Academic literature on the topic 'Histone-Lysine N-Methyltransferase'

Arginine methylation of histone H3 and H4 plays important roles in transcriptional regulation in eukaryotes such as yeasts, fruitflies, nematode worms, fish and mammals; however, less is known in plants. In the present paper, we report the identification and characterization of two Arabidopsis thaliana protein arginine N-methyltransferases, AtPRMT1a and AtPRMT1b, which exhibit high homology with human PRMT1. Both AtPRMT1a and AtPRMT1b methylated histone H4, H2A, and myelin basic protein in vitro. Site-directed mutagenesis of the third arginine (R3) on the N-terminus of histone H4 to lysine (H4R3N) completely abolished the methylation of histone H4. When fused to GFP (green fluorescent protein), both methyltransferases localized to the cytoplasm as well as to the nucleus. Consistent with their subcellular distribution, GST (glutathione transferase) pull-down assays revealed an interaction between the two methyltransferases, suggesting that both proteins may act together in a functional unit. In addition, we demonstrated that AtFib2 (Arabidopsis thaliana fibrillarin 2), an RNA methyltransferase, is a potential substrate for AtPRMT1a and AtPRMT1b, and, furthermore, uncovered a direct interaction between the protein methyltransferase and the RNA methyltransferase. Taken together, our findings implicate AtPRMT1a and AtPRMT1b as H4-R3 protein arginine N-methyltransferases in Arabidopsis and may be involved in diverse biological processes inside and outside the nucleus.

KMT2 (histone-lysine N-methyltransferase subclass 2) complexes methylate lysine 4 on the histone H3 tail at gene promoters and gene enhancers and, thus, control the process of gene transcription. These complexes not only play an essential role in normal development but have also been described as involved in the aberrant growth of tissues. KMT2 mutations resulting from the rearrangements of the KMT2A (MLL1) gene at 11q23 are associated with pediatric mixed-lineage leukemias, and recent studies demonstrate that KMT2 genes are frequently mutated in many types of human cancers. Moreover, other components of the KMT2 complexes have been reported to contribute to oncogenesis. This review summarizes the recent advances in our knowledge of the role of KMT2 complexes in cell transformation. In addition, it discusses the therapeutic targeting of different components of the KMT2 complexes.

Global patterns of histone methylation are remodelled during cleavage development. Of the five histone methyltransferases known to mediate methylation of the lysine 9 residue of histone H3 (H3K9), euchromatic histone-lysine N-methyltransferase 2 (EHMT2; also known as G9a) has been shown to be a primary mediator of H3K9 dimethylation; BIX-01294 has been shown to be a specific inhibitor of EHMT2. The objective of the present study was to determine the effect of BIX-01294 treatment on global H3K9 dimethylation in porcine embryos. We hypothesised that inhibition of EHMT2 by BIX-01294 would result in reduced levels of H3K9 dimethylation and compromised embryo development. Our results showed that incubation in 5 µM BIX-01294 markedly reduced global levels of H3K9 dimethylation at the pronuclear, 2-cell and 4-cell stages of development and resulted in developmental arrest before blastocyst formation. Although transient exposure of embryos to BIX-01294 did not alter in vitro development, embryos transiently exposed to BIX-01294 did not establish pregnancy. These data demonstrate that BIX-01294 is a potent inhibitor of H3K9 dimethylation and that transient alterations in global histone modifications can have profound effects on embryo developmental potential.

Most characterized protein methylation events encompass arginine and lysine N-methylation, and only a few cases of protein methionine thiomethylation have been reported. Newly discovered oncohistone mutations include lysine-to-methionine substitutions at positions 27 and 36 of histone H3.3. In these instances, the methionine substitution localizes to the active-site pocket of the corresponding histone lysine methyltransferase, thereby inhibiting the respective transmethylation activity. SET domain–containing 3 (SETD3) is a protein (i.e. actin) histidine methyltransferase. Here, we generated an actin variant in which the histidine target of SETD3 was substituted with methionine. As for previously characterized histone SET domain proteins, the methionine substitution substantially (76-fold) increased binding affinity for SETD3 and inhibited SETD3 activity on histidine. Unexpectedly, SETD3 was active on the substituted methionine, generating S-methylmethionine in the context of actin peptide. The ternary structure of SETD3 in complex with the methionine-containing actin peptide at 1.9 Å resolution revealed that the hydrophobic thioether side chain is packed by the aromatic rings of Tyr312 and Trp273, as well as the hydrocarbon side chain of Ile310. Our results suggest that placing methionine properly in the active site—within close proximity to and in line with the incoming methyl group of SAM—would allow some SET domain proteins to selectively methylate methionine in proteins.

