RESUMO
DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , DNA Ligase Dependente de ATP/metabolismo , Metilação de DNA , Replicação do DNA , DNA/biossíntese , Epigênese Genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas Estimuladoras de Ligação a CCAAT/genética , DNA/genética , DNA Ligase Dependente de ATP/química , DNA Ligase Dependente de ATP/genética , Células-Tronco Embrionárias/enzimologia , Células HEK293 , Células HeLa , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Humanos , Lisina , Metilação , Camundongos , Modelos Moleculares , Mimetismo Molecular , Mutação , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Transfecção , Domínio Tudor , Ubiquitina-Proteína LigasesRESUMO
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
Bacterial HEMK2 homologs initially had been proposed to be involved in heme biogenesis or to function as adenine DNA methyltransferase. Later it was shown that this family of enzymes has protein glutamine methyltransferase activity, and they methylate the glutamine residue in the GGQ motif of ribosomal translation termination factors. The murine HEMK2 enzyme methylates Gln(185) of the eukaryotic translation termination factor eRF1. We have employed peptide array libraries to investigate the peptide sequence recognition specificity of murine HEMK2. Our data show that HEMK2 requires a GQX3R motif for methylation activity. In addition, amino acid preferences were observed between the -3 and +7 positions of the peptide substrate (considering the target glutamine as 0), including a preference for Ser, Arg, and Gly at the +1 and a preference for Arg at the +7 position. Based on our specificity profile, we identified several human proteins that contain putative HEMK2 methylation sites and show that HEMK2 methylates 58 novel peptide substrates. After cloning, expression, and purification of the corresponding protein domains, we confirmed methylation for 11 of them at the protein level. Transfected CHD5 (chromodomain helicase DNA-binding protein 5) and NUT (nuclear protein in testis) were also demonstrated to be methylated by HEMK2 in human HEK293 cells. Our data expand the range of proteins potentially subjected to glutamine methylation significantly, but further investigation will be required to understand the function of HEMK2-mediated methylation in proteins other than eRF1.
Assuntos
Processamento de Proteína Pós-Traducional , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , DNA Helicases/metabolismo , Células HEK293 , Humanos , Metilação , Camundongos , Proteínas de Neoplasias , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/fisiologia , Especificidade por SubstratoRESUMO
Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies.
Assuntos
Anticorpos/metabolismo , Histonas/metabolismo , Mapeamento de Interação de Proteínas/métodos , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Anticorpos/imunologia , Sítios de Ligação/genética , Western Blotting , Imunoprecipitação da Cromatina , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Células HEK293 , Histonas/imunologia , Humanos , Lisina/metabolismo , Metilação , Peptídeos/metabolismo , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Análise Serial de Proteínas/métodos , Ligação Proteica , Reprodutibilidade dos TestesRESUMO
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
Methylation of lysine residues is an important post-translational modification of histone and non-histone proteins, which is introduced by protein lysine methyltransferases (PKMTs). An increasing number of reports demonstrate that aberrant lysine methylation plays a central role in carcinogenesis that is often correlated with abnormal expression of PKMTs. Recent whole genome and whole transcriptome sequencing projects have also discovered several somatic mutations in PKMTs that frequently appear in various tumors. These include chromosomal translocations that lead to aberrant expression or mistargeting of PKMTs and nonsense or frameshift mutations, which cause the loss of the protein function. Another type of mutations are missense mutations which may lead to the loss of enzyme activity, but may also alter the properties of PKMTs either by changing the product or substrate specificity or by increasing the enzymatic activity finally leading to a gain-of-function phenotype. In this review, we provide an overview of the roles of EZH2, SETD2, NSD family, SMYD family, MLL family and DOT1L PKMTs in cancer focusing on the effects of somatic cancer mutations in these enzymes. Investigation of the effect of somatic cancer mutations in PKMTs is pivotal to understand the general role of this important class of enzymes in carcinogenesis and to improve and develop more individualized cancer therapies.
Assuntos
Histona-Lisina N-Metiltransferase/genética , Mutação , Neoplasias/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Histona-Lisina N-Metiltransferase/fisiologia , Humanos , Complexo Repressor Polycomb 2/genéticaRESUMO
Mutations in the ATRX protein are associated with the alpha-thalassemia and mental retardation X-linked syndrome (ATR-X). Almost half of the disease-causing mutations occur in its ATRX-Dnmt3-Dnmt3L (ADD) domain. By employing peptide arrays, chromatin pull-down and peptide binding assays, we show specific binding of the ADD domain to H3 histone tail peptides containing H3K9me3. Peptide binding was disrupted by the presence of the H3K4me3 and H3K4me2 modification marks indicating that the ATRX-ADD domain has a combined readout of these two important marks (absence of H3K4me2 and H3K4me3 and presence of H3K9me3). Disease-causing mutations reduced ATRX-ADD binding to H3 tail peptides. ATRX variants, which fail in the H3K9me3 interaction, show a loss of heterochromatic localization in cells, which indicates the chromatin targeting function of the ADD domain of ATRX. Disruption of H3K9me3 binding may be a general pathogenicity pathway of ATRX mutations in the ADD domain which may explain the clustering of disease mutations in this part of the ATRX protein.
