Methylation-specific Antibodies Research Articles

Protein Arginine Methylation Patterns in Plasma Small Extracellular Vesicles Are Altered in Patients with Early-Stage Pancreatic Ductal Adenocarcinoma.

Small extracellular vesicles (sEVs) contain lipids, proteins and nucleic acids, which often resemble their cells of origin. Therefore, plasma sEVs are considered valuable resources for cancer biomarker development. However, previous efforts have been largely focused on the level of proteins and miRNAs in plasma sEVs, and the post-translational modifications of sEV proteins, such as arginine methylation, have not been explored. Protein arginine methylation, a relatively stable post-translational modification, is a newly described molecular feature of PDAC. The present study examined arginine methylation patterns in plasma sEVs derived from patients with early-stage PDAC (n = 23) and matched controls. By utilizing the arginine methylation-specific antibodies for western blotting, we found that protein arginine methylation patterns in plasma sEVs are altered in patients with early-stage PDAC. Specifically, we observed a reduction in the level of symmetric dimethyl arginine (SDMA) in plasma sEV proteins derived from patients with early- and late-stage PDAC. Importantly, immunoprecipitation followed by proteomics analysis identified a number of arginine-methylated proteins exclusively present in plasma sEVs derived from patients with early-stage PDAC. These results indicate that arginine methylation patterns in plasma sEVs are potential indicators of PDAC, a new concept meriting further investigation.

DeepPRMS: advanced deep learning model to predict protein arginine methylation sites.

Protein methylation is a form of post-translational modifications of protein, which is crucial for various cellular processes, including transcription activity and DNA repair. Correctly predicting protein methylation sites is fundamental for research and drug discovery. Some experimental techniques, such as methyl-specific antibodies, chromatin immune precipitation and mass spectrometry, exist for predicting protein methylation sites, but these techniques are time-consuming and costly. The ability to predict methylation sites using in silico techniques may help researchers identify potential candidate sites for future examination and make it easier to carry out site-specific investigations and downstream characterizations. In this research, we proposed a novel deep learning-based predictor, named DeepPRMS, to identify protein methylation sites in primary sequences. The DeepPRMS utilizes the gated recurrent unit (GRU) and convolutional neural network (CNN) algorithms to extract the sequential and spatial information from the primary sequences. GRU is used to extract sequential information, while CNN is used for spatial information. We combined the latent representation of GRU and CNN models to have a better interaction among them. Based on the independent test data set, DeepPRMS obtained an accuracy of 85.32%, a specificity of 84.94%, Matthew's correlation coefficient of 0.71 and a sensitivity of 85.80%. The results indicate that DeepPRMS can predict protein methylation sites with high accuracy and outperform the state-of-the-art models. The DeepPRMS is expected to effectively guide future research experiments for identifying potential methylated protein sites. The web server is available at http://deepprms.nitsri.ac.in/.

PRMxAI: protein arginine methylation sites prediction based on amino acid spatial distribution using explainable artificial intelligence

BackgroundProtein methylation, a post-translational modification, is crucial in regulating various cellular functions. Arginine methylation is required to understand crucial biochemical activities and biological functions, like gene regulation, signal transduction, etc. However, some experimental methods, including Chip–Chip, mass spectrometry, and methylation-specific antibodies, exist for the prediction of methylated proteins. These experimental methods are expensive and tedious. As a result, computational methods based on machine learning play an efficient role in predicting arginine methylation sites.ResultsIn this research, a novel method called PRMxAI has been proposed to predict arginine methylation sites. The proposed PRMxAI extract sequence-based features, such as dipeptide composition, physicochemical properties, amino acid composition, and information theory-based features (Arimoto, Havrda-Charvat, Renyi, and Shannon entropy), to represent the protein sequences into numerical format. Various machine learning algorithms are implemented to select the better classifier, such as Decision trees, Naive Bayes, Random Forest, Support vector machines, and K-nearest neighbors. The random forest algorithm is selected as the underlying classifier for the PRMxAI model. The performance of PRMxAI is evaluated by employing 10-fold cross-validation, and it yields 87.17% and 90.40% accuracy on mono-methylarginine and di-methylarginine data sets, respectively. This research also examines the impact of various features on both data sets using explainable artificial intelligence.ConclusionsThe proposed PRMxAI shows the effectiveness of the features for predicting arginine methylation sites. Additionally, the SHapley Additive exPlanation method is used to interpret the predictive mechanism of the proposed model. The results indicate that the proposed PRMxAI model outperforms other state-of-the-art predictors.

