DNA methylation patterns expose variations in enhancer-chromatin modifications during embryonic stem cell differentiation

English version

Autoři: Adi Alajem ^aff001; Hava Roth ^aff001; Sofia Ratgauzer ^aff001; Danny Bavli ^aff001; Alex Motzik ^aff001; Shlomtzion Lahav ^aff001; Itay Peled ^aff001; Oren Ram ^aff001
Působiště autorů: Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel ^aff001
Vyšlo v časopise: DNA methylation patterns expose variations in enhancer-chromatin modifications during embryonic stem cell differentiation. PLoS Genet 17(4): e1009498. doi:10.1371/journal.pgen.1009498
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1009498

Souhrn

In mammals, cellular identity is defined through strict regulation of chromatin modifications and DNA methylation that control gene expression. Methylation of cytosines at CpG sites in the genome is mainly associated with suppression; however, the reason for enhancer-specific methylation is not fully understood. We used sequential ChIP-bisulfite-sequencing for H3K4me1 and H3K27ac histone marks. By collecting data from the same genomic region, we identified enhancers differentially methylated between these two marks. We observed a global gain of CpG methylation primarily in H3K4me1-marked nucleosomes during mouse embryonic stem cell differentiation. This gain occurred largely in enhancer regions that regulate genes critical for differentiation. The higher levels of DNA methylation in H3K4me1- versus H3K27ac-marked enhancers, despite it being the same genomic region, indicates cellular heterogeneity of enhancer states. Analysis of single-cell RNA-seq profiles demonstrated that this heterogeneity correlates with gene expression during differentiation. Furthermore, heterogeneity of enhancer methylation correlates with transcription start site methylation. Our results provide insights into enhancer-based functional variation in complex biological systems.

Klíčová slova:

Cell differentiation – DNA methylation – Gene expression – Gene regulation – Histones – Chromatin – Mammalian genomics – Methylation

Zdroje

1. Creyghton MP, Cheng AW, Welstead GG, Kooistra T, Carey BW, Steine EJ, et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc Natl Acad Sci U S A. 2010;107: 21931–6. doi: 10.1073/pnas.1016071107 21106759

2. Calo E, Wysocka J. Modification of Enhancer Chromatin: What, How, and Why? Mol Cell. 2013;49: 825–837. doi: 10.1016/j.molcel.2013.01.038 23473601

3. Robertson G, Hirst M, Bainbridge M, Bilenky M, Zhao Y, Zeng T, et al. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nat Methods. 2007;4: 651. Available: doi: 10.1038/nmeth1068 17558387

4. Rotem A, Ram O, Shoresh N, Sperling RA, Goren A, Weitz DA, et al. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state. Nat Biotechnol. 2015;33: 1165–1172. doi: 10.1038/nbt.3383 26458175

5. Ai S, Xiong H, Li CC, Luo Y, Shi Q, Liu Y, et al. Profiling chromatin states using single-cell itChIP-seq. Nat Cell Biol. 2019;21: 1164–1172. doi: 10.1038/s41556-019-0383-5 31481796

6. Grosselin K, Durand A, Marsolier J, Poitou A, Marangoni E, Nemati F, et al. High-throughput single-cell ChIP-seq identifies heterogeneity of chromatin states in breast cancer. Nat Genet. 2019;51: 1060–1066. doi: 10.1038/s41588-019-0424-9 31152164

7. Dahl JA, Gilfillan GD. How low can you go? Pushing the limits of low-input ChIP-seq. Brief Funct Genomics. 2018;17: 89–95. doi: 10.1093/bfgp/elx037 29087438

8. Shema E, Jones D, Shoresh N, Donohue L, Ram O, Bernstein BE. Single-molecule decoding of combinatorially modified nucleosomes. Science. 2016;352: 717–21. doi: 10.1126/science.aad7701 27151869

9. Smith ZD, Meissner A. DNA methylation: roles in mammalian development. Nat Rev Genet. 2013;14: 204–220. doi: 10.1038/nrg3354 23400093

10. DeChiara TM, Robertson EJ, Efstratiadis A. Parental imprinting of the mouse insulin-like growth factor II gene. Cell. 1991;64: 849–59. doi: 10.1016/0092-8674(91)90513-x 1997210

