Jmjd2a and Jmjd2c regulate H3K9me3 and H3K36me3 at H3K4me3 positive TSSs being essential for ESC self-renewal and embryogenesis (microarray)

We have characterized the role of the Jmjd2/Kdm4 proteins in embryonic stem cell (ESC) biology, histone methylation and gene regulation. The Jmjd2 proteins are H3K9/H3K36 histone demethylases and three Jmjd2 family members are expressed in ESCs: Jmjd2a/Kdm4a, Jmjd2b/Kdm4b and Jmjd2c/Kdm4c/Gasc1. We find that specifically Jmjd2a and Jmjd2c exert redundant functions, which are essential for ESC self-renewal and early embryonic development. ChIP-seq studies show that Jmjd2a and Jmjd2c both localize to H3K4me3 marked regions, where they have general and widespread roles preventing the accumulation of especially H3K9me3, but also H3K36me3. Jmjd2 catalytic activity is required for ESC maintenance, and increased H3K9me3 levels in knockout ESCs compromise the expression of several Jmjd2a/c targets, including genes that are important for ESC self-renewal. Thus, continual removal of H3K9 promoter methylation by Jmjd2 demethylases represents a novel mechanism ensuring transcriptional competence and stability of the pluripotent cell identity. We generated conditional Jmjd2a;Jmjd2c (2ac) double knockout ESCs (Jmjd2a(f/f);Jmjd2c(f/f);Rosa26::CreERT2) and Jmjd2a:Jmjd2b;Jmjd2c (2abc) triple knockout ESCs (Jmjd2a(f/f);Jmjd2b(f/f);Jmjd2c(f/f);Rosa26::CreERT2). 3 independently established ESC lines of 2ac and 2abc ESCs, respectively, were grown in the absence or presence of OHT leading to activation of the Cre recombinase and subsequent loss of Jmjd2 expression. Upon recovery from the OHT treatment, RNA was extracted for gene expression analyses. ESCs were cultured in 2i serum-free medium.