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.

Globally altered epigenetic landscape and delayed osteogenic differentiation in H3.3-G34W-mutant giant cell tumor of bone.

The neoplastic stromal cells of giant cell tumor of bone (GCTB) carry a mutation in H3F3A, leading to a mutant histone variant, H3.3-G34W, as a sole recurrent genetic alteration. We show that in patient-derived stromal cells H3.3-G34W is incorporated into the chromatin and associates with massive epigenetic alterations on the DNA methylation, chromatin accessibility and histone modification level, that can be partially recapitulated in an orthogonal cell line system by the introduction of H3.3-G34W. These epigenetic alterations affect mainly heterochromatic and bivalent regions and provide possible explanations for the genomic instability, as well as the osteolytic phenotype of GCTB. The mutation occurs in differentiating mesenchymal stem cells and associates with an impaired osteogenic differentiation. We propose that the observed epigenetic alterations reflect distinct differentiation stages of H3.3 WT and H3.3 MUT stromal cells and add to H3.3-G34W-associated changes.

Sequence specificity analysis of the SETD2 protein lysine methyltransferase and discovery of a SETD2 super-substrate.

SETD2 catalyzes methylation at lysine 36 of histone H3 and it has many disease connections. We investigated the substrate sequence specificity of SETD2 and identified nine additional peptide and one protein (FBN1) substrates. Our data showed that SETD2 strongly prefers amino acids different from those in the H3K36 sequence at several positions of its specificity profile. Based on this, we designed an optimized super-substrate containing four amino acid exchanges and show by quantitative methylation assays with SETD2 that the super-substrate peptide is methylated about 290-fold more efficiently than the H3K36 peptide. Protein methylation studies confirmed very strong SETD2 methylation of the super-substrate in vitro and in cells. We solved the structure of SETD2 with bound super-substrate peptide containing a target lysine to methionine mutation, which revealed better interactions involving three of the substituted residues. Our data illustrate that substrate sequence design can strongly increase the activity of protein lysine methyltransferases.

Analysis of the Substrate Specificity of the SMYD2 Protein Lysine Methyltransferase and Discovery of Novel Non-Histone Substrates.

The SMYD2 protein lysine methyltransferase methylates various histone and non-histone proteins and is overexpressed in several cancers. Using peptide arrays, we investigated the substrate specificity of the enzyme, revealing a recognition of leucine (or weaker phenylalanine) at the -1 peptide site and disfavor of acidic residues at the +1 to +3 sites. Using this motif, novel SMYD2 peptide substrates were identified, leading to the discovery of 32 novel peptide substrates with a validated target site. Among them, 19 were previously reported to be methylated at the target lysine in human cells, strongly suggesting that SMYD2 is the protein lysine methyltransferase responsible for this activity. Methylation of some of the novel peptide substrates was tested at the protein level, leading to the identification of 14 novel protein substrates of SMYD2, six of which were more strongly methylated than p53, the best SMYD2 substrate described so far. The novel SMYD2 substrate proteins are involved in diverse biological processes such as chromatin regulation, transcription, and intracellular signaling. The results of our study provide a fundament for future investigations into the role of this important enzyme in normal development and cancer.

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.

The Legionella pneumophila Methyltransferase RomA Methylates Also Non-histone Proteins during Infection.

RomA is a SET-domain containing protein lysine methyltransferase encoded by the Gram-negative bacterium Legionella pneumophila. It is exported into human host cells during infection and has been previously shown to methylate histone H3 at lysine 14 [Rolando et al. (2013), Cell Host Microbe, 13, 395-405]. Here, we investigated the substrate specificity of RomA on peptide arrays showing that it mainly recognizes a G-K-X-(PA) sequence embedded in a basic amino acid sequence context. Based on the specificity profile, we searched for possible additional RomA substrates in the human proteome and identified 34 novel peptide substrates. For nine of these, the corresponding full-length protein or protein domains could be cloned and purified. Using radioactive and antibody-based methylation assays, we showed that seven of them are methylated by RomA, four of them strongly, one moderately, and two weakly. Mutagenesis confirmed for the seven methylated proteins that methylation occurs at target lysine residues fitting to the specificity profile. Methylation of one novel substrate (AROS) was investigated in HEK293 cells overexpressing RomA and during infection with L. pneumophila. Methylation could be detected in both conditions, confirming that RomA methylates non-histone proteins in human cells. Our data show that the bacterial methyltransferase RomA methylates also human non-histone proteins suggesting a multifaceted role in the infection process.