11. Riggs AD. X inactivation, differentiation, and DNA methylation. Cytogenet Genome Res. 1975;14: 9–25. doi: 10.1159/000130315 1093816

12. Kuramochi-Miyagawa S, Watanabe T, Gotoh K, Totoki Y, Toyoda A, Ikawa M, et al. DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes. Genes Dev. 2008;22: 908–917. doi: 10.1101/gad.1640708 18381894

13. Gujar H, Weisenberger D, Liang G, Gujar H, Weisenberger DJ, Liang G. The Roles of Human DNA Methyltransferases and Their Isoforms in Shaping the Epigenome. Genes (Basel). 2019;10: 172. doi: 10.3390/genes10020172 30813436

14. Ross SE, Bogdanovic O. TET enzymes, DNA demethylation and pluripotency. Biochem Soc Trans. 2019;47: 875–885. doi: 10.1042/BST20180606 31209155

15. Tsumura A, Hayakawa T, Kumaki Y, Takebayashi S, Sakaue M, Matsuoka C, et al. Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Genes to Cells. 2006;11: 805–814. doi: 10.1111/j.1365-2443.2006.00984.x 16824199

16. Dawlaty MM, Ganz K, Powell BE, Hu Y-C, Markoulaki S, Cheng AW, et al. Tet1 Is Dispensable for Maintaining Pluripotency and Its Loss Is Compatible with Embryonic and Postnatal Development. Cell Stem Cell. 2011;9: 166–175. doi: 10.1016/j.stem.2011.07.010 21816367

17. Dawlaty MM, Breiling A, Le T, Raddatz G, Barrasa MI, Cheng AW, et al. Combined Deficiency of Tet1 and Tet2 Causes Epigenetic Abnormalities but Is Compatible with Postnatal Development. Dev Cell. 2013;24: 310–323. doi: 10.1016/j.devcel.2012.12.015 23352810

18. Okano M, Bell DW, Haber DA, Li E. DNA Methyltransferases Dnmt3a and Dnmt3b Are Essential for De Novo Methylation and Mammalian Development. Cell. 1999;99: 247–257. doi: 10.1016/s0092-8674(00)81656-6 10555141

19. Dawlaty MM, Breiling A, Le T, Barrasa MI, Raddatz G, Gao Q, et al. Loss of Tet enzymes compromises proper differentiation of embryonic stem cells. Dev Cell. 2014;29: 102–11. doi: 10.1016/j.devcel.2014.03.003 24735881

20. Ficz G, Branco MR, Seisenberger S, Santos F, Krueger F, Hore TA, et al. Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature. 2011;473: 398–402. doi: 10.1038/nature10008 21460836

21. Liao J, Karnik R, Gu H, Ziller MJ, Clement K, Tsankov AM, et al. Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells. Nat Genet. 2015;47: 469–478. doi: 10.1038/ng.3258 25822089

22. Bock C, Tomazou EM, Brinkman AB, Müller F, Simmer F, Gu H, et al. Quantitative comparison of genome-wide DNA methylation mapping technologies. Nat Biotechnol. 2010;28: 1106–1114. doi: 10.1038/nbt.1681 20852634

23. Stadler MB, Murr R, Burger L, Ivanek R, Lienert F, Schöler A, et al. DNA-binding factors shape the mouse methylome at distal regulatory regions. 2011 [cited 1 Jul 2019]. doi: 10.1038/nature10716 22170606

24. Guo H, Zhu P, Wu X, Li X, Wen L, Tang F. Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing. Genome Res. 2013;23: 2126–35. doi: 10.1101/gr.161679.113 24179143

25. Hui T, Cao Q, Wegrzyn-Woltosz J, O’Neill K, Hammond CA, Knapp DJHF, et al. High-Resolution Single-Cell DNA Methylation Measurements Reveal Epigenetically Distinct Hematopoietic Stem Cell Subpopulations. Stem Cell Reports. 2018;11: 578–592. doi: 10.1016/j.stemcr.2018.07.003 30078558