Assuntos
DNA Helicases/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Proteínas Nucleares/metabolismo , Peptídeos/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Cromatina/metabolismo , Clonagem Molecular , DNA Helicases/genética , Expressão Gênica , Células HEK293 , Humanos , Lisina/genética , Deficiência Intelectual Ligada ao Cromossomo X/genética , Deficiência Intelectual Ligada ao Cromossomo X/patologia , Metilação , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Proteínas Nucleares/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteína Nuclear Ligada ao X , Talassemia alfa/genética , Talassemia alfa/patologiaRESUMO
The demand for practical and convenient enzyme assays for histone lysine methyltransferases (HKMTs) emerges along with the rapid development of this young class of enzymes. A supramolecular reporter pair composed of p-sulfonatocalix[4]arene (CX4) and the fluorescent dye lucigenin (LCG) has been used to monitor enzymatic trimethylation of lysine residues in peptide substrates. The assay affords a switch-ON fluorescence response and operates in a continuous, real-time, and label-free fashion. The underlying working principle relies on the higher affinity of the macrocycle towards the trimethylated product of the enzymatic reaction as compared to the substrate, which allows the assay to be carried out in the product-selective mode. The final product incorporates a trimethylammonium moiety, a known high-affinity binding motif for CX4. Two substrates corresponding to the H3 N-terminal tail, namely, S2 (RTKQTARKSTGGKAP) and S6 (QTARKSTGGS), were selected as model compounds for methylation with the Neurospora crassa Dim-5 enzyme and investigated by the newly developed supramolecular tandem HKMTs assay. Only the longer substrate S2 underwent methylation in solution. The potential of the assay for inhibitor screening was demonstrated by means of inhibition studies with 1,10-phenanthroline to afford an inhibition constant of (70±20)â µM.
Assuntos
Corantes Fluorescentes/química , Histona-Lisina N-Metiltransferase/química , Lisina/química , Compostos Macrocíclicos/química , Metiltransferases/química , Peptídeos/química , Soluções/química , Corantes Fluorescentes/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Cinética , Lisina/metabolismo , Compostos Macrocíclicos/metabolismo , Metiltransferases/metabolismo , Estrutura Molecular , Peptídeos/metabolismo , Especificidade por SubstratoRESUMO
BACKGROUND: Epigenetic reading domains are involved in the regulation of gene expression and chromatin state by interacting with histones in a post-translational modification specific manner. A detailed knowledge of the target modifications of reading domains, including enhancing and inhibiting secondary modifications, will lead to a better understanding of the biological signaling processes mediated by reading domains. RESULTS: We describe the application of Celluspots peptide arrays which contain 384 histone peptides carrying 59 post translational modifications in different combinations as an inexpensive, reliable and fast method for initial screening for specific interactions of reading domains with modified histone peptides. To validate the method, we tested the binding specificities of seven known epigenetic reading domains on Celluspots peptide arrays, viz. the HP1ß and MPP8 Chromo domains, JMJD2A and 53BP1 Tudor domains, Dnmt3a PWWP domain, Rag2 PHD domain and BRD2 Bromo domain. In general, the binding results agreed with literature data with respect to the primary specificity of the reading domains, but in almost all cases we obtained additional new information concerning the influence of secondary modifications surrounding the target modification. CONCLUSIONS: We conclude that Celluspots peptide arrays are powerful screening tools for studying the specificity of putative reading domains binding to modified histone peptides.
Assuntos
Epigênese Genética , Histonas/química , Histonas/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Análise Serial de Proteínas/métodos , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/metabolismo , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Especificidade por Substrato , Fatores de Transcrição , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
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
Eukaryotic elongation factor 1 alpha (eEF1A) delivers aminoacyl-tRNA to the ribosome and thereby plays a key role in protein synthesis. Human eEF1A is subject to extensive post-translational methylation, but several of the responsible enzymes remain unknown. Using a wide range of experimental approaches, we here show that human methyltransferase (MTase)-like protein 13 (METTL13) contains two distinct MTase domains targeting the N terminus and Lys55 of eEF1A, respectively. Our biochemical and structural analyses provide detailed mechanistic insights into recognition of the eEF1A N terminus by METTL13. Moreover, through ribosome profiling, we demonstrate that loss of METTL13 function alters translation dynamics and results in changed translation rates of specific codons. In summary, we here unravel the function of a human MTase, showing that it methylates eEF1A and modulates mRNA translation in a codon-specific manner.
