Histone methyltransferase G9a contributes to H3K27 methylation in vivo

Histone modifications play a vital role in the conformation and function of their associated chromatin templates ¹. Histone H3K27 methylation mediated by the PRC2 complex is critical for transcriptional regulation, Polycomb silencing, Drosophila segmentation, mammalian X inactivation and cancer ¹. Interestingly, H3K27me1 (H3 mono-methylated at residue K27) levels in vivo remain unaffected after PRC2 disruption ^{2, 3}, which is an indication for the existence of other contributing histone methyltransferase(s) to H3K27me1. However, in animals, histone methyltransferases responsible for H3K27me1 levels in vivo remain undefined. On the other hand, G9a is a histone methyltransferase that controls H3K9me2 in vivo and predominantly represses genes at euchromatic regions ⁴. It has also been reported to act on H3K27 in vitro on H3 tail fused with a GST tag ⁵, but its role in H3K27 methylation has not been tested on the nucleosomes, the native substrates for histone methyltransferases. In addition, G9a's contribution to H3K27 methylation in vivo remains obscure.

To directly detect the reaction products, wild-type nucleosomes were reacted with G9a and S-adenosyl methionine. The reaction products were chemically propionylated ⁸ prior to mass spectrometry analysis to ensure the detection of H3K9 methylation marks in subsequent LC-MS (liquid chromatograph-coupled mass spectrometry) procedures. G9a-treated nucleosomal H3 samples contained H3K9me1, H3K9me2 signals and low amounts of H3K9me3 as expected (Supplementary information, Figure S1). Moreover, they also contained robust signals for H3K27me1 and H3K27me2 (Figure 1B). In addition, another histone methyltransferase GLP, a close homologue of G9a, can also methylate H3K27 in addition to H3K9 in vitro (Supplementary information, Figure S2).

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