H3K9 methylation, transcriptome and Hi-C analysis in mouse ES cells and iMEFs

Histone H3 lysine 9 dimethylation (H3K9me2) is a highly conserved silencing epigenetic mark and mainly regulated by the lysine methyltranferase complex, G9a/GLP in mammals. Chromatin marked with H3K9me2 forms large domains and correlates well with lamina-associated domains and the B compartment in the nucleus. However, the role of H3K9me2 in 3-dimensional (3D) genome organization remains unclear. We investigated the genome-wide H3K9me2 distribution, the transcriptome and 3D genome organization in mouse embryonic stem cells and fibroblasts upon the inhibition or depletion of H3K9 methyltransferases (MTases) G9a/GLP, SETDB1, and SUV39H1/2. We found that H3K9me2 is regulated by these five MTases; however, H3K9me2 and transcription in the A and B compartments were largely regulated by different sets of the MTases: H3K9me2 in the A compartments were mainly regulated by G9a/GLP and SETDB1, while H3K9me2 in the B compartments were regulated by all five H3K9 MTases. Because G9a/GLP has strong impact on H3K9me2, regulation or a role of G9a/GLP-independent H3K9me2 remains to be elucidated. Our study clarified entire H3K9me2 deposition mechanism in mammals. Furthermore, decreased H3K9me2 correlated with the changes to the more active compartmental state that accompanied transcriptional activation. In conclusion, we showed that H3K9me2 domain formation is functionally linked to 3D genome organization and propose that H3K9me2 could contributes to heterochromatin compartment formation as one of the key players of determinant mechanisms.