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.
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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.).
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.
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)
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)
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