Assuntos
Códon/genética , Metiltransferases/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Humanos , Metiltransferases/química , Metiltransferases/genética , Fator 1 de Elongação de Peptídeos/química , Fator 1 de Elongação de Peptídeos/genética , Ligação Proteica , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Ribossomos/metabolismoRESUMO
DNA, RNA and histone methylation is implicated in various human diseases such as cancer or viral infections, playing a major role in cell process regulation, especially in modulation of gene expression. Here we developed a convergent synthetic pathway starting from a protected bromomethylcytosine derivative to synthesize transition state analogues of the DNA methyltransferases. This approach led to seven 5-methylcytosine-adenosine compounds that were, surprisingly, inactive against hDNMT1, hDNMT3Acat, TRDMT1 and other RNA human and viral methyltransferases. Interestingly, compound 4 and its derivative 2 showed an inhibitory activity against PRMT4 in the micromolar range. Crystal structures showed that compound 4 binds to the PRMT4 active site, displacing strongly the S-adenosyl-l-methionine cofactor, occupying its binding site, and interacting with the arginine substrate site through the cytosine moiety that probes the space filled by a substrate peptide methylation intermediate. Furthermore, the binding of the compounds induces important structural switches. These findings open new routes for the conception of new potent PRMT4 inhibitors based on the 5-methylcytosine-adenosine scaffold.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.
Assuntos
Domínio Catalítico , Metiltransferases/síntese química , Peptídeos/metabolismo , HumanosRESUMO
Somatic missense mutations in the mixed lineage leukemia 1 (MLL1) histone H3K4 methyltransferase are often observed in cancers. MLL1 forms a complex with WDR5, RBBP5, and ASH2L (WRA) which stimulates its activity. The MM-102 compound prevents the interaction between MLL1 and WDR5 and functions as an MLL1 inhibitor. We have studied the effects of four cancer mutations in the catalytic SET domain of MLL1 on the enzymatic activity of MLL1 and MLL1-WRA complexes. In addition, we studied the interaction of the MLL1 mutants with the WRA proteins and inhibition of MLL1-WRA complexes by MM-102. All four investigated mutations had strong effects on the activity of MLL1. R3903H was inactive and S3865F showed reduced activity both alone and in complex with WRA, but its activity was stimulated by the WRA complex. By contrast, R3864C and R3841W were both more active than wild-type MLL1, but still less active than the wild-type MLL1-WRA complex. Both mutants were not stimulated by complex formation with WRA, although no differences in the interaction with the complex proteins were observed. These results indicate that both mutants are in an active conformation even in the absence of the WRA complex and their normal control of activity by the WRA complex is altered. In agreement with this observation, the activity of R3864C and R3841W was not reduced by addition of the MM-102 inhibitor. We show that different cancer mutations in MLL1 lead to a loss or increase in activity, illustrating the complex and tumor-specific role of MLL1 in carcinogenesis. Our data exemplify that biochemical investigations of somatic tumor mutations are required to decipher their pathological role. Moreover, our data indicate that MM-102 may not be used as an MLL1 inhibitor if the R3864C and R3841W mutations are present. More generally, the efficacy of any enzyme inhibitor must be experimentally confirmed for mutant enzymes before an application can be considered.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Complexos Multiproteicos/metabolismo , Mutação/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Biocatálise , Dicroísmo Circular , Clonagem Molecular , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Metilação , Proteínas Mutantes/isolamento & purificação , Ligação ProteicaRESUMO
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
In fission yeast, the catalytic activity of the protein lysine methyltransferase (PKMT) Clr4, the sole homolog of the mammalian SUV39H1 and SUV39H2 enzymes, majorly contributes to the formation of heterochromatin. The enzyme introduces histone 3 lysine 9 (H3K9) di- and tri-methylation, a central heterochromatic histone modification, and later it was also found to methylate the Mlo3 protein, which has a role in heterochromatin formation as well. Herein, we have investigated the substrate specificity of Clr4 using custom made mutational scanning peptide arrays. Our data show, that Clr4 recognizes an RK core motif, showing high preference for R8. In addition, it exhibits specific contacts at the S10, T11, G12 and G13 positions of the H3 peptide recognizing an R-K-SKRT-TCS-G sequence. Based on the specificity profile and in vitro methyltransferase assay targeted searches, 11 putative methylation sites in S. pombe proteins were identified from reported Clr4 interacting proteins including Mlo3. Peptide methylation was observed on Mlo3 and 7 novel target sites with strongest methylation signals on Spbc28F2.11 (HMG box-containing protein) at lysine 292 and Hrp3 (Chromodomain ATP-dep DNA helicase) at lysine 89. These data suggest that Clr4 has additional methylation substrates and it will be important to study the biological function of these novel methylation events. Furthermore, the specificity profile of Clr4 has been used to develop a quantitative method to compare and cluster specificity profiles of PKMTs. It shows that the specificity profile of Clr4 is most similar to that of the SUV39H2 enzyme, one of its human homologs. This approach will be helpful in the comparison of the recognition profiles of other families of PKMTs as well.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Metiltransferases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , Histona-Lisina N-Metiltransferase , Metilação , Metiltransferases/genética , Processamento de Proteína Pós-Traducional , Schizosaccharomyces/enzimologia , Proteínas de Schizosaccharomyces pombe/genética , Especificidade por SubstratoRESUMO
