RESUMO
Recent evidence has documented the potential roles of histone-modifying enzymes in autism-spectrum disorder (ASD). Aberrant histone H3 lysine 9 (H3K9) dimethylation resulting from genetic variants in histone methyltransferases is known for neurodevelopmental and behavioral anomalies. However, a systematic examination of H3K9 methylation dynamics in ASD is lacking. Here we resequenced nine genes for histone methyltransferases and demethylases involved in H3K9 methylation in individuals with ASD and healthy controls using targeted next-generation sequencing. We identified a novel rare variant (A211S) in the SUV39H2, which was predicted to be deleterious. The variant showed strongly reduced histone methyltransferase activity in vitro. In silico analysis showed that the variant destabilizes the hydrophobic core and allosterically affects the enzyme activity. The Suv39h2-KO mice displayed hyperactivity and reduced behavioral flexibility in learning the tasks that required complex behavioral adaptation, which is relevant for ASD. The Suv39h2 deficit evoked an elevated expression of a subset of protocadherin ß (Pcdhb) cluster genes in the embryonic brain, which is attributable to the loss of H3K9 trimethylation (me3) at the gene promoters. Reduced H3K9me3 persisted in the cerebellum of Suv39h2-deficient mice to an adult stage. Congruently, reduced expression of SUV39H1 and SUV39H2 in the postmortem brain samples of ASD individuals was observed, underscoring the role of H3K9me3 deficiency in ASD etiology. The present study provides direct evidence for the role of SUV39H2 in ASD and suggests a molecular cascade of SUV39H2 dysfunction leading to H3K9me3 deficiency followed by an untimely, elevated expression of Pcdhb cluster genes during early neurodevelopment.
Assuntos
Transtorno Autístico , Histona-Lisina N-Metiltransferase/genética , Animais , Encéfalo/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Camundongos , ProtocaderinasRESUMO
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.
Assuntos
Histona-Lisina N-Metiltransferase/análise , Dicroísmo Circular , Células HEK293 , Histona-Lisina N-Metiltransferase/isolamento & purificação , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Metilação , Especificidade por SubstratoRESUMO
The SUV39H1 and SUV39H2 enzymes introduce H3K9me3, which is essential for the viability of mammalian cells. It was the aim of the present work to investigate the substrate specificity and product pattern of SUV39H2. Methylation of peptide SPOT arrays showed that SUV39H2 recognizes a long motif on H3 comprising T6-K14, with highly specific readout of R8, S10, T11 and G12 and partial specificity at T6, A7, G13 and K14. Modification of R8 and phosphorylation of S10 or T11 lead to a reduction or loss of SUV39H2 activity towards H3K9. The specificity of SUV39H2 differs from other H3K9 PKMTs, like Dim-5 or G9a, and these biochemical differences can be explained by the structures of the corresponding enzymes. Based on the specificity profile we identified additional non-histone candidate substrates in human proteins, but all of them were only weakly methylated by SUV39H2 at the peptide level. We conclude that SUV39H2 displays a high preference for the methylation of H3. Using the catalytic SET domain we show here that the enzyme prefers H3K9me0 as a substrate over H3K9me1 and H3K9me2 and it introduces the first two methyl groups into H3K9me0 in a processive reaction. SUV39H2 can transfer up to three methyl groups to lysine 9 of histone H3 but the last methylation reaction is much slower than the first two steps. We also demonstrate that the N324K mutant in the SET domain of SUV39H2 that has been shown to cause an inherited nasal skin disease in Labrador Retrievers renders SUV39H2 inactive. Differences in the circular dichroism spectra of wild type and mutant proteins indicated that the mutation causes slight structural changes.
Assuntos
Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Sequência de Aminoácidos , Dicroísmo Circular , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Proteínas Mutantes/química , Proteínas Mutantes/genética , Fosforilação , Conformação Proteica , Especificidade por SubstratoRESUMO
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.
