Mass spectrometry analysis of the variants of histone H3 and H4 of soybean and their post-translational modifications

Background

Functional genomics tools provide researchers with the ability to apply high-throughput techniques to determine the function and interaction of a diverse range of genes. Mutagenised plant populations are one such resource that facilitate gene characterisation. They allow complex physiological responses to be correlated with the expression of single genes in planta, through either reverse genetics where target genes are mutagenised to assay the affect, or through forward genetics where populations of mutant lines are screened to identify those whose phenotype diverges from wild type for a particular trait. One limitation of these types of populations is the prevalence of gene redundancy within plant genomes, which can mask the affect of individual genes. Activation or enhancer populations, which not only provide knock-out but also dominant activation mutations, can facilitate the study of such genes.

Results

We have developed a population of almost 50,000 activation tagged A. thaliana lines that have been archived as individual lines to the T₃ generation. The population is an excellent tool for both reverse and forward genetic screens and has been used successfully to identify a number of novel mutants. Insertion site sequences have been generated and mapped for 15,507 lines to enable further application of the population, while providing a clear distribution of T-DNA insertions across the genome. The population is being screened for a number of biochemical and developmental phenotypes, provisional data identifying novel alleles and genes controlling steps in proanthocyanidin biosynthesis and trichome development is presented.

Conclusion

This publicly available population provides an additional tool for plant researcher's to assist with determining gene function for the many as yet uncharacterised genes annotated within the Arabidopsis genome sequence http://aafc-aac.usask.ca/FST. The presence of enhancer elements on the inserted T-DNA molecule allows both knock-out and dominant activation phenotypes to be identified for traits of interest.     

Mass spectrometry analysis of the variants of histone H3 and H4 of soybean and their post-translational modifications

Background  

Histone modifications and histone variants are of importance in many biological processes. To understand the biological functions of the global dynamics of histone modifications and histone variants in higher plants, we elucidated the variants and post-translational modifications of histones in soybean, a legume plant with a much bigger genome than that of Arabidopsis thaliana.     

Aurora1 phosphorylation activity on histone H3 and its cross-talk with other post-translational histone modifications in Arabidopsis

The enzymological properties of AtAurora1, a kinase responsible for the cell cycle-dependent phosphorylation of histone H3 at S10, and its cross-talk with other post-translational histone modifications, were determined. In vitro phosphorylation of H3S10 by AtAurora1 is strongly increased by K9 acetylation, and decreased by K14 acetylation and T11 phosphorylation. However, S10 phosphorylation activity is unaltered by mono-, di- or trimethylation of K9. An interference of H3K9 dimethylation by SUVR4 occurs by a pre-existing phosphorylation at S10. Hence, cross-talk in plants exists between phosphorylation of H3S10 and methylation, acetylation or phosphorylation of neighbouring amino acid residues. AtAurora1 undergoes autophosphorylation in vivo regardless of the presence of substrate, and forms dimers in planta . Of the three ATP-competitive Aurora inhibitors tested, Hesperadin was most effective in reducing the in vivo kinase activity of AtAurora1. Hesperadin consistently inhibited histone H3S10 phosphorylation during mitosis in Arabidopsis cells, but did not affect other H3 post-translational modifications, suggesting a specific inhibition of AtAurora in vivo . Inactivation of AtAurora also caused lagging chromosomes in a number of anaphase cells, but, unlike the situation in mammalian cells, Hesperadin did not influence the microtubule dynamics in dividing cells.     

Characterization of post-translational modifications of histone H2B-variants isolated from Arabidopsis thaliana

Eukaryotic DNA is structurally packed into chromatin by the basic histone proteins H2A, H2B, H3, and H4. There is increasing evidence that incorporation and post-translational modifications of histone variants have a fundamental role in gene regulation. While modifications of H3 and H4 histones are now well-established, considerably less is known about H2B modifications. Here, we present the first detailed characterization of H2B-variants isolated from the model plant Arabidopsis thaliana. We combined reversed-phase chromatography with tandem mass spectrometry to identify post-translational modifications of the H2B-variants HTB1, HTB2, HTB4, HTB9, and HTB11, isolated from total chromatin and euchromatin-enriched fractions. The HTB9-variant has acetylation sites at lysines 6, 11, 27, 32, 38, and 39, while Lys-145 can be ubiquitinated. Analogous modifications and an additional methylation of Lys-3 were identified for HTB11. HTB2 shows similar acetylation and ubiquitination sites and an additional methylation at Lys-11. Furthermore, the N-terminal alanine residues of HTB9 and HTB11 were found to be mono-, di-, or trimethylated or unmodified. No methylation of arginine residues was detected. The data suggest that most of these modification sites are only partially occupied. Our study significantly expands the map of covalent Arabidopsis histone modifications and is the first step to unraveling the histone code in higher plants.     

