Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Dnmt3a is essential for hematopoietic stem cell differentiation

Abstract

Loss of the de novo DNA methyltransferases Dnmt3a and Dnmt3b in embryonic stem cells obstructs differentiation; however, the role of these enzymes in somatic stem cells is largely unknown. Using conditional ablation, we show that Dnmt3a loss progressively impairs hematopoietic stem cell (HSC) differentiation over serial transplantation, while simultaneously expanding HSC numbers in the bone marrow. Dnmt3a-null HSCs show both increased and decreased methylation at distinct loci, including substantial CpG island hypermethylation. Dnmt3a-null HSCs upregulate HSC multipotency genes and downregulate differentiation factors, and their progeny exhibit global hypomethylation and incomplete repression of HSC-specific genes. These data establish Dnmt3a as a critical participant in the epigenetic silencing of HSC regulatory genes, thereby enabling efficient differentiation.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Dnmt3a is highly expressed in HSCs and its ablation has profound functional effects.
Figure 2: Cellular kinetics of Dnmt3a-null HSCs.
Figure 3: Dnmt3a-null HSCs show inhibition of long-term differentiation in serial competitive transplantation of HSCs.
Figure 4: Dnmt3a loss in HSCs results in both hyper- and hypo-methylation.
Figure 5: Dnmt3a loss in HSCs leads to higher expression of HSC multipotency genes.
Figure 6: Dnmt3a is required to suppress the stem cell program in HSCs to permit differentiation.
Figure 7: Exogenous Dnmt3a partially restores function and methylation patterns.
Figure 8: Model for Dnmt3a action in HSCs.

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Attwood, J.T., Yung, R.L. & Richardson, B.C. DNA methylation and the regulation of gene transcription. Cell. Mol. Life Sci. 59, 241–257 (2002).

    Article  CAS  Google Scholar 

  2. Okano, M., Xie, S. & Li, E. Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat. Genet. 19, 219–220 (1998).

    Article  CAS  Google Scholar 

  3. 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).

    Article  CAS  Google Scholar 

  4. Lei, H. et al. De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells. Development 122, 3195–3205 (1996).

    CAS  Google Scholar 

  5. Chen, T., Ueda, Y., Dodge, J.E., Wang, Z. & Li, E. Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b. Mol. Cell. Biol. 23, 5594–5605 (2003).

    Article  CAS  Google Scholar 

  6. Wu, H. et al. Dnmt3a-dependent nonpromoter DNA methylation facilitates transcription of neurogenic genes. Science 329, 444–448 (2010).

    Article  CAS  Google Scholar 

  7. Trowbridge, J.J., Snow, J.W., Kim, J. & Orkin, S.H. DNA methyltransferase 1 is essential for and uniquely regulates hematopoietic stem and progenitor cells. Cell Stem Cell 5, 442–449 (2009).

    Article  CAS  Google Scholar 

  8. Bröske, A.M. et al. DNA methylation protects hematopoietic stem cell multipotency from myeloerythroid restriction. Nat. Genet. 41, 1207–1215 (2009).

    Article  Google Scholar 

  9. Tadokoro, Y., Ema, H., Okano, M., Li, E. & Nakauchi, H. De novo DNA methyltransferase is essential for self-renewal, but not for differentiation, in hematopoietic stem cells. J. Exp. Med. 204, 715–722 (2007).

    Article  CAS  Google Scholar 

  10. Ley, T.J. et al. DNMT3A mutations in acute myeloid leukemia. N. Engl. J. Med. 363, 2424–2433 (2010).

    Article  CAS  Google Scholar 

  11. Yan, X.J. et al. Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic leukemia. Nat. Genet. 43, 309–315 (2011).

    Article  CAS  Google Scholar 

  12. Yamashita, Y. et al. Array-based genomic resequencing of human leukemia. Oncogene 29, 3723–3731 (2010).

    Article  CAS  Google Scholar 

  13. Walter, M.J. et al. Recurrent DNMT3A mutations in patients with myelodysplastic syndromes. Leukemia 25, 1153–1158 (2011).

    Article  CAS  Google Scholar 

  14. Kaneda, M. et al. Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting. Nature 429, 900–903 (2004).

    Article  CAS  Google Scholar 

  15. Baldridge, M.T., King, K.Y., Boles, N.C., Weksberg, D.C. & Goodell, M.A. Quiescent haematopoietic stem cells are activated by IFN-γ in response to chronic infection. Nature 465, 793–797 (2010).