Assuntos
Histona-Lisina N-Metiltransferase , Histonas , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Metilação , Mutação , Processamento de Proteína Pós-TraducionalRESUMO
Post-translational methylation plays a crucial role in regulating and optimizing protein function. Protein histidine methylation, occurring as the two isomers 1- and 3-methylhistidine (1MH and 3MH), was first reported five decades ago, but remains largely unexplored. Here we report that METTL9 is a broad-specificity methyltransferase that mediates the formation of the majority of 1MH present in mouse and human proteomes. METTL9-catalyzed methylation requires a His-x-His (HxH) motif, where "x" is preferably a small amino acid, allowing METTL9 to methylate a number of HxH-containing proteins, including the immunomodulatory protein S100A9 and the NDUFB3 subunit of mitochondrial respiratory Complex I. Notably, METTL9-mediated methylation enhances respiration via Complex I, and the presence of 1MH in an HxH-containing peptide reduced its zinc binding affinity. Our results establish METTL9-mediated 1MH as a pervasive protein modification, thus setting the stage for further functional studies on protein histidine methylation.
Assuntos
Metilistidinas/metabolismo , Metiltransferases/metabolismo , Proteoma/metabolismo , Motivos de Aminoácidos , Animais , Células Cultivadas , Histidina/metabolismo , Humanos , Mamíferos/classificação , Mamíferos/genética , Mamíferos/metabolismo , Metilação , Metiltransferases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mutação , Processamento de Proteína Pós-Traducional , Proteoma/química , Especificidade por Substrato , Zinco/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
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.
Assuntos
Histona-Lisina N-Metiltransferase/economia , Processamento de Proteína Pós-Traducional/genética , Especificidade por Substrato/genética , Sequência de Aminoácidos/genética , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Humanos , Lisina , Metilação , Mutação/genética , Peptídeos/genéticaRESUMO
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.
Assuntos
Neoplasias Ósseas/genética , Tumor de Células Gigantes do Osso/genética , Histonas/genética , Osteogênese , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/fisiopatologia , Metilação de DNA , Epigênese Genética , Epigenômica , Tumor de Células Gigantes do Osso/metabolismo , Tumor de Células Gigantes do Osso/fisiopatologia , Histonas/metabolismo , Humanos , Mutação de Sentido IncorretoRESUMO
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.
Assuntos
Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Mutação , Neoplasias/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Reparo do DNA , Fibroblastos , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/química , Histonas/metabolismo , Humanos , Metilação , Modelos Moleculares , Mutagênese Sítio-Dirigida , Nucleossomos , S-Adenosilmetionina/metabolismoRESUMO
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.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Legionella pneumophila/patogenicidade , Doença dos Legionários/metabolismo , Proteínas Nucleares/metabolismo , Peptídeos/análise , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Células HEK293 , Células HeLa , Humanos , Legionella pneumophila/enzimologia , Lisina/metabolismo , Metilação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Peptídeos/química , Peptídeos/genética , Processamento de Proteína Pós-Traducional , Especificidade por Substrato , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
SUV39H1 is an H3K9 methyltransferase involved in the formation of heterochromatin. We investigated its substrate specificity profile and show recognition of H3 residues between K4 and G12 with highly specific readout of R8. The specificity profile of SUV39H1 is distinct from its paralog SUV39H2, indicating that they can have different additional substrates. Using the specificity profile, several novel SUV39H1 candidate substrates were identified. We observed methylation of 19 novel substrates at the peptide level and for six of them at the protein level. Methylation of RAG2, SET8, and DOT1L was confirmed in cells, which all have important roles in chromatin regulation. Methylation of SET8 allosterically stimulates its H4K20 monomethylation activity connecting SUV39H1 to the generation of increased H4K20me3 levels, another heterochromatic modification. Methylation of RAG2 alters its subnuclear localization, indicating that SUV39H1 might regulate VDJ recombination. Taken together, our results indicate that beyond the generation of H3K9me3, SUV39H1 has additional roles in chromatin biology by direct stimulation of the establishment of H4K20me3 and the regulation of chromatin binding of RAG2.
Assuntos
Cromatina/metabolismo , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Recombinação V(D)J , Animais , Núcleo Celular/enzimologia , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Metilação , Camundongos , Especificidade por SubstratoRESUMO
The H3K9 protein lysine methyltransferase SUV39H2 was reported to methylate K134 of H2AX and stimulate H2AX phosphorylation during DNA damage response [Sone K et al. (2014) Nat Commun 5, 5691]. However, the sequence context of H2AX-K134 differs from the specificity of SUV39H2. We performed in vitro methylation reactions with SUV39H2 (and its homolog SUV39H1) using H2AX protein and peptides, but no methylation at K134 or any other lysine in H2AX was detected. Positive controls demonstrated the functionality of the assays. While our data cannot finally exclude H2AX methylation of SUV39H2 in cells, additional experimental evidence is required to validate this claim.