Histone posttranslational modifications are among the epigenetic mechanisms that modulate chromatin structure and gene transcription. Histone methylation and demethylation are dynamic processes controlled respectively by histone methyltransferases (HMTs) and demethylases (HDMs). Several HMTs and HDMs have been implicated in cancer, inflammation, and diabetes, making them attractive targets for drug therapy. Hence, the discovery of small-molecule modulators for these two enzyme classes has drawn significant attention from the pharmaceutical industry. Herein, the authors describe the development and optimization of homogeneous LANCE Ultra and AlphaLISA antibody-based assays for measuring the catalytic activity of two epigenetic enzymes acting on lysine 4 of histone H3: SET7/9 methyltransferase and LSD1 demethylase. Both the SET7/9 and LSD1 assays were designed as signal-increase assays using biotinylated peptides derived from the N-terminus of histone H3. In addition, the SET7/9 assay was demonstrated using full-length histone H3 protein as substrate in the AlphaLISA format. Optimized assays in 384-well plates are robust (Z′ factors ≥0.7) and sensitive, requiring only nanomolar concentrations of enzyme and substrate. All assays allowed profiling of known SET7/9 and LSD1 inhibitors. The results demonstrate that the optimized LANCE Ultra and AlphaLISA assay formats provide a relevant biochemical screening approach toward the identification of small-molecule inhibitors of HMTs and HDMs that could lead to novel epigenetic therapies.

Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)–PKMT–substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (SN2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MS-based method to measure KIEs and binding isotope effects (BIEs) of the cofactorS-adenosyl-l-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic13C KIE of 1.04, an inverse intrinsic α-secondary CD3KIE of 0.90, and a small but statistically significant inverse CD3BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical SN2 transition state with the C-N and C-S distances of 2.35–2.40 Å and 2.00–2.05 Å, respectively. This transition state is further supported by the KIEs, BIEs, and steady-state kinetics with the SAM analogSe-adenosyl-l-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors.

The following work demonstrates that chromatin regulators play far more pronounced roles in dynamic gene expression than they do in steady-state. Histone modifications have been associated with transcription activity. However, previous analyses of gene expression in mutants affecting histone modifications show limited alteration. I systematically dissected the effects of 83 histone mutants and 119 gene deletion mutants on gene induction/repression in response to diamide stress in yeast. Importantly, I observed far more changes in gene induction/repression than changes in steady-state gene expression. The extensive dynamic gene expression profile of histone mutants and gene deletion mutants also allowed me to identify specific interactions between histone modifications and chromatin modifiers. Furthermore, by combining these functional results with genome-wide mapping of several histone modifications in the same time course, I was able to investigate the correspondence between histone modification occurrence and function. One such observation was the role of Set1-dependent H3K4 methylation in the repression of ribosomal protein genes (RPGs) during multiple stresses. I found that proper repression of RPGs in stress required the presence, but not the specific sequence, of an intron, an element which is almost unique to this gene class in Saccharomyces cerevisiae. This repression may be related to Set1’s role in antisense RNA-mediated gene silencing. Finally, I found a potential role for Set1 in producing or maintaining uncapped mRNAs in cells through a mechanism that does not involved nuclear exoribonucleases. Thus, deletion of Set1 in xrn1Δ suppresses the accumulation of uncapped transcripts observed in xrn1Δ. These findings reveal that Set1, along with other chromatin regulators, plays important roles in dynamic gene expression through diverse mechanisms and thus provides a coherent means of responding to environmental cues.

