DNA microarray analysis of Jmjd1a and/or Jmjd1b knockout embryonic stem cells

Histone H3 lysine 9 (H3K9) methylation is an epigenetic mark of transcriptionally repressed chromatin. During mammalian development, H3K9 methylation levels seem to be spatiotemporally regulated by the opposing activities of methyltransferases and demethylases to govern correct gene expression. However, the combination(s) of H3K9 methyltransferase(s) and demethylase(s) that contribute to this regulation and the genes regulated by them remain unclear. Herein, we demonstrate the essential roles of H3K9 demethylases Jmjd1a and Jmjd1b in the embryogenesis and viability control of embryonic stem (ES) cells. Mouse embryos lacking Jmjd1a/Jmjd1b died after implantation. Depletion of Jmjd1a/Jmjd1b in mouse ES cells induced rapid cell death accompanied with a massive increase in H3K9 methylation. Jmjd1a/Jmjd1b depletion induced an increase in H3K9 methylation in the gene-rich regions of the chromosomes, indicating that Jmjd1a/Jmjd1b removes H3K9 methylation marks in the euchromatin. Importantly, the additional disruption of the H3K9 methyltransferase G9a in a Jmjd1a/Jmjd1b-deficient background rescued not only the H3K9 hypermethylation phenotype but also the cell death phenotype. We also found that Jmjd1a/Jmjd1b removes H3K9 methylation marks deposited by G9a in the Oct4 and Ccnd1 loci to activate transcription. In conclusion, Jmjd1a/Jmjd1b ensures ES cell viability by antagonizing G9a-mediated H3K9 hypermethylation in the gene-rich euchromatin. Jmjd1b-conditional targeting vector was constructed using the BAC recombineering technique and introduced into the Jmjd1bΔ/+ ES cell line 2b-11. Homologous recombination was confirmed by Southern blot analysis with the aforementioned probe. The homologous recombinant ES cell line YY90 (genotype: Jmjd1bΔ/2lox2FRT) was used for further evaluation. The Jmjd1b-deficient ES cell lines, D23.6 and D4.1 (genotype: Jmjd1bΔ/1lox2FRT), were established from a pool of YY90 cells infected with Cre-expressing recombinant adenoviruses. Jmjd1b conditional targeting vector was introduced into the Jmjd1a-deficient ES line 31-1 (Inagaki et al. 2009, Genes Cells 14: 991-1001). The expression plasmid for MerCreMer was then introduced into the recombinant ES cell lines #117 and #131 (genotype: Jmjd1aΔ/Δ; Jmjd1b2lox1FRT/2lox2FRT).