Global mapping of post-translational modifications on histone H3 variants in mouse testes

Mass spectrometry (MS)-based characterization is important in proteomic research for verification of structural features and functional understanding of gene expression. Post-translational modifications (PTMs) such as methylation and acetylation have been reported to be associated with chromatin remodeling during spermatogenesis. Although antibody- and MS-based approaches have been applied for characterization of PTMs on H3 variants during spermatogenesis, variant-specific PTMs are still underexplored. We identified several lysine modifications in H3 variants, including testis-specific histone H3 (H3t), through their successful separation with MS-based strategy, based on differences in masses, retention times, and presence of immonium ions. Besides methylation and acetylation, we detected formylation as a novel PTM on H3 variants in mouse testes. These patterns were also observed in H3t. Our data provide high-throughput structural information about PTMs on H3 variants in mouse testes and show possible applications of this strategy in future proteomic studies on histone PTMs.     

Characterization of histone H2A and H2B variants and their post-translational modifications by mass spectrometry

The nucleosome, the fundamental structural unit of chromatin, contains an octamer of core histones H3, H4, H2A, and H2B. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function, analysis of histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2A and H2B variants derived from Jurkat cells. A combination of mass spectrometric techniques, HPLC separations, and enzymatic digestions using endoproteinase Glu-C, endoproteinase Arg-C, and trypsin were used to identify histone H2A and H2B subtypes and their modifications. We identified nine histone H2A and 11 histone H2B subtypes, among them proteins that only had been postulated at the gene level. The two main H2A variants, H2AO and H2AC, as well as H2AL were either acetylated at Lys-5 or phosphorylated at Ser-1. For the replacement histone H2AZ, acetylation at Lys-4 and Lys-7 was found. The main histone H2B variant, H2BA, was acetylated at Lys-12, -15, and -20. The analysis of core histone subtypes with their modifications provides a first step toward an understanding of the functional significance of the diversity of histone structures.     

Application of mass spectrometry to the identification and quantification of histone post-translational modifications

The core histones are the primary protein component of chromatin, which is responsible for the packaging of eukaryotic DNA. The NH(2)-terminal tail domains of the core histones are the sites of numerous post-translational modifications that have been shown to play an important role in the regulation of chromatin structure. In this study, we discuss the recent application of modern analytical techniques to the study of histone modifications. Through the use of mass spectrometry, a large number of new sites of histone modification have been identified, many of which reside outside of the NH(2)-terminal tail domains. In addition, techniques have been developed that allow mass spectrometry to be effective for the quantitation of histone post-translational modifications. Hence, the use of mass spectrometry promises to dramatically alter our view of histone post-translational modifications.     

Mass spectrometric analysis of histone posttranslational modifications

The uses of tandem and Fourier transform mass spectrometric methodologies for assignment of the posttranslational sites and occupancies of histones and their isoforms is described employing several illustrative examples. A comparison of information that can be obtained from intact protein sequencing and proteolytic digestion is presented.     

Enhanced top-down characterization of histone post-translational modifications

Post-translational modifications (PTMs) of core histones work synergistically to fine tune chromatin structure and function, generating a so-called histone code that can be interpreted by a variety of chromatin interacting proteins. We report a novel online two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) platform for high-throughput and sensitive characterization of histone PTMs at the intact protein level. The platform enables unambiguous identification of 708 histone isoforms from a single 2D LC-MS/MS analysis of 7.5 µg purified core histones. The throughput and sensitivity of comprehensive histone modification characterization is dramatically improved compared with more traditional platforms.     

Recent advances in mass spectrometry analysis of histone post-translational modifications: potential clinical impact of the PAT-H-MS approach

Histone post-translational modifications (hPTMs) contribute to the regulation of gene expression and increasing evidence links them to the development of various pathologies, highlighting their potential as biomarkers for prognostic, diagnostic and therapeutic applications. Mass spectrometry (MS) has emerged as a powerful analytical tool for hPTM analysis, which has also been applied to the analysis of epigenetic aberrations in diseases. However, the potential offered by the MS-based hPTM analysis of clinical samples for epigenetic biomarker discovery has been left largely unexploited. This article summarizes the contribution of MS-based approaches to clinical epigenetics, with a special focus on the PAThology tissue analysis of Histones by Mass Spectrometry (PAT-H-MS) approach – which represents the first application of MS-based hPTM analysis to formalin-fixed paraffin-embedded clinical samples – discussing its strengths and limitations, as well as possible implementations.     

Pan-cancer analysis of post-translational modifications reveals shared patterns of protein regulation

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Combined bottom-up and top-down mass spectrometry analyses of the pattern of post-translational modifications of Drosophila melanogaster linker histone H1