    Article  CAS  Google Scholar 

  16. Gu, H. et al. Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution. Nat. Methods 7, 133–136 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Chambers, S.M. et al. Hematopoietic fingerprints: an expression database of stem cells and their progeny. Cell Stem Cell 1, 578–591 (2007).

    Article  CAS  Google Scholar 

  19. Chen, D. & Zhang, G. Enforced expression of the GATA-3 transcription factor affects cell fate decisions in hematopoiesis. Exp. Hematol. 29, 971–980 (2001).

    Article  CAS  Google Scholar 

  20. Okuda, T., van Deursen, J., Hiebert, S.W., Grosveld, G. & Downing, J.R. AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell 84, 321–330 (1996).

    Article  CAS  Google Scholar 

  21. Ficara, F., Murphy, M.J., Lin, M. & Cleary, M.L. Pbx1 regulates self-renewal of long-term hematopoietic stem cells by maintaining their quiescence. Cell Stem Cell 2, 484–496 (2008).

    Article  CAS  Google Scholar 

  22. Cheng, T. et al. Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science 287, 1804–1808 (2000).

    Article  CAS  Google Scholar 

  23. Mackarehtschian, K. et al. Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors. Immunity 3, 147–161 (1995).

    Article  CAS  Google Scholar 

  24. Georgopoulos, K. et al. The ikaros gene is required for the development of all lymphoid lineages. Cell 79, 143–156 (1994).

    Article  CAS  Google Scholar 

  25. Scott, E.W., Simon, M.C., Anastasi, J. & Singh, H. Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science 265, 1573–1577 (1994).

    Article  CAS  Google Scholar 

  26. Stehling-Sun, S., Dade, J., Nutt, S.L., DeKoter, R.P. & Camargo, F.D. Regulation of lymphoid versus myeloid fate 'choice' by the transcription factor Mef2c. Nat. Immunol. 10, 289–296 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  28. Challen, G.A. & Goodell, M.A. Runx1 isoforms show differential expression patterns during hematopoietic development but have similar functional effects in adult hematopoietic stem cells. Exp. Hematol. 38, 403–416 (2010).

    Article  CAS  Google Scholar 

  29. Sirin, O., Lukov, G.L., Mao, R., Conneely, O.M. & Goodell, M.A. The orphan nuclear receptor Nurr1 restricts the proliferation of haematopoietic stem cells. Nat. Cell Biol. 12, 1213–1219 (2010).

    Article  CAS  Google Scholar 

  30. Jelinek, J. et al. Digital restriction enzyme analysis of methylation (DREAM) by next generation sequencing yields high resolution maps of DNA methylation. Blood 114, 567 (2009).

    Google Scholar 

  31. Venezia, T.A. et al. Molecular signatures of proliferation and quiescence in hematopoietic stem cells. PLoS Biol. 2, e301 (2004).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  33. Fahrner, J.A., Eguchi, S., Herman, J.G. & Baylin, S.B. Dependence of histone modifications and gene expression on DNA hypermethylation in cancer. Cancer Res. 62, 7213–7218 (2002).

    CAS  PubMed  Google Scholar 

  34. Wilson, N.K. et al. Combinatorial transcriptional control in blood stem/progenitor cells: genome-wide analysis of ten major transcriptional regulators. Cell Stem Cell 7, 532–544 (2010).

    Article  CAS  Google Scholar 

  35. Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935 (2009).

    Article  CAS  Google Scholar 

  36. Gonzalo, S. et al. DNA methyltransferases control telomere length and telomere recombination in mammalian cells. Nat. Cell Biol. 8, 416–424 (2006).

    Article  CAS  Google Scholar 

  37. Allsopp, R.C., Morin, G.B., DePinho, R., Harley, C.B. & Weissman, I.L. Telomerase is required to slow telomere shortening and extend replicative lifespan of HSCs during serial transplantation. Blood 102, 517–520 (2003).

    Article  CAS  Google Scholar 

  38. Ehrlich, M. et al. Hypomethylation and hypermethylation of DNA in Wilms tumors. Oncogene 21, 6694–6702 (2002).

    Article  CAS  Google Scholar 

  39. Frigola, J. et al. Differential DNA hypermethylation and hypomethylation signatures in colorectal cancer. Hum. Mol. Genet. 14, 319–326 (2005).

