Distinct and stage-specific contributions of TET1 and TET2 to stepwise cytosine oxidation in the transition from naive to primed pluripotency.
Animals
CRISPR-Cas Systems
Cell Differentiation
Chromatography, High Pressure Liquid
Cytosine
/ metabolism
DNA Methylation
DNA-Binding Proteins
/ genetics
Dioxygenases
Embryonic Stem Cells
/ cytology
Epigenesis, Genetic
Mice
Mice, Knockout
Oxidation-Reduction
Proteome
Proteomics
Proto-Oncogene Proteins
/ genetics
Tandem Mass Spectrometry
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
21 07 2020
21 07 2020
Historique:
received:
27
01
2020
accepted:
29
06
2020
entrez:
23
7
2020
pubmed:
23
7
2020
medline:
22
12
2020
Statut:
epublish
Résumé
Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contributions of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity that may constitute another layer of epigenetic regulation.
Identifiants
pubmed: 32694513
doi: 10.1038/s41598-020-68600-3
pii: 10.1038/s41598-020-68600-3
pmc: PMC7374584
doi:
Substances chimiques
DNA-Binding Proteins
0
Proteome
0
Proto-Oncogene Proteins
0
TET1 protein, mouse
0
Cytosine
8J337D1HZY
Dioxygenases
EC 1.13.11.-
Tet2 protein, mouse
EC 1.13.11.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
12066Références
Smith, Z. D. & Meissner, A. DNA methylation: Roles in mammalian development. Nat. Rev. Genet. 14, 204–220 (2013).
pubmed: 23400093
Seisenberger, S. et al. Reprogramming DNA methylation in the mammalian life cycle: Building and breaking epigenetic barriers. Philos. Trans. R. Soc. Lond. B Biol Sci. 368, 20110330 (2013).
pubmed: 23166394
pmcid: 3539359
Greenberg, M. V. C. & Bourc’his, D. The diverse roles of DNA methylation in mammalian development and disease. Nat. Rev. Mol. Cell Biol. https://doi.org/10.1038/s41580-019-0159-6 (2019).
doi: 10.1038/s41580-019-0159-6
pubmed: 31399642
Lee, H. J., Hore, T. A. & Reik, W. Reprogramming the methylome: erasing memory and creating diversity. Cell Stem Cell 14, 710–719 (2014).
pubmed: 24905162
pmcid: 4051243
Monk, M., Boubelik, M. & Lehnert, S. Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 99, 371–382 (1987).
pubmed: 3653008
Smith, Z. D. et al. A unique regulatory phase of DNA methylation in the early mammalian embryo. Nature 484, 339–344 (2012).
pubmed: 22456710
pmcid: 3331945
Wang, L. et al. Programming and inheritance of parental DNA methylomes in mammals. Cell 157, 979–991 (2014).
pubmed: 24813617
pmcid: 4096154
Borgel, J. et al. Targets and dynamics of promoter DNA methylation during early mouse development. Nat. Genet. 42, 1093–1100 (2010).
pubmed: 21057502
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).
pubmed: 25476147
pmcid: 4295324
Seisenberger, S. et al. The dynamics of genome-wide DNA methylation reprogramming in mouse primordial germ cells. Mol. Cell 48, 849–862 (2012).
pubmed: 23219530
pmcid: 3533687
Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935 (2009).
pubmed: 19372391
pmcid: 2715015
Iyer, L. M., Tahiliani, M., Rao, A. & Aravind, L. Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids. Cell Cycle 8, 1698–1710 (2009).
pubmed: 19411852
pmcid: 2995806
Ito, S. et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303 (2011).
pubmed: 21778364
pmcid: 3495246
He, Y.-F. et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333, 1303–1307 (2011).
pubmed: 21817016
pmcid: 3462231
Pfaffeneder, T. et al. The discovery of 5-formylcytosine in embryonic stem cell DNA. Angew. Chem. Int. Ed Engl. 50, 7008–7012 (2011).
pubmed: 21721093
Maiti, A. & Drohat, A. C. Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: Potential implications for active demethylation of CpG sites. J. Biol. Chem. 286, 35334–35338 (2011).
pubmed: 21862836
pmcid: 3195571
Hashimoto, H. et al. Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation. Nucleic Acids Res. 40, 4841–4849 (2012).
pubmed: 22362737
pmcid: 3367191
Otani, J. et al. Cell cycle-dependent turnover of 5-hydroxymethyl cytosine in mouse embryonic stem cells. PLoS ONE 8, e82961 (2013).
pubmed: 24340069
pmcid: 3858372
Bachman, M. et al. 5-Hydroxymethylcytosine is a predominantly stable DNA modification. Nat. Chem. 6, 1049–1055 (2014).
pubmed: 25411882
pmcid: 4382525
Bachman, M. et al. 5-Formylcytosine can be a stable DNA modification in mammals. Nat. Chem. Biol. 11, 555–557 (2015).
pubmed: 26098680
pmcid: 5486442
Wu, H., Wu, X., Shen, L. & Zhang, Y. Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing. Nat. Biotechnol. 32, 1231–1240 (2014).