Magnetic Immunosensor Coupled to Enzymatic Signal for Determination of Genomic DNA Methylation.

Aberrations of genomic DNA methylation have been confirmed to be involved in the evolution of human cancer and have thus gained the potential to be depicted as biomarkers for cancer diagnostics and prognostic predictions, which implicates an urgent need for detection of total genomic DNA methylation. In this work, we suggested an assay for the quantification of global DNA methylation, utilizing methylation specific antibody (5mC) modified magnetic beads (MBs) for immunorecognition and affinity enrichment. Subsequently, the captured DNA on the surface of MBs interacted with the glucose oxidase-conjugated DNA antibody whose catalytic reaction product was engaged in electrochemical detection of the overall level of DNA methylation on a PB-doped screen-printed electrode. With 15 pg of input DNA, which, to our best knowledge, is the lowest required amount of DNA without sodium bisulfite treatment or amplification, this test strategy was able to perceive as low as 5% methylation level within 70 min including the preparation of anti-5mC-MBs. We believe this detection technique offers a promising option to detect global DNA methylation in both academic and clinical scenarios.

Detection and Quantification of Histone Methyltransferase Activity In Vitro.

Histone methyltransferases (HMTs) catalyze the methylation of lysine and arginine residues in histone as well as nonhistone substrates. In vitro histone methyltransferase assays have been instrumental in identifying HMTs, and they continue to be invaluable tools for the study of these important enzymes, revealing novel substrates and modes of regulation.Here we describe a universal protocol to examine HMT activity in vitro that can be adapted to a range of HMTs, substrates, and experimental objectives. We provide protocols for the detection of activity based on incorporation of 3H-labeled methyl groups from S-adenosylmethionine (SAM), methylation-specific antibodies, and quantification of the reaction product S-adenosylhomocysteine (SAH).

Single-Nucleotide-Resolution Sequencing of N6-Methyldeoxyadenosine.

Use of methylation-specific antibodies with methylated-DNA-immunoprecipitation sequencing allows for the mapping of methylated DNA, such as N6-methyldeoxyadenosine (6mA). However, such mapping methods only detect methylated DNA at low resolution. Here, we describe 6mA Cross-linking Exonuclease sequencing (6mACE-seq), which utilizes 6mA-specific antibodies cross-linked to 6mA sites to protect 6mA-DNA fragments from subsequent exonuclease treatment. This allowed 6mACE-seq to map human-genome-wide 6mA at single-nucleotide resolution.

Identification of Rpl29 as a major substrate of the lysine methyltransferase Set7/9

Set7/9 (also known as Set7, Set9, Setd7, and Kmt7) is a lysine methyltransferase that catalyzes the methylation of multiple substrates, including histone H3 and non-histone proteins. Although not essential for normal development and physiology, Set7/9-mediated methylation events play important roles in regulating cellular pathways involved in various human diseases, making Set7/9 a promising therapeutic target. Multiple Set7/9 inhibitors have been developed, which exhibit varying degrees of potency and selectivity in vitro However, validation of these compounds in vivo has been hampered by the lack of a reliable cellular biomarker for Set7/9 activity. Here, we report the identification of Rpl29, a ribosomal protein abundantly expressed in all cell types, as a major substrate of Set7/9. We show that Rpl29 lysine 5 (Rpl29K5) is methylated exclusively by Set7/9 and can be demethylated by Lsd1 (also known as Kdm1a). Rpl29 is not a core component of the ribosome translational machinery and plays a regulatory role in translation efficiency. Our results indicate that Rpl29 methylation has no effect on global protein synthesis but affects Rpl29 subcellular localization. Using an Rpl29 methylation-specific antibody, we demonstrate that Rpl29K5 methylation is present ubiquitously and validate that (R)-PFI-2, a Set7/9 inhibitor, efficiently reduces Rpl29K5 methylation in cell lines. Thus, Rpl29 methylation can serve as a specific cellular biomarker for measuring Set7/9 activity.