Linker histone H1 is a major chromatin component that binds internucleosomal DNA and mediates the folding of nucleosomes into a higher-order structure, namely the 30-nm chromatin fiber. Multiple post-translational modifications (PTMs) of core histones H2A, H2B, H3 and H4 have been identified and their important contribution to the regulation of chromatin structure and function is firmly established. In contrast, little is known about histone H1 modifications and their function. Here we address this question in Drosophila melanogaster, which, in contrast to most eukaryotic species, contains a single histone H1 variant, dH1. For this purpose, we combined bottom-up and top-down mass-spectrometry strategies. Our results indicated that dH1 is extensively modified by phosphorylation, methylation, acetylation and ubiquitination, with most PTMs falling in the N-terminal domain. Interestingly, several dH1 N-terminal modifications have also been reported in specific human and/or mouse H1 variants, suggesting that they have conserved functions. In this regard, we also provide evidence for the contribution of one of such conserved PTMs, dimethylation of K27, to heterochromatin organization during mitosis. Furthermore, our results also identified multiple dH1 isoforms carrying several phosphorylations and/or methylations, illustrating the high structural heterogeneity of dH1. In particular, we identified several non-CDK sites at the N-terminal domain that appear to be hierarchically phosphorylated. This study provides the most comprehensive PTM characterization of any histone H1 variant to date.     

Mapping post-translational modifications of the histone variant MacroH2A1 using tandem mass spectrometry

Post-translational histone modifications modulate chromatin-templated processes and therefore affect cellular proliferation, growth, and development. Although post-translational modifications on the core histones have been under intense investigation for several years, the modifications on variant histones are poorly understood. We used tandem mass spectrometry to identify covalent modifications on a histone H2A variant, macroH2A1.2. MacroH2A1.2 can be monoubiquitinated; however, the site of monoubiquitination has not been documented. In this study we used green fluorescent protein-tagged macroH2A1.2 to determine that Lys(115) is a site of ubiquitination. In addition, we found that this variant H2A is methylated on the epsilon amino group of lysine residues Lys(17), Lys(122), and Lys(238) and phosphorylated on Thr(128). Three of these modifications were also found to be present in the endogenous protein by mass spectrometric analysis. These results provide the first direct evidence that multiple post-translational modifications are imposed on macroH2A1.2, suggesting that, like canonical H2A, this variant H2A is subject to regulation by combinatorial use of covalent modifications.     

Delicate analysis of post-translational modifications on Dishevelled 3

The Wnt/β-catenin signaling pathway plays a critical role in multiple developmental events during embryogenesis and adult tissue homeostasis. Dishevelled (Dvl) is an important component of Wnt/β-catenin signaling pathway, although the modification, especially phosphorylation, seems closely related to the activity and stability of Dvl, the overall modification status of Dvl is still poorly understood. In this study, we focused on low abundant Dvl3, one of the three Dvl isoforms. Using affinity purification of different sources of Dvl3, followed by digestion with both trypsin and chymotrypsin, we systematically analyzed the overall modification status of Dvl3 with liquid chromatography coupled LTQ-Orbitrap. Altogether, we confidently identified Dvl3 with more than 50% sequence coverage, including 6 phosphorylation, 4 methylation, and 1 dimethylation sites. Most of the identified modification sites were novel and located in the linker region between different motifs. Subsequently, the validity of modified peptides was confirmed by synthetic modified peptides. Finally, SRM analysis was performed to verify and quantify the successive change of one dimethylation site on Dvl3 in vivo during Wnt signaling. Taken together, our study elucidated the existence of a novel post-translational modification on Dvl3 and provided a delicate experimental procedure for analysis of modification on low abundant proteins.     

Mapping protein post-translational modifications with mass spectrometry

Post-translational modifications of proteins control many biological processes, and examining their diversity is critical for understanding mechanisms of cell regulation. Mass spectrometry is a fundamental tool for detecting and mapping covalent modifications and quantifying their changes. Modern approaches have made large-scale experiments possible, screening complex mixtures of proteins for alterations in chemical modifications. By profiling protein chemistries, biologists can gain deeper insight into biological control. The aim of this review is introduce biologists to current strategies in mass spectrometry-based proteomics that are used to characterize protein post-translational modifications, noting strengths and shortcomings of various approaches.     

Tissue-specific expression and post-translational modification of histone H3 variants

Analyses of histone H3 from 10 rat tissues using a Middle Down proteomics platform revealed tissue-specific differences in their expression and global PTM abundance. ESI/FTMS with electron capture dissociation showed that, in general, these proteins were hypomodified in heart, liver and testes. H3.3 was hypermodified compared to H3.2 in some, but not all tissues. In addition, a novel rat testes-specific H3 protein was identified with this approach.     

The impact of histone post-translational modifications in neurodegenerative diseases

Every year, neurodegenerative disorders take more than 5000 lives in the US alone. Cures have not yet been found for many of the multitude of neuropathies. The majority of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Parkinson's disease (PD) cases have no known genetic basis. Thus, it is evident that contemporary genetic approaches have failed to explain the etiology or etiologies of ALS/FTD and PD. Recent investigations have explored the potential role of epigenetic mechanisms in disease development. Epigenetics comprises heritable changes in gene utilization that are not derived from changes in the genome. A main epigenetic mechanism involves the post-translational modification of histones. Increased knowledge of the epigenomic landscape of neurodegenerative diseases would not only further our understanding of the disease pathologies, but also lead to the development of treatments able to halt their progress. Here, we review recent advances on the association of histone post-translational modifications with ALS, FTD, PD and several ataxias.