26. Smallwood SA, Lee HJ, Angermueller C, Krueger F, Saadeh H, Peat J, et al. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat Methods. 2014;11: 817–820. doi: 10.1038/nmeth.3035 25042786

27. Karemaker ID, Vermeulen M. Single-Cell DNA Methylation Profiling: Technologies and Biological Applications. Trends Biotechnol. 2018;36: 952–965. doi: 10.1016/j.tibtech.2018.04.002 29724495

28. Song Y, van den Berg PR, Markoulaki S, Soldner F, Dall’Agnese A, Henninger JE, et al. Dynamic Enhancer DNA Methylation as Basis for Transcriptional and Cellular Heterogeneity of ESCs. Mol Cell. 2019;75: 905–920.e6. doi: 10.1016/j.molcel.2019.06.045 31422875

29. Kundu S, Ji F, Sunwoo H, Jain G, Lee JT, Sadreyev RI, et al. Polycomb Repressive Complex 1 Generates Discrete Compacted Domains that Change during Differentiation. Mol Cell. 2017;65: 432–446.e5. doi: 10.1016/j.molcel.2017.01.009 28157505

30. Sim Y-J, Kim M-S, Nayfeh A, Yun Y-J, Kim S-J, Park K-T, et al. 2i Maintains a Naive Ground State in ESCs through Two Distinct Epigenetic Mechanisms. Stem Cell Reports. 2017;8: 1312–1328. doi: 10.1016/j.stemcr.2017.04.001 28457889

31. Strübing C, Ahnert-Hilger G, Shan J, Wiedenmann B, Hescheler J, Wobus AM. Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons. Mech Dev. 1995;53: 275–287. doi: 10.1016/0925-4773(95)00446-8 8562428

32. Semrau S, Goldmann JE, Soumillon M, Mikkelsen TS, Jaenisch R, Van Oudenaarden A. Dynamics of lineage commitment revealed by single-cell transcriptomics of differentiating embryonic stem cells. Nat Commun. 2017;8: 1–16. doi: 10.1038/s41467-016-0009-6 28232747

33. Brinkman AB, Gu H, Bartels SJJ, Zhang Y, Matarese F, Simmer F, et al. Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk. Genome Res. 2012;22: 1128–38. doi: 10.1101/gr.133728.111 22466170

34. Initial sequencing and analysis of the human genome. Nature. 2001;409: 860–921. doi: 10.1038/35057062 11237011

35. Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, et al. Simple Combinations of Lineage-Determining Transcription Factors Prime cis-Regulatory Elements Required for Macrophage and B Cell Identities. Mol Cell. 2010;38: 576–589. Available: https://linkinghub.elsevier.com/retrieve/pii/S1097276510003667 doi: 10.1016/j.molcel.2010.05.004 20513432

36. Neri F, Rapelli S, Krepelova A, Incarnato D, Parlato C, Basile G, et al. Intragenic DNA methylation prevents spurious transcription initiation. Nature. 2017;543: 72–77. doi: 10.1038/nature21373 28225755

37. Jjingo D, Conley AB, Yi S V., Lunyak V V., King Jordan I. On the presence and role of human gene-body DNA methylation. Oncotarget. 2012;3: 462–474. doi: 10.18632/oncotarget.497 22577155

38. Shen Y, Yue F, McCleary DF, Ye Z, Edsall L, Kuan S, et al. A map of the cis-regulatory sequences in the mouse genome. Nature. 2012;488: 116–120. doi: 10.1038/nature11243 22763441

39. Valouev A, Johnson DS, Sundquist A, Medina C, Anton E, Batzoglou S, et al. Genome-wide analysis of transcription factor binding sites based on ChIP-Seq data. Nat Methods. 2008;5: 829–834. doi: 10.1038/nmeth.1246 19160518

40. Soudais C, Bielinska M, Heikinheimo M, MacArthur CA, Narita N, Saffitz JE, et al. Targeted mutagenesis of the transcription factor GATA-4 gene in mouse embryonic stem cells disrupts visceral endoderm differentiation in vitro. Development. 1995;121. 8582296

41. Koutsourakis M, Langeveld A, Patient R, Beddington R, Grosveld F. The transcription factor GATA6 is essential for early extraembryonic development. Development. 1999;126: 723–732.

