KDM4B-mediated reduction of H3K9me3 levels improves somatic cell reprogramming into pluripotency

Correct reprogramming of epigenetic marks is crucial for somatic cells to regain pluripotency. Repressive histone lysine methylation marks are known to be very stable and difficult to reprogram. In this study, we generated transgenic mice and mouse embryonic fibroblasts (MEFs) for the inducible expression of KDM4B, a demethylase that primarily removes the histone 3 lysine 9 trimethylation (H3K9me3) mark. Upon inducing Kdm4b, H3K9me3 levels dropped ~100-fold compared to non-induced controls. Concurrently, H3K9me1 levels significantly increased, while. H3K9me2 and H3K27me3 remained unchanged. The global transcriptional impact of Kdm4b-mediated reduction in H3K9me3 levels was examined by comparative microarray analysis and mRNA-sequencing of three independent transgenic MEF lines. We identified 15 genes as commonly misregulated, including the up-regulated heterochromatin-associated zinc finger protein 37. Following somatic nuclear transfer, reduced H3K9me3 levels were rapidly restored. Nevertheless, hypo-methylated KDM4B-MEF donors reprogrammed six-fold better into cloned blastocysts than non-induced donors. Using a complementary functional assay, they also reprogrammed nine-fold better into induced pluripotent stem cells (iPSCs). These transgenic Kdm4-iPSCs gave rise to teratomas and coat-colour chimeras, demonstrating their pluripotency. In summary, we firmly established H3K9me3 as a major roadblock to somatic cell reprogramming in mouse and identified transcriptional targets of derestricted chromatin.