Stem Cell Reviews and Reports

, Volume 10, Issue 5, pp 643–652 | Cite as

Mll2 Controls Cardiac Lineage Differentiation of Mouse Embryonic Stem Cells by Promoting H3K4me3 Deposition at Cardiac-Specific Genes

  • Xiaoling Wan
  • Lulu Liu
  • Xiaodan Ding
  • Peipei Zhou
  • Xiujie Yuan
  • Zhongwen Zhou
  • Ping Hu
  • Hong Zhou
  • Qiang Li
  • Shenghai Zhang
  • Sidong XiongEmail author
  • Yan ZhangEmail author


Trithorax group (TrxG) proteins play critical roles in transcriptional activation by promoting methylation of histone H3 Lysine 4 (H3K4), but the precise functions of the individual TrxG members during embryonic differentiation are not fully understood. Here we show that Mll2, a TrxG member, is required for proliferation but is dispensable for maintaining the pluripotency of mouse embryonic stem cells (ESCs). In addition, differentiation of ESCs toward mesodermal and endodermal lineages is severely altered and, in particular, the cardiac lineage differentiation of ESCs is completely abolished in the absence of Mll2. Moreover, the expression of core cardiac transcription factors and the levels of H3K4 tri-methylation of these cardiac-specific promoters are significantly decreased by the loss of Mll2. Taken together, our results reveal a critical role for Mll2 in proliferation and cardiac lineage differentiation of mouse ESCs, and provide novel molecular insight into the mechanisms of cardiac development and disease.


Histone methylation Cardiac differentiation Embryonic stem cells 



We thank Drs. Bin Zhou and Huangtian Yang for helpful comments and advice. We gratefully acknowledge the assistance of the Flow Cytometry Facility and Transgenic Core Facility, Institut Pasteur of Shanghai, Chinese Academy of Sciences. This work was supported by National Basic Research Program of China Grants 2010CB945600 and 2011CB966300, National Natural Science Foundation of China Grants 30971672, 81090410, and 81270618, and grant from the 100 Talent Program of the Chinese Academy of Sciences (to Y. Z.). National Natural Science Foundation of China grant 31171420 and Science and Technology Commission of Shanghai Municipality grant 12PJ1409700 (to P. H.).

Author contributions

X.W., L.L., S.X., and Y.Z. designed research, X.W., L.L., X.D., P.Z. and X.Y. performed research, H.Z., S.Z. and S.X. contributed new reagents/analytic tools; X.W., L.L., Z.Z., P.H., Q.L. and Y.Z. analyzed data; and X.W. and Y.Z. wrote the paper.

Conflict of interest

The authors declare no potential conflict of interest.

Supplementary material

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Fig. S1

In vivo teratoma formation of Mll2-knockdown cells. (A) Teratomas formed by Mll2-knockdown ESCs were much smaller than those from control cells. Five animals in each group were injected subcutaneously with 2.5×106 of control and either of Mll2-knockdown ESCs (Sh-RNA1 or Sh-RNA2) under both sides of the armpit, respectively. The experiment was stoped after one month. (B) H&E staining of teratomas. Arrowhead indicates muscle cells in the teratoma form by control ESCs (Left), and no muscle cells were found in the teratomas formed by Mll2-knockdown ESCs (Middle and Right). (JPEG 3125 kb)

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Fig. S2

Microarray analysis. (A) Genome-wide analysis of gene expression in Mll2-knockdown and control ESCs. The plot shows normalized (log2) hybridization signals for individual features on the microarrays probed with Mll2-knockdown or control labeled cRNA. (B) Gene ontology analysis for more than 2-fold up- or down-regulated genes in Mll2-knockdown ESCs compared to control ESCs. The most highly represented categories are presented with ontology terms on the y-axis and p-values for the significance of enrichment are shown on the x-axis. Microarray data have been deposited in the Gene Expression Omnibus database (GSE54382). (JPEG 2353 kb)