pubmed: 25362244
pmcid: 4269366
Spruijt, C. G. et al. Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell 152, 1146–1159 (2013).
pubmed: 23434322
Dawlaty, M. M. et al. Loss of Tet enzymes compromises proper differentiation of embryonic stem cells. Dev. Cell 29, 102–111 (2014).
pubmed: 24735881
pmcid: 4035811
Kang, J. et al. Simultaneous deletion of the methylcytosine oxidases Tet1 and Tet3 increases transcriptome variability in early embryogenesis. Proc. Natl. Acad. Sci. USA 112, E4236–E4245 (2015).
pubmed: 26199412
Dai, H.-Q. et al. TET-mediated DNA demethylation controls gastrulation by regulating Lefty-Nodal signalling. Nature 538, 528 (2016).
pubmed: 27760115
Li, X. et al. Tet proteins influence the balance between neuroectodermal and mesodermal fate choice by inhibiting Wnt signaling. Proc. Natl. Acad. Sci. USA 113, E8267–E8276 (2016).
pubmed: 27930333
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).
pubmed: 21816367
pmcid: 3154739
Khoueiry, R. et al. Lineage-specific functions of TET1 in the postimplantation mouse embryo. Nat. Genet. https://doi.org/10.1038/ng.3868 (2017).
doi: 10.1038/ng.3868
pubmed: 28504700
pmcid: 6033328
Moran-Crusio, K. et al. Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation. Cancer Cell 20, 11–24 (2011).
pubmed: 21723200
pmcid: 3194039
Zhang, Q. et al. Differential regulation of the ten-eleven translocation (TET) family of dioxygenases by O-linked β-N-acetylglucosamine transferase (OGT). J. Biol. Chem. 289, 5986–5996 (2014).
pubmed: 24394411
pmcid: 3937666
Huang, Y. et al. Distinct roles of the methylcytosine oxidases Tet1 and Tet2 in mouse embryonic stem cells. Proc. Natl. Acad. Sci. USA 111, 1361–1366 (2014).
pubmed: 24474761
Xiong, J. et al. Cooperative action between SALL4A and TET proteins in stepwise oxidation of 5-methylcytosine. Mol. Cell 64, 913–925 (2016).
pubmed: 27840027
Shen, L. et al. Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics. Cell 153, 692–706 (2013).
pubmed: 23602152
pmcid: 3687516
Crawford, D. J. et al. Tet2 catalyzes stepwise 5-methylcytosine oxidation by an iterative and de novo mechanism. J. Am. Chem. Soc. 138, 730–733 (2016).
pubmed: 26734843
pmcid: 4762542
Tamanaha, E., Guan, S., Marks, K. & Saleh, L. Distributive processing by the iron(II)/α-ketoglutarate-dependent catalytic domains of the TET enzymes is consistent with epigenetic roles for oxidized 5-methylcytosine bases. J. Am. Chem. Soc. 138, 9345–9348 (2016).
pubmed: 27362828
Xu, L. et al. Pyrene-based quantitative detection of the 5-formylcytosine loci symmetry in the CpG duplex content during TET-dependent demethylation. Angew. Chem. Int. Ed Engl. 53, 11223–11227 (2014).
pubmed: 25159856
pmcid: 4227401
Ying, Q.-L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008).
pubmed: 18497825
pmcid: 5328678
Weinberger, L., Ayyash, M., Novershtern, N. & Hanna, J. H. Dynamic stem cell states: naive to primed pluripotency in rodents and humans. Nat. Rev. Mol. Cell Biol. 17, 155–169 (2016).
pubmed: 26860365
Leitch, H. G. et al. Naive pluripotency is associated with global DNA hypomethylation. Nat. Struct. Mol. Biol. 20, 311–316 (2013).
pubmed: 23416945
pmcid: 3591483
Ficz, G. et al. FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency. Cell Stem Cell 13, 351–359 (2013).
pubmed: 23850245
pmcid: 3765959
Habibi, E. et al. Whole-genome bisulfite sequencing of two distinct interconvertible DNA methylomes of mouse embryonic stem cells. Cell Stem Cell 13, 360–369 (2013).
pubmed: 23850244
Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S. & Saitou, M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146, 519–532 (2011).
pubmed: 21820164
Shirane, K. et al. Global landscape and regulatory principles of DNA methylation reprogramming for germ cell specification by mouse pluripotent stem cells. Dev. Cell 39, 87–103 (2016).
pubmed: 27642137
Okano, M., Bell, D. W., Haber, D. A. & Li, E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247–257 (1999).
pubmed: 10555141
Watanabe, D., Suetake, I., Tada, T. & Tajima, S. Stage- and cell-specific expression of Dnmt3a and Dnmt3b during embryogenesis. Mech. Dev. 118, 187–190 (2002).
pubmed: 12351185
Ito, S. et al. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466, 1129–1133 (2010).