De manière concomitante à leur synthèse, les ARNm néotranscrits sont sujets à différentes étapes de maturation conduisant à la formation de complexes ribonucléoprotéiques (RNPm) compétents pour l'export. Ces RNPm matures sont reconnus par le récepteur d'export, Mex67 chez la levure, TAP chez les métazoaires, qui assure leur translocation à travers le pore nucléaire. De récentes études ont montré que l'ubiquitination joue un rôle régulateur majeur dans l'export des ARNm. De manière intéressante, Mex67/TAP présente dans sa région C-terminale un domaine UBA (Ubiquitin associated) jusqu'à présent caractérisé pour son interaction avec les nucléoporines. L'objectif de cette thèse a été d'approfondir les propriétés structurales et fonctionnelles du domaine UBA de Mex67 en vue de mieux comprendre le rôle de l'ubiquitination dans l'export nucléaire des ARNm. Nous avons tout d'abord montré que le domaine UBA de Mex67 est capable de lier l'ubiquitine ainsi que des partenaires spécifiques ubiquitinés par des mécanismes que nous avons identifiés, tels que Hpr1, facteur impliqué dans l'élongation de la transcription ou encore Swd2 participant à la méthylation de l'histone H3 et à la maturation des ARNm à leur extrémité 3'. D'un point de vue structural, nous avons mis en évidence la particularité de ce domaine qui, en plus des trois hélices caractéristiques des domaines UBA, présente une hélice supplémentaire (hélice H4). Nos résultats montrent que cette hélice H4 interfère avec le site de liaison à l'ubiquitine d'une part et est nécessaire à la reconnaissance des substrats spécifiques tel que d'Hprl d'autre part. Plus précisément, l'interaction entre le domaine UBA de Mex67 et Hpr1 via l'hélice H4 provoque un changement conformationnel qui permet de rétablir le site de liaison à l'ubiquitine coordonnant ainsi la reconnaissance simultanée de l'ubiquitine et de substrats spécifiques. In w'tfo, le domaine UBA de Mex67 est important pour le recrutement co-transcriptionnel du récepteur et joue un rôle crucial dans l'export des ARNm. Nous avons par ailleurs montré que l'interaction entre le domaine UBA de Mex67 et Hpr1 ubiquitiné facilite le recrutement du récepteur couplant ainsi export des ARNm et élongation de la transcription. De manière plus générale, nous proposons un modèle de travail selon lequel Mex67 pourrait participer à la coordination des machineries de biogenèse et d'export des ARNm grâce à la capacité de son domaine UBA à interagir spécifiquement et de façon dynamique avec des partenaires ubiquitinés impliqués dans différentes étapes de la biogenèse des ARNm
Concomitantly to their transcription, newly synthesised mRNAs undergo several processing steps leading to export competent ribonucleoprotein particles (mRNP). Fully mature mRNPs are recognised by the essential mRNA export receptor Mex67 in yeast, TAP in metazoans, which promotes their translocation through the nuclear pore complex. Recent studies have shown that the ubiquitin pathway is involved in the regulation of mRNA nuclear export. Interestingly, the essential mRNA export receptor Mex67/TAP harbours in its C-terminus a UBA (Ubiquitin associated) domain so far described to interact with nucleoporins. The aim of this thesis was to analyse the structural and functional properties of the UBA domain of Mex67 in order to better understand the role of ubiquitylation in mRNA export. Our studies first showed that the UBA of Mex67 is able to bind ubiquitin and led to the identification of distinct and ubiquitinylated spécifie UBA-Mex67 interacting proteins implicated in different steps of transcription, including Hpr1, a transcription elongation factor and Swd2 which participates in histone H3 methylation and 3' end processing of mRNAs. The determination by NMR of the structure of the UBA domain of Mex67 has revealed a classical UBA-fold consisting of three alpha helices. However, it differs from other studied UBA domains by the presence of an additional C-terminal helix (helix H4). We found that helix H4 interferes with the ability of the UBA domain to interact with ubiquitin. However, H4 is also essential for the interaction with specific substrates like Hpr1 and once engaged in such interaction, a conformational change occurs that unmasks the ubiquitin binding site and restores the ability of UBA-Mex67 to interact ubiquitin. Altogether, these results led us to propose that the additional fourth helix of the UBA domain of Mex67 acts as a molecular switch that restricts UBA-Mex67/ubiquitin interaction to specific targets. In vivo, we reported that the UBA domain of Mex67 is not only required for proper nuclear export but also contributes to early co-transcriptional recruitment of the receptor. We showed that the interaction between UBA of Mex67 and Hpr1 facilitates the recruitment of the receptor coupling by this way nuclear export to transcription elongation. More generally, we proposed a working hypothesis in which Mex67 could participate in the coordination of biogenesis and export machineries due to the dynamic interaction of its UBA domain with specific partners implicated in different steps of mRNAs biogenesis

博士
亞洲大學
生物資訊與醫學工程學系
104
Leukemiais the most common leading cause of childhood malignancies.A recently research in nature genetics indicates that the SETD2 gene is associated with acute lymphoblastic leukemia. This study aims to identify the potent lead compounds from traditional Chinese medicine using virtual screening for SETD2 proteinagainst acute lymphoblastic leukemia. We also employed the molecular dynamics (MD) simulation to discuss the stability of docking poses of SETD2 proteins complexes with top three TCM candidates and control. According to the result in docking and MD simulation, coniselin and coniferylferulate have high binding affinity and stable interactions with SETD2 protein. Coniselin is isolated from the alcoholic extract of ConiselinumvaginatumThell.Coniferylferulate can be isolated from Angelica sinensis, Poriacocos (Schw.) Wolf, and Notopterygiumforbesii. Although SAH have more stable interactions with key residues in the binding domain than coniselin and coniferylferulate during MD simulation,the TCM compounds, coniselin and coniferylferulate, are still thepotential candidates as lead compounds for further study in drug development process with the SETD2 protein against acute lymphoblastic leukemia. Keywords: Leukemia; Histone-lysine N-methyltransferase SETD2; traditional Chinese medicine (TCM); virtual Screening; molecular dynamics (MD)