    Article  CAS  Google Scholar 

  40. Cho, Y.H. et al. Aberrant promoter hypermethylation and genomic hypomethylation in tumor, adjacent normal tissues and blood from breast cancer patients. Anticancer Res. 30, 2489–2496 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Goodell, M.A., Brose, K., Paradis, G., Conner, A.S. & Mulligan, R.C. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J. Exp. Med. 183, 1797–1806 (1996).

    Article  CAS  Google Scholar 

  42. Challen, G.A., Boles, N., Lin, K.K. & Goodell, M.A. Mouse hematopoietic stem cell identification and analysis. Cytometry A 75, 14–24 (2009).

    Article  Google Scholar 

  43. Feng, C.G., Weksberg, D.C., Taylor, G.A., Sher, A. & Goodell, M.A. The p47 GTPase Lrg-47 (Irgm1) links host defense and hematopoietic stem cell proliferation. Cell Stem Cell 2, 83–89 (2008).

    Article  CAS  Google Scholar 

  44. Challen, G.A., Boles, N.C., Chambers, S.M. & Goodell, M.A. Distinct hematopoietic stem cell subtypes are differentially regulated by TGF-β1. Cell Stem Cell 6, 265–278 (2010).

    Article  CAS  Google Scholar 

  45. Song, L., James, S.R., Kazim, L. & Karpf, A.R. Specific method for the determination of genomic DNA methylation by liquid chromatography–electrospray ionization tandem mass spectrometry. Anal. Chem. 77, 504–510 (2005).

    Article  CAS  Google Scholar 

  46. Dahl, J.A. & Collas, P. A rapid micro chromatin immunoprecipitation assay (microChIP). Nat. Protoc. 3, 1032–1045 (2008).

    Article  CAS  Google Scholar 

  47. Smith, Z.D., Gu, H., Bock, C., Gnirke, A. & Meissner, A. High-throughput bisulfite sequencing in mammalian genomes. Methods 48, 226–232 (2009).

    Article  CAS  Google Scholar 

  48. Xi, Y. & Li, W. BSMAP: whole genome bisulfite sequence MAPping program. BMC Bioinformatics 10, 232 (2009).

    Article  Google Scholar 

  49. Kroeger, H. et al. Aberrant CpG island methylation in acute myeloid leukemia is accentuated at relapse. Blood 112, 1366–1373 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors would like to thank all members of the Goodell laboratory for scientific advice, A. Rosen, Y. Zheng and L. Yang for mouse management and technical support, C. Threeton for flow cytometry, L. White (microarray core) and A. Gnirke for assistance with the RRBS technique. G.A.C. was supported by a grant from the US National Institutes of Health (NIH K99 DK084259-01A1) and is an American Society of Hematology Scholar. This work was also supported by NIH grants (AG036562, HL086223, CA100632, CA129831, P50CA126752, DK092883, CA125123 and DK58192), the Ellison foundation, the American Heart Association (CPRIT grant RP110028) and the Functional Genomics Core (P30 DK056338).

Author information

Authors and Affiliations

Authors

Contributions

G.A.C. designed and performed experiments, analyzed data and wrote the manuscript. Experiments were also designed by J.S.B., J.-P.J.I., L.A.G., H.G., C.B., W.L. and M.A.G. and were performed by M.J., M.L., A.V. and J.J. Data were additionally analyzed and interpreted by M.J., D.S., M.L., C.B., A.V., J.J., S.L., Y.L., A.M., J.-P.J.I., L.A.G., W.L. and M.A.G. D.S., C.B., Y.X., S.L. and Y.L. developed critical software. The manuscript was written or edited by G.J.D., W.L., L.A.G., J.-P.J.I., J.S.B., C.B. and M.A.G.

Corresponding authors

Correspondence to Wei Li or Margaret A Goodell.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Tables 1–5 and 9. (PDF 4648 kb)

Supplementary Table 6

Annotation of differentially methylated regions (DMRs) (XLSX 498 kb)

Supplementary Table 7

Microarray transcriptional profiling comparison of secondarily-transplanted control and Dnmt3a-KO HSCs (XLS 31014 kb)

Supplementary Table 8

DREAM sequencing of secondary transplant control and Dnmt3a-KO B-cells (XLSX 149 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Challen, G., Sun, D., Jeong, M. et al. Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet 44, 23–31 (2012). https://doi.org/10.1038/ng.1009

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng.1009

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing