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.

The H3.3 G34W oncohistone mutation increases K36 methylation by the protein lysine methyltransferase NSD1.

The H3.3 G34W mutation has been observed in 90% of the patients affected by giant cell tumor of bone (GCTB). It had been shown to reduce the activity of the SETD2 H3K36 protein lysine methyltransferase (PKMT) and lead to genome wide changes in epigenome modifications including a global reduction in DNA methylation. Here, we investigated the effect of the H3.3 G34W mutation on the activity of the H3K36me2 methyltransferase NSD1, because NSD1 is known to play an important role in the differentiation of chondrocytes and osteoblasts. Unexpectedly, we observed that H3.3 G34W has a gain-of-function effect and it stimulates K36 methylation by NSD1 by about 2.3-fold with peptide substrates and 6.3-fold with recombinant nucleosomal substrates. This effect is specific for NSD1, as NSD2 shows only a mild stimulation on G34W substrates. The potential downstream effects of the G34W induced hyperactivity of NSD1 on DNA methylation, H3K27me3, histone acetylation and splicing are discussed.

Somatic Cancer Mutations in the SUV420H1 Protein Lysine Methyltransferase Modulate Its Catalytic Activity.

SUV420H1 is a protein lysine methyltransferase that introduces di- and trimethylation of H4K20 and is frequently mutated in human cancers. We investigated the functional effects of eight somatic cancer mutations on SUV420H1 activity in vitro and in cells. One group of mutations (S255F, K258E, A269V) caused a reduction of the catalytic activity on peptide and nucleosome substrates. The mutated amino acids have putative roles in AdoMet binding and recognition of H4 residue D24. Group 2 mutations (E238V, D249N, E320K) caused a reduction of activity on peptide substrates, which was partially recovered when using nucleosomal substrates. The corresponding residues could have direct or indirect roles in peptide and AdoMet binding, but the effects of the mutations can be overcome by additional interactions between SUV420H1 and the nucleosome substrate. The third group of mutations (S283L, S304Y) showed enhanced activity with peptide substrates when compared with nucleosomal substrates, suggesting that these residues are involved in nucleosomal interaction or allosteric activation of SUV420H1 after nucleosome binding. Group 2 and 3 mutants highlight the role of nucleosomal contacts for SUV420H1 regulation in agreement with the high activity of this enzyme on nucleosomal substrates. Strikingly, seven of the somatic cancer mutations studied here led to a reduction of the catalytic activity of SUV420H1 in cells, suggesting that SUV420H1 activity might have a tumor suppressive function. This could be explained by the role of H4K20me2/3 in DNA repair, suggesting that loss or reduction of SUV420H1 activity could contribute to a mutator phenotype in cancer cells.