Defining the chromatin compaction state of all histone post-translational modifications
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE125384 | GEO | 2019/01/22
REPOSITORIES: GEO
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Project description:Trypanosome histone N-terminal sequences are very divergent from the other eukaryotes, although they are still decorated by post-translational modifications (PTMs). Here, we used a highly robust workflow to analyze histone PTMs in the parasite Trypanosoma cruzi using mass spectrometry-based data-independent acquisition (DIA). We adapted the workflow for the analysis of the parasite’s histone sequences by modifying the software EpiProfile 2.0, improving peptide and PTM quantification accuracy. This workflow could now be applied to the study of 141 T. cruzi modified histone peptides, which we used to investigate the dynamics of histone PTMs along the metacyclogenesis and the life cycle of T. cruzi.
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Project description:How eukaryotic chromosomes are compacted during mitosis has been a leading question in cell biology since the nineteenth century. Non-histone proteins such as condensin complexes contribute to chromosome shaping, but appear not to be necessary for mitotic chromatin compaction. Histone modifications are known to affect chromatin structure. As histones undergo major changes in their post-translational modifications during mitotic entry, we speculated that the spectrum of cell-cycle-specific histone modifications might contribute to chromosome compaction during mitosis. To test this hypothesis, we isolated core histones from interphase and mitotic cells and reconstituted chromatin with them. We used mass spectrometry to show that key post-translational modifications remained intact during our isolation procedure. Light, atomic force and transmission electron microscopy analysis showed that chromatin assembled from mitotic histones has a much greater tendency to aggregate than chromatin assembled from interphase histones, even under low magnesium conditions where interphase chromatin remains as separate beads-on-a-string structures. These observations are consistent with the hypothesis that mitotic chromosome formation is a two-stage process with changes in the spectrum of histone post-translational modifications driving mitotic chromatin compaction, while the action of non-histone proteins such as condensin may then shape the condensed chromosomes into their classic mitotic morphology.
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Project description:Defining the chromatin compaction state of all histone post-translational modifications
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Project description:Recombinant antibodies to histone post-translational modifications (PTMs), with their essentially infinite renewability, could fundamentally eliminate a major source of low reproducibility in epigenetics research. Here, we report new recombinant antibodies to trimethylated Lys4 and Lys9, respectively, on histone H3. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications, including ChIP. These results demonstrate the feasibility of generating recombinant antibodies to a range of histone marks, which will accelerate epigenetics research. We characterized recombinant antibodies with native ChIP using HEK293 cells followed by deep sequencing.
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Project description:Post-translational modifications (PTMs) of proteins play critical roles in regulating many cellular events. Antibodies targeting site-specific PTMs are essential tools for detecting and enriching PTMs at sites of interest. However, fundamental difficulties in molecular recognition of both PTM and surrounding peptide sequence have hindered the efficient generation of highly specific anti-PTM antibodies. Furthermore, the widespread use of potentially inconsistent, non-renewable and molecularly undefined antibodies presents experimental challenges thought to contribute to the reproducibility crisis in biomedical research. In this study, we describe the structure-guided development of a platform that efficiently generates potent and selective recombinant antibodies to PTMs that are molecularly-defined and infinitely renewable. Our platform is built on our previous discovery of an unconventional binding mode of anti-PTM antibodies, antigen clasping, where two antigen binding sites cooperatively clasp a single antigen, creating extensive interactions with the antigen and leading to high selectivity and potency. We designed the platform that generates clasping antibodies with two distinct binding units, resulting in efficient generation of antibodies to a set of trimethylated histone H3 with high levels of specificity and affinity. Performance comparison in chromatin immunoprecipitation (ChIP), a common application in epigenomics, revealed that an anti-H3K27me3 clasping antibody exhibited superior specificity to a widely-used conventional antibody and captured symmetric and asymmetric nucleosomes in a less biased manner. We further generated clasping antibodies to phosphotyrosine antigens by using the same principle. These results suggest the broad applicability of our platform to generating high-performance clasping antibodies to diverse PTMs.
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FOXO transcription factors are an important, conserved family of regulators of cellular processes including metabolism, cell-cycle progression, apoptosis and stress resistance. They are required for the efficacy of several of the genetic interventions that modulate lifespan. FOXO activity is regulated by multiple post-translational modifications (PTMs) that affect its subcellular localization, half-life, DNA binding and transcriptional activity. Here, we show how a mathematical modelling approach can be used to simulate the effects, singly and in combination, of these PTMs. Our model is implemented using the Systems Biology Markup Language (SBML), generated by an ancillary program and simulated in a stochastic framework. The use of the ancillary program to generate the SBML is necessary because the possibility that many regulatory PTMs may be added, each independently of the others, means that a large number of chemically distinct forms of the FOXO molecule must be taken into account, and the program is used to generate them. Although the model does not yet include detailed representations of events upstream and downstream of FOXO, we show how it can qualitatively, and in some cases quantitatively, reproduce the known effects of certain treatments that induce various single and multiple PTMs, and allows for a complex spatiotemporal interplay of effects due to the activation of multiple PTM-inducing treatments. Thus, it provides an important framework to integrate current knowledge about the behaviour of FOXO. The approach should be generally applicable to other proteins experiencing multiple regulations.
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Project description:As histones undergo major changes in their post-translational modifications during mitotic entry, we speculated that the spectrum of cell cycle-specific histone modifications might contribute to chromosome compaction during mitosis. To test this hypothesis, we isolated core histones from interphase and mitotic cells and reconstituted chromatin with them. We used mass spectrometry to show that key post-translational modifications remained intact during our isolation procedure.
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Project description:Proteins of all living cells undergo a myriad of posttranslational modifications (PTMs) that are critical to multifarious life processes. In this study, we describe the first comprehensive multiple PTM-omics atlas in parallel with quantitative proteome analyses of two representative species of trypanosomes, Trypanosoma brucei and T. evansi. Approximately 40,000 modified sites and 150 histone marks were identified in each species. Proteins involved in the redox system were mainly regulated in T. brucei, while proteins associated with motility were predominantly modified in T. evansi. The establishment of a database of multiple PTMs in the two parasites provides us with insight into the biological mechanisms that underpin life processes in trypanosomes with huge different life cycles.
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Project description:Graham R. Smith & Daryl P. Shanley. Modelling the response of FOXO transcription factors to multiple post-translational modifications made by ageing-related signalling pathways. PLoS ONE 5, 6 (2010).
FOXO transcription factors are an important, conserved family of regulators of cellular processes including metabolism, cell-cycle progression, apoptosis and stress resistance. They are required for the efficacy of several of the genetic interventions that modulate lifespan. FOXO activity is regulated by multiple post-translational modifications (PTMs) that affect its subcellular localization, half-life, DNA binding and transcriptional activity. Here, we show how a mathematical modelling approach can be used to simulate the effects, singly and in combination, of these PTMs. Our model is implemented using the Systems Biology Markup Language (SBML), generated by an ancillary program and simulated in a stochastic framework. The use of the ancillary program to generate the SBML is necessary because the possibility that many regulatory PTMs may be added, each independently of the others, means that a large number of chemically distinct forms of the FOXO molecule must be taken into account, and the program is used to generate them. Although the model does not yet include detailed representations of events upstream and downstream of FOXO, we show how it can qualitatively, and in some cases quantitatively, reproduce the known effects of certain treatments that induce various single and multiple PTMs, and allows for a complex spatiotemporal interplay of effects due to the activation of multiple PTM-inducing treatments. Thus, it provides an important framework to integrate current knowledge about the behaviour of FOXO. The approach should be generally applicable to other proteins experiencing multiple regulations.
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