42. Cho Y-S, Kim E-J, Park U-H, Sin H-S, Um S-J. Additional sex comb-like 1 (ASXL1), in cooperation with SRC-1, acts as a ligand-dependent coactivator for retinoic acid receptor. J Biol Chem. 2006;281: 17588–98. doi: 10.1074/jbc.M512616200 16606617

43. Liu C, Peng G, Jing N. TGF-β signaling pathway in early mouse development and embryonic stem cells. Acta Biochim Biophys Sin (Shanghai). 2018;50: 68–73. doi: 10.1093/abbs/gmx120 29190317

44. Ngondo RP, Cohen-Tannoudji M, Ciaudo C. Fast In Vitro Procedure to Identify Extraembryonic Differentiation Defect of Mouse Embryonic Stem Cells. STAR Protoc. 2020;1: 100127. doi: 10.1016/j.xpro.2020.100127 33377021

45. Klein AM, Mazutis L, Akartuna I, Tallapragada N, Veres A, Li V, et al. Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells. Cell. 2015;161: 1187–1201. doi: 10.1016/j.cell.2015.04.044 26000487

46. Satija R, Farrell JA, Gennert D, Schier AF, Regev A. Spatial reconstruction of single-cell gene expression data. Nat Biotechnol. 2015;33: 495–502. doi: 10.1038/nbt.3192 25867923

47. Silva J, Nichols J, Theunissen TW, Guo G, van Oosten AL, Barrandon O, et al. Nanog is the gateway to the pluripotent ground state. Cell. 2009;138: 722–37. doi: 10.1016/j.cell.2009.07.039 19703398

48. Bhattacharya B, Miura T, Brandenberger R, Mejido J, Luo Y, Yang AX, et al. Gene expression in human embryonic stem cell lines: unique molecular signature. Blood. 2004;103: 2956–2964. doi: 10.1182/blood-2003-09-3314 15070671

49. Campbell PA, Rudnicki MA. Oct4 interaction with Hmgb2 regulates Akt signaling and pluripotency. Stem Cells. 2013;31: 1107–20. doi: 10.1002/stem.1365 23495099

50. Lau KX, Mason EA, Kie J, De Souza DP, Kloehn J, Tull D, et al. Unique properties of a subset of human pluripotent stem cells with high capacity for self-renewal. Nat Commun. 2020;11. doi: 10.1038/s41467-020-16214-8 32415101

51. Morrisey EE, Ip HS, Lu MM, Parmacek MS. GATA-6: A Zinc Finger Transcription Factor That Is Expressed in Multiple Cell Lineages Derived from Lateral Mesoderm. Dev Biol. 1996;177: 309–322. doi: 10.1006/dbio.1996.0165 8660897

52. Kanai-Azuma M, Kanai Y, Gad JM, Tajima Y, Taya C, Kurohmaru M, et al. Depletion of definitive gut endoderm in Sox17-null mutant mice. Development. 2002;129. 11973269

53. Gao Y, Jammes H, Rasmussen MA, Oestrup O, Beaujean N, Hall V, et al. Epigenetic regulation of gene expression in porcine epiblast, hypoblast, trophoectoderm and epiblast-derived neural progenitor cells. Epigenetics. 2011;6: 1149–1161. doi: 10.4161/epi.6.9.16954 21975513

54. Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, et al. A Bivalent Chromatin Structure Marks Key Developmental Genes in Embryonic Stem Cells. Cell. 2006;125: 315–326. doi: 10.1016/j.cell.2006.02.041 16630819

55. King AD, Huang K, Rubbi L, Liu S, Wang C-Y, Wang Y, et al. Reversible Regulation of Promoter and Enhancer Histone Landscape by DNA Methylation in Mouse Embryonic Stem Cells. Cell Rep. 2016;17: 289–302. doi: 10.1016/j.celrep.2016.08.083 27681438

56. Schultz MD, He Y, Whitaker JW, Hariharan M, Mukamel EA, Leung D, et al. Human body epigenome maps reveal noncanonical DNA methylation variation. Nature. 2015;523: 212–216. doi: 10.1038/nature14465 26030523