  1. 1.
    Surani, M. A., Hayashi, K., & Hajkova, P. (2007). Genetic and epigenetic regulators of pluripotency. Cell, 128, 747–762.PubMedCrossRefGoogle Scholar
  2. 2.
    Kooistra, S. M., & Helin, K. (2012). Molecular mechanisms and potential functions of histone demethylases. Nature reviews, 13, 297–311.PubMedCrossRefGoogle Scholar
  3. 3.
    Schuettengruber, B., Chourrout, D., Vervoort, M., Leblanc, B., & Cavalli, G. (2007). Genome regulation by polycomb and trithorax proteins. Cell, 128, 735–745.PubMedCrossRefGoogle Scholar
  4. 4.
    Schuettengruber, B., Martinez, A. M., Iovino, N., & Cavalli, G. (2011). Trithorax group proteins: switching genes on and keeping them active. Nature reviews, 12, 799–814.PubMedCrossRefGoogle Scholar
  5. 5.
    Shilatifard, A. (2012). The COMPASS family of histone H3K4 methylases: mechanisms of regulation in development and disease pathogenesis. Annual Review of Biochemistry, 81, 65–95.PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Yu, B. D., Hess, J. L., Horning, S. E., Brown, G. A., & Korsmeyer, S. J. (1995). Altered Hox expression and segmental identity in Mll-mutant mice. Nature, 378, 505–508.PubMedCrossRefGoogle Scholar
  7. 7.
    Lubitz, S., Glaser, S., Schaft, J., Stewart, A. F., & Anastassiadis, K. (2007). Increased apoptosis and skewed differentiation in mouse embryonic stem cells lacking the histone methyltransferase mll2. Molecular Biology of the Cell, 18, 2356–2366.PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Glaser, S., Schaft, J., Lubitz, S., et al. (2006). Multiple epigenetic maintenance factors implicated by the loss of Mll2 in mouse development. Development (Cambridge, England), 133, 1423–1432.CrossRefGoogle Scholar
  9. 9.
    Lee, J., Saha, P. K., Yang, Q. H., et al. (2008). Targeted inactivation of MLL3 histone H3-Lys-4 methyltransferase activity in the mouse reveals vital roles for MLL3 in adipogenesis. Proceedings of the National Academy of Sciences of the United States of America, 105, 19229–19234.PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Prasad, R., Zhadanov, A. B., Sedkov, Y., et al. (1997). Structure and expression pattern of human ALR, a novel gene with strong homology to ALL-1 involved in acute leukemia and to drosophila trithorax. Oncogene, 15, 549–560.PubMedCrossRefGoogle Scholar
  11. 11.
    Ng, S. B., Bigham, A. W., Buckingham, K. J., et al. (2010). Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome. Nature Genetics, 42, 790–793.PubMedCrossRefPubMedCentralGoogle Scholar
  12. 12.
    Parsons, D. W., Li, M., Zhang, X., et al. (2011). The genetic landscape of the childhood cancer medulloblastoma. Science (New York, NY), 331, 435–439.CrossRefGoogle Scholar
  13. 13.
    Chapman, M. A., Lawrence, M. S., Keats, J. J., et al. (2011). Initial genome sequencing and analysis of multiple myeloma. Nature, 471, 467–472.PubMedCrossRefPubMedCentralGoogle Scholar
  14. 14.
    Grasso, C. S., Wu, Y. M., Robinson, D. R., et al. (2012). The mutational landscape of lethal castration-resistant prostate cancer. Nature, 487, 239–243.PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    Pasqualucci, L., Trifonov, V., Fabbri, G., et al. (2011). Analysis of the coding genome of diffuse large B-cell lymphoma. Nature Genetics, 43, 830–837.PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Zhou, P., Wang, Z., Yuan, X., et al. (2013). Mixed lineage leukemia 5 (MLL5) protein regulates cell cycle progression and E2F1-responsive gene expression via association with host cell factor-1 (HCF-1). The Journal of Biological Chemistry, 288, 17532–17543.PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Issaeva, I., Zonis, Y., Rozovskaia, T., et al. (2007). Knockdown of ALR (MLL2) reveals ALR target genes and leads to alterations in cell adhesion and growth. Molecular and Cellular Biology, 27, 1889–1903.PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Lessard, J. A., & Crabtree, G. R. (2010). Chromatin regulatory mechanisms in pluripotency. Annual Review of Cell and Developmental Biology, 26, 503–532.PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Li, V. C., Ballabeni, A., & Kirschner, M. W. (2012). Gap 1 phase length and mouse embryonic stem cell self-renewal. Proceedings of the National Academy of Sciences of the United States of America, 109, 12550–12555.PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Paulussen, A. D., Stegmann, A. P., Blok, M. J., et al. (2010). MLL2 mutation spectrum in 45 patients with Kabuki syndrome. Human Mutation, 32, E2018–E2025.PubMedCrossRefGoogle Scholar
  21. 21.
    Agger, K., Cloos, P. A., Christensen, J., et al. (2007). UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development. Nature, 449, 731–734.PubMedCrossRefGoogle Scholar
  22. 22.
    Lee, S., Lee, J. W., & Lee, S. K. (2012). UTX, a histone H3-lysine 27 demethylase, acts as a critical switch to activate the cardiac developmental program. Developmental Cell, 22, 25–37.PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Xiaoling Wan
    • 1
    • 2
  • Lulu Liu
    • 1
    • 2
  • Xiaodan Ding
    • 2
    • 3
  • Peipei Zhou
    • 2
  • Xiujie Yuan
    • 2
  • Zhongwen Zhou
    • 4
  • Ping Hu
    • 5
  • Hong Zhou
    • 3
  • Qiang Li
    • 6
  • Shenghai Zhang
    • 7
  • Sidong Xiong
    • 1
    Email author
  • Yan Zhang
    • 2
    Email author
  1. 1.Institute of Biology and Medical SciencesSoochow UniversityNanjingChina
  2. 2.Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of ShanghaiChinese Academy of SciencesShanghaiChina
  3. 3.Department of Microbiology & ImmunologyNanjing Medical UniversityNanjingChina
  4. 4.Department of pathologyHuanshan Hospital of Fudan UniversityShanghaiChina
  5. 5.Institute of Biochemistry and Cell BiologyChinese Academy of SciencesShanghaiChina
  6. 6.Children’s Hospital of Fudan UniversityShanghaiChina
  7. 7.Institute of OphthalmologyEye and ENT Hospital of Fudan UniversityShanghaiChina

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