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Identification of genetic elements that autonomously determine DNA methylation states

Abstract

Cytosine methylation is a repressive, epigenetically propagated DNA modification. Although patterns of DNA methylation seem tightly regulated in mammals, it is unclear how these are specified and to what extent this process entails genetic or epigenetic regulation. To dissect the role of the underlying DNA sequence, we sequentially inserted over 50 different DNA elements into the same genomic locus in mouse stem cells. Promoter sequences of approximately 1,000 bp autonomously recapitulated correct DNA methylation in pluripotent cells. Moreover, they supported proper de novo methylation during differentiation. Truncation analysis revealed that this regulatory potential is contained within small methylation-determining regions (MDRs). MDRs can mediate both hypomethylation and de novo methylation in cis, and their activity depends on developmental state, motifs for DNA-binding factors and a critical CpG density. These results demonstrate that proximal sequence elements are both necessary and sufficient for regulating DNA methylation and reveal basic constraints of this regulation.

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Figure 1: Ectopic Nanog promoter recapitulates the methylation state of the endogenous promoter.
Figure 2: One-kb elements autonomously set DNA methylation state.
Figure 3: Truncation experiments identify methylation-determining regions.
Figure 4: MDR function depends on CpG density and DNA-binding motifs.
Figure 5: MDRs control de novo methylation and function in cis on heterologous DNA.

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References

  1. Law, J.A. & Jacobsen, S.E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11, 204–220 (2010).

    Article  CAS  Google Scholar 

  2. Li, E., Bestor, T.H. & Jaenisch, R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915–926 (1992).

    Article  CAS  Google Scholar 

  3. Bird, A. DNA methylation patterns and epigenetic memory. Genes Dev. 16, 6–21 (2002).

    Article  CAS  Google Scholar 

  4. Lister, R. et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462, 315–322 (2009).

    Article  CAS  Google Scholar 

  5. Weber, M. et al. Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat. Genet. 39, 457–466 (2007).

    Article  CAS  Google Scholar 

  6. Brandeis, M. et al. Sp1 elements protect a CpG island from de novo methylation. Nature 371, 435–438 (1994).

    Article  CAS  Google Scholar 

  7. Macleod, D., Charlton, J., Mullins, J. & Bird, A.P. Sp1 sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island. Genes Dev. 8, 2282–2292 (1994).

    Article  CAS  Google Scholar 

  8. Dickson, J. et al. VEZF1 elements mediate protection from DNA methylation. PLoS Genet. 6, e1000804 (2010).

    Article  Google Scholar 

  9. Meissner, A. et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454, 766–770 (2008).

    Article  CAS  Google Scholar 

  10. Hawkins, R.D. et al. Distinct epigenomic landscapes of pluripotent and lineage-committed human cells. Cell Stem Cell 6, 479–491 (2010).

    Article  CAS  Google Scholar 

  11. Tamaru, H. & Selker, E.U. A histone H3 methyltransferase controls DNA methylation in Neurospora crassa. Nature 414, 277–283 (2001).

    Article  CAS  Google Scholar 

  12. Jackson, J.P., Lindroth, A.M., Cao, X. & Jacobsen, S.E. Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 416, 556–560 (2002).

    Article  CAS  Google Scholar 

  13. Cedar, H. & Bergman, Y. Linking DNA methylation and histone modification: patterns and paradigms. Nat. Rev. Genet. 10, 295–304 (2009).

    Article  CAS  Google Scholar 

  14. Farthing, C.R. et al. Global mapping of DNA methylation in mouse promoters reveals epigenetic reprogramming of pluripotency genes. PLoS Genet. 4, e1000116 (2008).

    Article  Google Scholar 

  15. Mohn, F. et al. Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol. Cell 30, 755–766 (2008).

    Article  CAS  Google Scholar 

  16. Brenner, C. et al. Myc represses transcription through recruitment of DNA methyltransferase corepressor. EMBO J. 24, 336–346 (2005).

    Article  CAS  Google Scholar 

  17. Suzuki, M. et al. Site-specific DNA methylation by a complex of PU.1 and Dnmt3a/b. Oncogene 25, 2477–2488 (2006).

    Article  CAS  Google Scholar 

  18. Sato, N., Kondo, M. & Arai, K. The orphan nuclear receptor GCNF recruits DNA methyltransferase for Oct-3/4 silencing. Biochem. Biophys. Res. Commun. 344, 845–851 (2006).

    Article  CAS  Google Scholar 

  19. Velasco, G. et al. Dnmt3b recruitment through E2F6 transcriptional repressor mediates germ-line gene silencing in murine somatic tissues. Proc. Natl. Acad. Sci. USA 107, 9281–9286 (2010).

    Article  Google Scholar 

  20. Zhao, Q. et al. PRMT5-mediated methylation of histone H4R3 recruits DNMT3A, coupling histone and DNA methylation in gene silencing. Nat. Struct. Mol. Biol. 16, 304–311 (2009).

    Article  CAS  Google Scholar 

  21. Viré, E. et al. The Polycomb group protein EZH2 directly controls DNA methylation. Nature 439, 871–874 (2006).

    Article  Google Scholar 

  22. Robertson, K.D. DNA methylation and human disease. Nat. Rev. Genet. 6, 597–610 (2005).

    Article  CAS  Google Scholar 

  23. Dulac, C. Brain function and chromatin plasticity. Nature 465, 728–735 (2010).

    Article  CAS  Google Scholar 

  24. Borgel, J. et al. Targets and dynamics of promoter DNA methylation during early mouse development. Nat. Genet. 42, 1-93–1100 (2010).

    Article  Google Scholar 

  25. Bibel, M. et al. Differentiation of mouse embryonic stem cells into a defined neuronal lineage. Nat. Neurosci. 7, 1003–1009 (2004).

    Article  CAS  Google Scholar 

  26. Fromm, G. & Bulger, M. A spectrum of gene regulatory phenomena at mammalian beta-globin gene loci. Biochem. Cell Biol. 87, 781–790 (2009).

    Article  CAS  Google Scholar 

  27. Lienert, F. et al. Genomic prevalence of heterochromatic H3K9me2 and transcription do not discriminate pluripotent from terminally differentiated cells. PLoS Genet. 7, e1002090 (2011).

    Article  CAS  Google Scholar 

  28. Feng, Y.Q. et al. Site-specific chromosomal integration in mammalian cells: highly efficient CRE recombinase-mediated cassette exchange. J. Mol. Biol. 292, 779–785 (1999).

    Article  CAS  Google Scholar 

  29. Schübeler, D. et al. Genomic targeting of methylated DNA: influence of methylation on transcription, replication, chromatin structure, and histone acetylation. Mol. Cell. Biol. 20, 9103–9112 (2000).

    Article  Google Scholar 

  30. Lorincz, M.C., Schubeler, D., Hutchinson, S.R., Dickerson, D.R. & Groudine, M. DNA methylation density influences the stability of an epigenetic imprint and Dnmt3a/b-independent de novo methylation. Mol. Cell. Biol. 22, 7572–7580 (2002).

    Article  CAS  Google Scholar 

  31. Chambers, I. et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643–655 (2003).

    Article  CAS  Google Scholar 

  32. Mitsui, K. et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113, 631–642 (2003).

    Article  CAS  Google Scholar 

  33. Deb-Rinker, P., Ly, D., Jezierski, A., Sikorska, M. & Walker, P.R. Sequential DNA methylation of the Nanog and Oct-4 upstream regions in human NT2 cells during neuronal differentiation. J. Biol. Chem. 280, 6257–6260 (2005).

    Article  CAS  Google Scholar 

  34. Levasseur, D.N., Wang, J., Dorschner, M.O., Stamatoyannopoulos, J.A. & Orkin, S.H. Oct4 dependence of chromatin structure within the extended Nanog locus in ES cells. Genes Dev. 22, 575–580 (2008).

    Article  CAS  Google Scholar 

  35. Illingworth, R. et al. A novel CpG island set identifies tissue-specific methylation at developmental gene loci. PLoS Biol. 6, e22 (2008).

    Article  Google Scholar 

  36. Kim, M. et al. Regulatory factor interactions and somatic silencing of the germ cell–specific ALF gene. J. Biol. Chem. 281, 34288–34298 (2006).

    Article  CAS  Google Scholar 

  37. Pant, V. et al. The nucleotides responsible for the direct physical contact between the chromatin insulator protein CTCF and the H19 imprinting control region manifest parent of origin-specific long-distance insulation and methylation-free domains. Genes Dev. 17, 586–590 (2003).

    Article  CAS  Google Scholar 

  38. Horvath, G.C., Kistler, M.K. & Kistler, W.S. RFX2 is a candidate downstream amplifier of A-MYB regulation in mouse spermatogenesis. BMC Dev. Biol. 9, 63 (2009).

    Article  Google Scholar 

  39. Sharif, J. et al. The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA. Nature 450, 908–912 (2007).

    Article  CAS  Google Scholar 

  40. Rauch, T.A., Wu, X., Zhong, X., Riggs, A.D. & Pfeifer, G.P. A human B cell methylome at 100–base pair resolution. Proc. Natl. Acad. Sci. USA 106, 671–678 (2009).

    Article  CAS  Google Scholar 

  41. Deaton, A.M. & Bird, A. CpG islands and the regulation of transcription. Genes Dev. 25, 1010–1022 (2011).

    Article  CAS  Google Scholar 

  42. Thomson, J.P. et al. CpG islands influence chromatin structure via the CpG-binding protein Cfp1. Nature 464, 1082–1086 (2010).

    Article  CAS  Google Scholar 

  43. Cohen, N.M., Kenigsberg, E. & Tanay, A. Primate CpG islands are maintained by heterogeneous evolutionary regimes involving minimal selection. Cell 145, 773–786 (2011).

    Article  CAS  Google Scholar 

  44. Schilling, E. & Rehli, M. Global, comparative analysis of tissue-specific promoter CpG methylation. Genomics 90, 314–323 (2007).

    Article  CAS  Google Scholar 

  45. Doi, A. et al. Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts. Nat. Genet. 41, 1350–1353 (2009).

    Article  CAS  Google Scholar 

  46. Irizarry, R.A. et al. The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores. Nat. Genet. 41, 178–186 (2009).

    Article  CAS  Google Scholar 

  47. Ji, H. et al. Comprehensive methylome map of lineage commitment from haematopoietic progenitors. Nature 467, 338–342 (2010).

    Article  CAS  Google Scholar 

  48. Gebhard, C. et al. General transcription factor binding at CpG islands in normal cells correlates with resistance to de novo DNA methylation in cancer cells. Cancer Res. 70, 1398–1407 (2010).

    Article  CAS  Google Scholar 

  49. Pachkov, M., Erb, I., Molina, N. & van Nimwegen, E. SwissRegulon: a database of genome-wide annotations of regulatory sites. Nucleic Acids Res. 35, D127–D131 (2007).

    Article  CAS  Google Scholar 

  50. Bibel, M., Richter, J., Lacroix, E. & Barde, Y.A. Generation of a defined and uniform population of CNS progenitors and neurons from mouse embryonic stem cells. Nat. Protoc. 2, 1034–1043 (2007).

    Article  CAS  Google Scholar 

  51. Bock, C. et al. BiQ Analyzer: visualization and quality control for DNA methylation data from bisulfite sequencing. Bioinformatics 21, 4067–4068 (2005).

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to M. Pietrzak for sequencing. We thank M. Lorincz of the University of British Columbia–Vancouver for providing plasmids for RMCE and S. Fiering for advice. We would also like to thank members of the Schübeler group and S. Gasser for critical comments on the manuscript. F.L. is supported by a PhD fellowship of the Boehringer Ingelheim Fonds. Research in the laboratory of A.D. is supported by the Intramural Program of National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health. Research in the laboratory of D.S. is supported by the Novartis Research Foundation, by the European Union (NoE “EpiGeneSys” FP7-HEALTH-2010-257082, LSHG-CT-2006-037415), the European Research Council (ERC-204264) and by the RTD “Cellplasticity” of the Swiss initiative in Systems Biology (SystemsX.ch).

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F.L. and C.W. performed experiments. I.S. and A.D. generated the target ES cell line. F.L., F.M. and D.S. designed the study, analyzed data and wrote the manuscript.

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Correspondence to Dirk Schübeler.

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The authors declare no competing financial interests.

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Lienert, F., Wirbelauer, C., Som, I. et al. Identification of genetic elements that autonomously determine DNA methylation states. Nat Genet 43, 1091–1097 (2011). https://doi.org/10.1038/ng.946

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