57. Weigel C, Veldwijk MR, Oakes CC, Seibold P, Slynko A, Liesenfeld DB, et al. Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis. Nat Commun. 2016;7: 10893. doi: 10.1038/ncomms10893 26964756

58. Ziller MJ, Gu H, Müller F, Donaghey J, Tsai LT-Y, Kohlbacher O, et al. Charting a dynamic DNA methylation landscape of the human genome. Nature. 2013;500: 477–481. doi: 10.1038/nature12433 23925113

59. Schübeler D. Function and information content of DNA methylation. Nature. 2015;517: 321–326. doi: 10.1038/nature14192 25592537

60. Blattler A, Yao L, Witt H, Guo Y, Nicolet CM, Berman BP, et al. Global loss of DNA methylation uncovers intronic enhancers in genes showing expression changes. Genome Biol. 2014;15: 469. doi: 10.1186/s13059-014-0469-0 25239471

61. Cheow LF, Quake SR, Burkholder WF, Messerschmidt DM. Multiplexed locus-specific analysis of DNA methylation in single cells. Nat Protoc. 2015;10: 619–31. doi: 10.1038/nprot.2015.041 25811896

62. Thurman RE, Rynes E, Humbert R, Vierstra J, Maurano MT, Haugen E, et al. The accessible chromatin landscape of the human genome. Nature. 2012;489: 75–82. doi: 10.1038/nature11232 22955617

63. Meissner A, Mikkelsen TS, Gu H, Wernig M, Hanna J, Sivachenko A, et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature. 2008;454: 766–770. doi: 10.1038/nature07107 18600261

64. Luu P-L, Scholer HR, Arauzo-Bravo MJ. Disclosing the crosstalk among DNA methylation, transcription factors, and histone marks in human pluripotent cells through discovery of DNA methylation motifs. Genome Res. 2013;23: 2013–2029. doi: 10.1101/gr.155960.113 24149073

65. Sharifi-Zarchi A, Gerovska D, Adachi K, Totonchi M, Pezeshk H, Taft RJ, et al. DNA methylation regulates discrimination of enhancers from promoters through a H3K4me1-H3K4me3 seesaw mechanism. BMC Genomics. 2017;18: 964. doi: 10.1186/s12864-017-4353-7 29233090

66. Meissner A, Gnirke A, Bell GW, Ramsahoye B, Lander ES, Jaenisch R. Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis. Nucleic Acids Res. 2005;33: 5868–5877. doi: 10.1093/nar/gki901 16224102

67. Liberzon A, Birger C, Thorvaldsdóttir H, Ghandi M, Mesirov JP, Tamayo P. The Molecular Signatures Database Hallmark Gene Set Collection. Cell Syst. 2015;1: 417–425. doi: 10.1016/j.cels.2015.12.004 26771021

Článek Functional assessment of the “two-hit” model for neurodevelopmental defects in Drosophila and X. laevis

Článek Analysis of cell-type-specific chromatin modifications and gene expression in Drosophila neurons that direct reproductive behavior

Článek Ubiquitin-protein ligase Ubr5 cooperates with hedgehog signalling to promote skeletal tissue homeostasis

Článek Virus-derived variation in diverse human genomes

Článek Aurora kinase A is essential for meiosis in mouse oocytes

Článek Pathways and signatures of mutagenesis at targeted DNA nicks

Článek LuxT controls specific quorum-sensing-regulated behaviors in Vibrionaceae spp. via repression of qrr1, encoding a small regulatory RNA

Článek A complex genetic architecture in zebrafish relatives Danio quagga and D. kyathit underlies development of stripes and spots

Článek Genetic analysis of the septal peptidoglycan synthase FtsWI complex supports a conserved activation mechanism for SEDS-bPBP complexes

Článek Selfish chromosomal drive shapes recent centromeric histone evolution in monkeyflowers

Článek Toxic Y chromosome: Increased repeat expression and age-associated heterochromatin loss in male Drosophila with a young Y chromosome

Článek MRLocus: Identifying causal genes mediating a trait through Bayesian estimation of allelic heterogeneity