Distinct specificities of the HEMK2 protein methyltransferase in methylation of glutamine and lysine residues.

The HEMK2 protein methyltransferase has been described as glutamine methyltransferase catalyzing ERF1-Q185me1 and lysine methyltransferase catalyzing H4K12me1. Methylation of two distinct target residues is unique for this class of enzymes. To understand the specific catalytic adaptations of HEMK2 allowing it to master this chemically challenging task, we conducted a detailed investigation of the substrate sequence specificities of HEMK2 for Q- and K-methylation. Our data show that HEMK2 prefers methylation of Q over K at peptide and protein level. Moreover, the ERF1 sequence is strongly preferred as substrate over the H4K12 sequence. With peptide SPOT array methylation experiments, we show that Q-methylation preferentially occurs in a G-Q-X3 -R context, while K-methylation prefers S/T at the first position of the motif. Based on this, we identified novel HEMK2 K-methylation peptide substrates with sequences taken from human proteins which are methylated with high activity. Since H4K12 methylation by HEMK2 was very low, other protein lysine methyltransferases were examined for their ability to methylate the H4K12 site. We show that SETD6 has a high H4K12me1 methylation activity (about 1000-times stronger than HEMK2) and this enzyme is mainly responsible for H4K12me1 in DU145 prostate cancer cells.

Methylation of recombinant mononucleosomes by DNMT3A demonstrates efficient linker DNA methylation and a role of H3K36me3.

Recently, the structure of the DNMT3A2/3B3 heterotetramer complex bound to a mononucleosome was reported. Here, we investigate DNA methylation of recombinant unmodified, H3KC4me3 and H3KC36me3 containing mononucleosomes by DNMT3A2, DNMT3A catalytic domain (DNMT3AC) and the DNMT3AC/3B3C complex. We show strong protection of the nucleosomal bound DNA against methylation, but efficient linker-DNA methylation next to the nucleosome core. High and low methylation levels of two specific CpG sites next to the nucleosome core agree well with details of the DNMT3A2/3B3-nucleosome structure. Linker DNA methylation next to the nucleosome is increased in the absence of H3K4me3, likely caused by binding of the H3-tail to the ADD domain leading to relief of autoinhibition. Our data demonstrate a strong stimulatory effect of H3K36me3 on linker DNA methylation, which is independent of the DNMT3A-PWWP domain. This observation reveals a direct functional role of H3K36me3 on the stimulation of DNA methylation, which could be explained by hindering the interaction of the H3-tail and the linker DNA. We propose an evolutionary model in which the direct stimulatory effect of H3K36me3 on DNA methylation preceded its signaling function, which could explain the evolutionary origin of the widely distributed "active gene body-H3K36me3-DNA methylation" connection.