Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Surani, M. A., Hayashi, K., & Hajkova, P. (2007). Genetic and epigenetic regulators of pluripotency. Cell, 128, 747–762.
Kooistra, S. M., & Helin, K. (2012). Molecular mechanisms and potential functions of histone demethylases. Nature reviews, 13, 297–311.
Schuettengruber, B., Chourrout, D., Vervoort, M., Leblanc, B., & Cavalli, G. (2007). Genome regulation by polycomb and trithorax proteins. Cell, 128, 735–745.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Chapman, M. A., Lawrence, M. S., Keats, J. J., et al. (2011). Initial genome sequencing and analysis of multiple myeloma. Nature, 471, 467–472.
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.
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.
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.
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.
Lessard, J. A., & Crabtree, G. R. (2010). Chromatin regulatory mechanisms in pluripotency. Annual Review of Cell and Developmental Biology, 26, 503–532.
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.
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.
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.
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.
Acknowledgments
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.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Xiaoling Wan and Lulu Liu contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(DOCX 14 kb)
ESM 2
(DOC 60 kb)
ESM 3
(XLS 1467 kb)
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)
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)
Rights and permissions
About this article
Cite this article
Wan, X., Liu, L., Ding, X. et al. Mll2 Controls Cardiac Lineage Differentiation of Mouse Embryonic Stem Cells by Promoting H3K4me3 Deposition at Cardiac-Specific Genes. Stem Cell Rev and Rep 10, 643–652 (2014). https://doi.org/10.1007/s12015-014-9527-y
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12015-014-9527-y