Arginine methylation of the C-terminus RGG motif promotes TOP3B topoisomerase activity and stress granule localization.

DNA topoisomerase 3B (TOP3B) is unique among all mammalian topoisomerases for its dual activities that resolve both DNA and RNA topological entanglements to facilitate transcription and translation. However, the mechanism by which TOP3B activity is regulated is still elusive. Here, we have identified arginine methylation as an important post-translational modification (PTM) for TOP3B activity. Protein arginine methyltransferase (PRMT) 1, PRMT3 and PRMT6 all methylate TOP3B in vitro at its C-terminal arginine (R) and glycine (G)-rich motif. Site-directed mutagenesis analysis identified R833 and R835 as the major methylation sites. Using a methylation-specific antibody, we confirmed that TOP3B is methylated in cells and that mutation of R833 and R835 to lysine (K) significantly reduces TOP3B methylation. The methylation-deficient TOP3B (R833/835K) is less active in resolving negatively supercoiled DNA, which consequently lead to accumulation of co-transcriptionally formed R-loops in vitro and in cells. Additionally, the methylation-deficient TOP3B (R833/835K) shows reduced stress granule localization, indicating that methylation is critical for TOP3B function in translation regulation. Mechanistically, we found that R833/835 methylation is partially involved in the interaction of TOP3B with its auxiliary factor, the Tudor domain-containing protein 3 (TDRD3). Together, our findings provide the first evidence for the regulation of TOP3B activity by PTM.

Lysine Methylation of the Valosin-Containing Protein (VCP) Is Dispensable for Development and Survival of Mice.

Valosin-containing protein (VCP) is a homohexameric ATPase involved in a multitude cellular processes and it was recently shown that VCP is trimethylated at lysine 315 by the VCP lysine methyltransferase (VCPKMT). Here, we generated and validated a constitutive knockout mouse by targeting exon 1–4 of the Vcpkmt gene. We show that Vcpkmt is ubiquitously expressed in all tissues examined and confirm the sub-cellular localization to the cytoplasm. We show by (I) mass spectrometric analysis, (II) VCPKMT-mediated in vitro methylation of VCP in cell extracts and (III) immunostaining with a methylation specific antibody, that in Vcpkmt -/- mice the methylation of lysine 315 in VCP is completely abolished. In contrast, VCP is almost exclusively trimethylated in wild-type mice. Furthermore, we investigated the specificity of VCPKMT with in vitro methylation assays using as source of substrate protein extracts from Vcpkmt -/- mouse organs or three human Vcpkmt -/- cell lines. The results show that VCPKMT is a highly specific enzyme, and suggest that VCP is its sole substrate. The Vcpkmt -/- mice were viable, fertile and had no obvious pathological phenotype. Their body weight, life span and acute endurance capacity were comparable to wild-type controls. Overall the results show that VCPKMT is an enzyme required for methylation of K315 of VCP in vivo, but VCPKMT is not essential for development or survival under unstressed conditions.