pubmed: 20639862
pmcid: 3491567
Wossidlo, M. et al. 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming. Nat. Commun. 2, 241 (2011).
pubmed: 21407207
Boroviak, T. et al. Lineage-specific profiling delineates the emergence and progression of naive pluripotency in mammalian embryogenesis. Dev. Cell 35, 366–382 (2015).
pubmed: 26555056
pmcid: 4643313
Kohli, R. M. & Zhang, Y. TET enzymes, TDG and the dynamics of DNA demethylation. Nature 502, 472–479 (2013).
pubmed: 24153300
pmcid: 4046508
Wu, X. & Zhang, Y. TET-mediated active DNA demethylation: Mechanism, function and beyond. Nat. Rev. Genet. https://doi.org/10.1038/nrg.2017.33 (2017).
doi: 10.1038/nrg.2017.33
pubmed: 28555658
Pfaffeneder, T. et al. Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA. Nat. Chem. Biol. 10, 574–581 (2014).
pubmed: 24838012
Fidalgo, M. et al. Zfp281 coordinates opposing functions of Tet1 and Tet2 in pluripotent states. Cell Stem Cell 19, 355–369 (2016).
pubmed: 27345836
pmcid: 5010473
Sohni, A. et al. Dynamic switching of active promoter and enhancer domains regulates Tet1 and Tet2 expression during cell state transitions between pluripotency and differentiation. Mol. Cell. Biol. 35, 1026–1042 (2015).
pubmed: 25582196
pmcid: 4333094
Raiber, E.-A. et al. 5-Formylcytosine alters the structure of the DNA double helix. Nat. Struct. Mol. Biol. 22, 44–49 (2015).
pubmed: 25504322
Ji, S., Shao, H., Han, Q., Seiler, C. L. & Tretyakova, N. Y. Reversible DNA-protein cross-linking at epigenetic DNA marks. Angew. Chem. Int. Ed. Engl. 56, 14130–14134 (2017).
pubmed: 28898504
pmcid: 5796521
Li, F. et al. 5-Formylcytosine yields DNA-protein cross-links in nucleosome core particles. J. Am. Chem. Soc. 139, 10617–10620 (2017).
pubmed: 28742335
pmcid: 5649621
Raiber, E. A. et al. 5-Formylcytosine controls nucleosome positioning through covalent histone-DNA interaction. bioRxiv https://doi.org/10.1101/224444 (2017).
doi: 10.1101/224444
Raiber, E.-A. et al. 5-Formylcytosine organizes nucleosomes and forms Schiff base interactions with histones in mouse embryonic stem cells. Nat. Chem. 10, 1258–1266 (2018).
pubmed: 30349137
Hayashi, K. & Saitou, M. Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells. Nat. Protoc. 8, 1513–1524 (2013).
pubmed: 23845963
Mulholland, C. B. et al. A modular open platform for systematic functional studies under physiological conditions. Nucleic Acids Res. 43, e112 (2015).
pubmed: 26007658
pmcid: 4787826
Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).
pubmed: 23643243
pmcid: 3969854
Bauer, C. et al. Phosphorylation of TET proteins is regulated via O-GlcNAcylation by the O-linked N-acetylglucosamine transferase (OGT). J. Biol. Chem. 290, 4801–4812 (2015).
pubmed: 25568311
pmcid: 4335217
Soumillon, M., Cacchiarelli, D., Semrau, S., van Oudenaarden, A. & Mikkelsen, T. S. Characterization of directed differentiation by high-throughput single-cell RNA-Seq. bioRxiv https://doi.org/10.1101/003236 (2014).
doi: 10.1101/003236
Ziegenhain, C. et al. Comparative analysis of single-cell RNA sequencing methods. Mol. Cell 65, 631-643.e4 (2017).
pubmed: 28212749
Bagnoli, J. W. et al. Sensitive and powerful single-cell RNA sequencing using mcSCRB-seq. Nat. Commun. 9, 2937 (2018).
pubmed: 30050112
pmcid: 6062574
Parekh, S., Ziegenhain, C., Vieth, B., Enard, W. & Hellmann, I. zUMIs—A fast and flexible pipeline to process RNA sequencing data with UMIs. Gigascience 7, giy059 (2018).
pmcid: 6007394
Rau, A., Gallopin, M., Celeux, G. & Jaffrézic, F. Data-based filtering for replicated high-throughput transcriptome sequencing experiments. Bioinformatics 29, 2146–2152 (2013).
pubmed: 23821648
pmcid: 3740625
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
pubmed: 4302049
pmcid: 4302049
Wagner, M. et al. Age-dependent levels of 5-methyl-, 5-hydroxymethyl-, and 5-formylcytosine in human and mouse brain tissues. Angew. Chem. Int. Ed. Engl. 54, 12511–12514 (2015).
pubmed: 26137924
pmcid: 4643189
Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).
pubmed: 17703201
Muntel, J. et al. Comparison of protein quantification in a complex background by DIA and TMT workflows with fixed instrument time. J. Proteome Res. 18, 1340–1351 (2019).
pubmed: 30726097