of developmental constraints in vertebrates. PLoS Genet. 9, e1003476 (2013).
Nord, A.S. et al. Rapid and pervasive changes in genome-wide enhancer usage during mammalian development. Cell 155, 1521–1531 (2013).
Stergachis, A.B. et al. Developmental fate and cellular maturity encoded in human regulatory DNA landscapes. Cell 154, 888–903 (2013).
Barreto, G. et al. Gadd45a promotes epigenetic gene activation by repair-mediated DNA demethylation. Nature 445, 671–675 (2007).
Rai, K. et al. DNA demethylation in zebrafish involves the coupling of a deaminase, a glycosylase, and Gadd45. Cell 135, 1201–1212 (2008).
Almeida, R.D. et al. 5-hydroxymethyl-cytosine enrichment of non-committed cells is not a universal feature of vertebrate development. Epigenetics 7, 383–389 (2012).
Kamstra, J.H., Løken, M., Aleström, P. & Legler, J. Dynamics of DNA hydroxymethylation in zebrafish. Zebrafish 12, 230–237 (2015).
Williams, K. et al. TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity. Nature 473, 343–348 (2011).
Wu, H. et al. Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells. Nature 473, 389–393 (2011).
Xu, Y. et al. Tet3 CXXC domain and dioxygenase activity cooperatively regulate key genes for Xenopus eye and neural development. Cell 151, 1200–1213 (2012).
Ge, L. et al. TET2 plays an essential role in erythropoiesis by regulating lineage-specific genes via DNA oxidative demethylation in a zebrafish model. Mol. Cell. Biol. 34, 989–1002 (2014).
Li, C. et al. Overlapping requirements for Tet2 and Tet3 in normal development and hematopoietic stem cell emergence. Cell Rep. 12, 1133–1143 (2015).
Buenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y. & Greenleaf, W.J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).
Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).
Vastenhouw, N.L. et al. Chromatin signature of embryonic pluripotency is established during genome activation. Nature 464, 922–926 (2010).
Lindeman, L.C. et al. Prepatterning of developmental gene expression by modified histones before zygotic genome activation. Dev. Cell 21, 993–1004 (2011).
Lee, H.J. et al. Developmental enhancers revealed by extensive DNA methylome maps of zebrafish early embryos. Nat. Commun. 6, 6315 (2015).
Perera, A. et al. TET3 is recruited by REST for context-specific hydroxymethylation and induction of gene expression. Cell Rep. 11, 283–294 (2015).
Ding, J. et al. Tex10 coordinates epigenetic control of super-enhancer activity in pluripotency and reprogramming. Cell Stem Cell 16, 653–668 (2015).
Gu, T.P. et al. The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature 477, 606–610 (2011).
Dawlaty, M.M. et al. Tet1 is dispensable for maintaining pluripotency and its loss is compatible with embryonic and postnatal development. Cell Stem Cell 9, 166–175 (2011).
Ko, M. et al. Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice. Proc. Natl. Acad. Sci. USA 108, 14566–14571 (2011).
Moran-Crusio, K. et al. Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation. Cancer Cell 20, 11–24 (2011).
Dawlaty, M.M. et al. Combined deficiency of Tet1 and Tet2 causes epigenetic abnormalities but is compatible with postnatal development. Dev. Cell 24, 310–323 (2013).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Lister, R. et al. Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 471, 68–73 (2011).
Schultz, M.D. et al. Human body epigenome maps reveal noncanonical DNA methylation variation. Nature 523, 212–216 (2015).
Quinlan, A.R. & Hall, I.M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
Bray, N., Pimentel, H., Melsted, P. & Pachter, L. Near-optimal RNA-Seq quantification. arXiv http://arxiv.org/abs/1505.02710 (2015).
Vilella, A.J. et al. EnsemblCompara GeneTrees: complete, duplication-aware phylogenetic trees in vertebrates. Genome Res. 19, 327–335 (2009).
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003).
Schwanhäusser, B. et al. Global quantification of mammalian gene expression control. Nature 473, 337–342 (2011).
Wis´niewski, J.R., Zougman, A. & Mann, M. Combination of FASP and StageTip-based fractionation allows in-depth analysis of the hippocampal membrane proteome. J. Proteome Res. 8, 5674–5678 (2009).
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.
The authors declare no competing financial interests.
Supplementary Figures 1–18. (PDF 2738 kb)
Overview of MethylC-seq, TAB-seq and ATAC-seq data used in this study. (XLSX 11 kb)
Genomic positions and directionality of DMRs. (XLSX 17421 kb)
Genomic positions of DMRs (FDR = 0.05 and minimum ΔmCG = 0.2). (XLSX 11756 kb)
Conserved gene ontology enrichments. (XLSX 23 kb)
Genomic positions of VISTA enhancers overlapping phylo(–)DMRs identified in mouse and zebrafish. (XLSX 9 kb)
Orthologous genes associated with phylo(–)DMRs in zebrafish, Xenopus and mouse. (XLSX 11 kb)
Label-free quantification (LFQ) intensities for zebrafish dome and 24 h.p.f. samples. (XLSX 380 kb)
Differentially expressed genes in the tet1-tet2-tet3 morphant. (XLSX 173 kb)
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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