Abstract 5156: The oncogenic polycomb histone methyltransferase EZH2 methylates lysine 120 on histone H2B and competes ubiquitination in human cancer

Abstract The histone methyltransferase EZH2 is known to be a polycomb protein homologus to Drosophila enhancer of zeste and catalyzes the addition of methyl groups to histone H3 at lysine 27 (H3K27). We previously reported that EZH2 was overexpressed in various types of cancer and plays a crucial role in the cell cycle regulation of cancer cells. In this study, we demonstrated that EZH2 has the function to mono-methylated lysine 120 on histone H2B (H2BK120). EZH2-dependent H2BK120 methylation in cancer cells was confirmed with an H2BK120 methylation-specific antibody. Overexpression of EZH2 significantly attenuated the ubiquitination of H2BK120, a key post-translational modification of histones for transcriptional regulation. Concordantly, knockdown of EZH2 increased the ubiquitination level of H2BK120, suggesting that the methylation of H2BK120 by EZH2 may competitively inhibit the ubiquitination of H2BK120. Subsequent ChIP-Seq and microarray analyses identified downstream candidate genes regulated by EZH2 through the methylation of H2BK120. This is the first report to describe a novel substrate of EZH2, H2BK120, unveiling a new aspect of EZH2 functions in human carcinogenesis. Citation Format: Ryuji Hamamoto, Yusuke Nakamura. The oncogenic polycomb histone methyltransferase EZH2 methylates lysine 120 on histone H2B and competes ubiquitination in human cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5156. doi:10.1158/1538-7445.AM2014-5156

The Oncogenic Polycomb Histone Methyltransferase EZH2Methylates Lysine 120 on Histone H2B and Competes Ubiquitination

The histone methyltransferase enhancer of zeste 2 (EZH2) is known to be a polycomb protein homologous to Drosophila enhancer of zeste and catalyzes the addition of methyl groups to histone H3 at lysine 27 (H3K27). We previously reported that EZH2 was overexpressed in various types of cancer and plays a crucial role in the cell cycle regulation of cancer cells. In the present study, we demonstrated that EZH2 has the function to monomethylate lysine 120 on histone H2B (H2BK120). EZH2-dependent H2BK120 methylation in cancer cells was confirmed with an H2BK120 methylation-specific antibody. Overexpression of EZH2 significantly attenuated the ubiquitination of H2BK120, a key posttranslational modification of histones for transcriptional regulation. Concordantly, knockdown of EZH2 increased the ubiquitination level of H2BK120, suggesting that the methylation of H2BK120 by EZH2 may competitively inhibit the ubiquitination of H2BK120. Subsequent chromatin immunoprecipitation- Seq and microarray analyses identified downstream candidate genes regulated by EZH2 through the methylation of H2BK120. This is the first report to describe a novel substrate of EZH2, H2BK120, unveiling a new aspect of EZH2 functions in human carcinogenesis.

Non‐radioactive Protein Lysine Methyltransferase Microplate Assay Based on Reading Domains

New protein lysine methyltransferase (PKMT) assays are needed to facilitate screening for improved PKMT inhibitors, because PKMTs are mutated or overexpressed in several cancers. In cells, methylated lysine residues are recognized by reading domains such as the chromodomain of HP1β, which bind to target proteins in a lysine-methylation-specific manner. Herein we describe a sensitive, robust, and non-radioactive high-throughput PKMT assay that employs the HP1β chromodomain to detect the methylation of peptide substrates by the human SUV39H1 and SUV39H2 PKMTs. The assay has a very good dynamic range and high signal-to-noise ratio. It can be used to screen for PKMT inhibitors, as illustrated by analyzing the inhibition of SUV39H1 by chaetocin. The IC50 value of this inhibition was found to be 480 nM, which is close to its published value. Our data indicate that natural reading domains can be used as alternates to methyl-specific antibodies in PKMT assays. Reading domains can be produced recombinantly in E. coli at low cost and consistent quality, and they are accessible to protein design.