SETDB1 is an essential H3K9 methyltransferase involved in silencing of retroviruses and gene regulation. We show here that its triple Tudor domain (3TD) specifically binds to doubly modified histone H3 containing K14 acetylation and K9 methylation. Crystal structures of 3TD in complex with H3K14ac/K9me peptides reveal that peptide binding and K14ac recognition occurs at the interface between Tudor domains (TD) TD2 and TD3. Structural and biochemical data demonstrate a pocket switch mechanism in histone code reading, because K9me1 or K9me2 is preferentially recognized by the aromatic cage of TD3, while K9me3 selectively binds to TD2. Mutations in the K14ac/K9me binding sites change the sub-nuclear localization of 3TD. ChIP-seq analyses show that SETDB1 is enriched at H3K9me3 regions and K9me3/K14ac is enriched at SETDB1 binding sites overlapping with LINE elements, suggesting that recruitment of the SETDB1 complex to K14ac/K9me regions has a role in silencing of active genomic regions.
Assuntos
Histonas/metabolismo , Elementos Nucleotídeos Longos e Dispersos/fisiologia , Proteínas Metiltransferases/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Domínio Tudor/fisiologia , Acetilação , Animais , Sítios de Ligação/fisiologia , Cristalografia por Raios X , Células HEK293 , Histona-Lisina N-Metiltransferase , Histonas/genética , Humanos , Metilação , Camundongos , Células-Tronco Embrionárias Murinas , Ligação Proteica/fisiologia , Proteínas Metiltransferases/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Protein lysine methylation is emerging as a general post-translational modification (PTM) with essential functions regulating protein stability, activity, and protein-protein interactions. One of the outstanding challenges in this field is linking protein lysine methyltransferases (PKMTs) with specific substrates and lysine methylation events in a systematic manner. Inability to validate reported PKMT substrates delayed progress in the field and cast unnecessary doubt about protein lysine methylation as a truly general PTM. Here, we aim to provide a concise guide to help avoid some of the most common pitfalls in studies searching for new PKMT substrates and propose a set of seven basic biochemical rules: (1) include positive controls; (2) use target lysine mutations of substrate proteins as negative controls; (3) use inactive enzyme variants as negative controls; (4) report quantitative methylation data; (5) consider PKMT specificity; (6) validate methyl lysine antibodies; and (7) connect cellular and in vitro results. We explain the logic behind them and discuss how they should be implemented in the experimental work.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Lisina/metabolismo , Humanos , Metilação , Modelos Moleculares , Reprodutibilidade dos Testes , Especificidade por SubstratoRESUMO
The SUV4-20H1 and SUV4-20H2 enzymes methylate histone H4 at K20, and they have overlapping and distinct biological effects. Here, by in vitro methylation studies we confirmed that both the murine SUV4-20H enzymes strongly favor the monomethylated H4K20 peptide substrate. We also show that both enzymes only generate dimethylated H4K20 products. We determined the substrate sequence recognition motif of both enzymes using SPOT peptide arrays showing that SUV4-20H1 recognizes an (RY)-Kme1-(IVLM)-(LFI)-X-D sequence. In contrast, SUV4-20H2 shows less specificity and recognizes an X-Kme1-(IVLMK)-(LVFI)-X-(DEV) sequence, which is partially overlapping with SUV4-20H1 but has relaxed specificity at the -1 and +4 positions (if the target H4K20me1 is positon 0). Based on our data, we identify novel peptide substrates for SUV4-20H1 (K1423 of Zinc finger protein castor homolog 1) and SUV4-20H2 (K1423 of Zinc finger protein castor homolog 1, K215 of Protein Mis18-beta and K308 of Centromere protein U). All these lysine residues were already identified to be methylated in human cells, but the responsible PKMT was not known. In addition, we also tested the activity of SUV4-20H enzymes on ERK1, which was recently reported to be methylated by SUV4-20H1 at K302 and K361. However the sequences surrounding both methylation sites do not fit to the specificity profile of SUV4-20H1 and we could not detect methylation of ERK1 by any of the SUV4-20H enzymes. The possible reasons of this discrepancy and its consequences are discussed.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Lisina/metabolismo , Linhagem Celular , Células HEK293 , Histonas/metabolismo , Humanos , MetilaçãoRESUMO
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.