Methylation by Set9 modulates FoxO3 stability and transcriptional activity

The FoxO family of transcription factors plays an important role in longevity and tumor suppression by regulating the expression of a wide range of target genes. FoxO3 has recently been found to be associated with extreme longevity in humans and to regulate the homeostasis of adult stem cell pools in mammals, which may contribute to longevity. The activity of FoxO3 is controlled by a variety of post-translational modifications that have been proposed to form a 'code' affecting FoxO3 subcellular localization, DNA binding ability, protein-protein interactions and protein stability. Lysine methylation is a crucial post-translational modification on histones that regulates chromatin accessibility and is a key part of the 'histone code'. However, whether lysine methylation plays a role in modulating FoxO3 activity has never been examined. Here we show that the methyltransferase Set9 directly methylates FoxO3 in vitro and in cells. Using a combination of tandem mass spectrometry and methyl-specific antibodies, we find that Set9 methylates FoxO3 at a single residue, lysine 271, a site previously known to be deacetylated by Sirt1. Methylation of FoxO3 by Set9 decreases FoxO3 protein stability, while moderately increasing FoxO3 transcriptional activity. The modulation of FoxO3 stability and activity by methylation may be critical for fine-tuning cellular responses to stress stimuli, which may in turn affect FoxO3's ability to promote tumor suppression and longevity.

M-Track: detecting short-lived protein-protein interactions in vivo

We developed a protein-proximity assay in yeast based on fusing a histone lysine methyltransferase onto a bait and its substrate onto a prey. Upon binding, the prey is stably methylated and detected by methylation-specific antibodies. We applied this approach to detect varying interaction affinities among proteins in a mitogen-activated protein kinase pathway and to detect short-lived interactions between protein phosphatase 2A and its substrates that have so far escaped direct detection.

Histone H4 Lysine 20 of Saccharomyces cerevisiae Is Monomethylated and Functions in Subtelomeric Silencing

Histones undergo post-translational modifications that are linked to important biological processes. Previous studies have indicated that lysine methylation correlating with closed or repressive chromatin is absent in the budding yeast Saccharomyces cerevisiae, including at H4 lysine 20 (K20). Here we provide functional evidence for H4 K20 monomethylation (K20me1) in budding yeast. H4 K20me1 is detectable on endogenous H4 by western analysis using methyl-specific antibodies, and the signal is abrogated by H4 K20 substitutions and by competition with H4 K20me1 peptides. Using chromatin immunoprecipitation, we show that H4 K20me1 levels are highest at heterochromatic locations, including subtelomeres, the silent mating type locus, and rDNA repeats, and lowest at centromeres within euchromatin. Further, an H4 K20A substitution strongly reduced heterochromatic reporter silencing at telomeres and the silent mating type locus and led to an increase in subtelomeric endogenous gene expression. The correlation between the location of H4 K20me1 and the effect of the H4 K20A substitution suggests that this modification plays a repressive function. Our findings reveal the first negative regulatory histone methylation in budding yeast and indicate that H4 K20me1 is evolutionarily conserved from simple to complex eukaryotes.

Abstract LB-172: Role of post-translational modification of the RUNX2 transcription factor in osteosarcoma

Abstract Background-Several transcription factors or co-factors have recently been reported to be methylated at certain arginine residues, a post-translational modification mediated by two classes of protein arginine methyltransferases (PRMT). Moreover, some PRMTs have been shown to be key regulators of mesenchymal differentiation, and one of the PRMTs is involved in the epigenetic programming of early embryo development. RUNX2 is major transcription factor in osteoblast differentiation but little is known about its activity or function in osteosarcoma (OS). RUNX1, another member of RUNT family, is known to methylated at R206/R210, within the Runt domain, a region that has considerable homology with RUNX2. Here, we examined the possible role of PRMTs in OS in relation to the post-translational modification of RUNX2 and its possible impact on cell proliferation and differentiation. Method-We generated arginine methylation-specific antibodies to RUNX2. The specificity of this antibody was confirmed by detecting immunoprecipitated RUNX2 from Saos2 cells by Western blot. We also confirmed the expression of RUNX2 and arginine methylated RUNX2 in Saos2 cell by immunofluorescence. We also examined the frequency of arginine methylation of RUNX2 in OS patient samples, compared to total RUNX2, by Western blot. Finally, we examined the influence of arginine methylation on the transcriptional activity of RUNX2 by luciferase assay using osteocalcin promoter plasmid. Results-We found that PRMTs can methylate the arginine residue in the Runt domain of RUNX2. This arginine methylation of RUNX2 alters its transcriptional activity at the osteocalcin promoter. Immunofluorescence with arginine methylation-specific antibody showed weak nuclear positivity in the SAOS-2 cell line. Patient tumor samples also show the expression of arginine methylated RUNX2, as well as total RUNX2. Conclusion- Our results confirm that arginine methylation of RUNX2 affects its function as a transcription factor. Further study of the possible role of arginine methylation of RUNX2 in regulating its effect on osteosarcoma proliferation and differentiation could be of translational interest, given the therapeutic potential of PRMT inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-172. doi:10.1158/1538-7445.AM2011-LB-172

Autocatalytic differentiation of epigenetic modifications within the Arabidopsis genome

In diverse eukaryotes, constitutively silent sequences, such as transposons and repeats, are marked by methylation at histone H3 lysine 9 (H3K9me). Although selective H3K9me is critical for maintaining genome integrity, mechanisms to exclude H3K9me from active genes remain largely unexplored. Here, we show in Arabidopsis that the exclusion depends on a histone demethylase gene, IBM1 (increase in BONSAI methylation). Loss-of-function ibm1 mutation results in ectopic H3K9me and non-CG methylation in thousands of genes. The ibm1-induced genic H3K9me depends on both histone methylase KYP/SUVH4 and DNA methylase CMT3, suggesting interdependence of two epigenetic marks--H3K9me and non-CG methylation. Notably, IBM1 enhances loss of H3K9me in transcriptionally de-repressed sequences. Furthermore, disruption of transcription in genes induces ectopic non-CG methylation, which mimics the loss of IBM1 function. We propose that active chromatin is stabilized by an autocatalytic loop of transcription and H3K9 demethylation. This process counteracts a similarly autocatalytic accumulation of silent epigenetic marks, H3K9me and non-CG methylation.

Characterization of HIV Tat modifications using novel methyl-lysine-specific antibodies

Modification-specific antibodies are important tools to examine the dynamics and functions of posttranslational protein modifications in cells. Here, we describe in detail the generation of polyclonal antibodies specific for mono-, di-, and trimethylated lysine 51 within the HIV transactivator Tat. Lysine 51 is a highly conserved residue located in the RNA-binding region of Tat and the target of lysine methyltransferases KMT1E (SETDB1) and KMT7 (Set7/9). Using affinity-purified methyl-specific antibodies of Tat, we find that cellular Tat is predominantly monomethylated at lysine 51, a modification enhanced by coexpression of KMT7.

Chemical Approaches for Studying Histone Modifications

Histones form the protein core around which genomic DNA is wrapped in eukaryotic chromatin. Numerous genetic studies have established that the structure and transcriptional state of chromatin are closely related to histone post-translational modifications. Further elucidation of the precise mechanistic roles for individual histone modifications requires the ability to isolate and study homogeneously modified histones. However, the highly heterogeneous nature of histone modifications in vivo poses a significant challenge for such studies. Chemical tools that have enabled biochemical and biophysical studies of site-specifically modified histones are the focus of this minireview.

The Retinoblastoma Binding Protein RBP2 Is an H3K4 Demethylase

Changes in histone methylation status regulate chromatin structure and DNA-dependent processes such as transcription. Recent studies indicate that, analogous to other histone modifications, histone methylation is reversible. Retinoblastoma binding protein 2 (RBP2), a nuclear protein implicated in the regulation of transcription and differentiation by the retinoblastoma tumor suppressor protein, contains a JmjC domain recently defined as a histone demethylase signature motif. Here we report that RBP2 is a demethylase that specifically catalyzes demethylation on H3K4, whose methylation is normally associated with transcriptionally active genes. RBP2-/- mouse cells displayed enhanced transcription of certain cytokine genes, which, in the case of SDF1, was associated with increased H3K4 trimethylation. Furthermore, RBP2 specifically demethylated H3K4 in biochemical and cell-based assays. These studies provide mechanistic insights into transcriptional regulation by RBP2 and provide the first example of a mammalian enzyme capable of erasing trimethylated H3K4.