Application of Celluspots peptide arrays for the analysis of the binding specificity of epigenetic reading domains to modified histone tails

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>We conclude that Celluspots peptide arrays are powerful screening tools for studying the specificity of putative reading domains binding to modified histone peptides.</p

Real‐Time SWMF at CCMC: Assessing the Dst Output From Continuous Operational Simulations

The ground‐based magnetometer index of Dst is a commonly used measure of near‐Earth current systems, in particular the storm time inner magnetospheric current systems. The ability of a large‐scale, physics‐based model to reproduce, or even predict, this index is therefore a tangible measure of the overall validity of the code for space weather research and space weather operational usage. Experimental real‐time simulations of the Space Weather Modeling Framework (SWMF) are conducted at the Community Coordinated Modeling Center (CCMC). Presently, two configurations of the SWMF are running in real time at CCMC, both focusing on the geospace modules, using the Block Adaptive Tree Solar wind‐type Roe Upwind Solver magnetohydrodynamic model, the Ridley Ionosphere Model, and with and without the Rice Convection Model. While both have been running for several years, nearly continuous results are available since April 2015. A 27‐month interval through July 2017 is used for a quantitative assessment of Dst from the model output compared against the Kyoto real‐time Dst. Quantitative measures are presented to assess the goodness of fit including contingency tables and a receiver operating characteristic curve. It is shown that the SWMF run with the inner magnetosphere model is much better at reproducing storm time values, with a correlation coefficient of 0.69, a prediction efficiency of 0.41, and Heidke skill score of 0.57 (for a −50‐nT threshold). A comparison of real‐time runs with and without the inner magnetospheric drift physics model reveals that nearly all of the storm time Dst signature is from current systems related to kinetic processes on closed magnetic field lines.Plain Language SummaryAs society becomes more dependent on technologies susceptible to adverse space weather, it is becoming increasingly critical to have numerical models capable of running in real time to nowcast/forecast the conditions in the near‐Earth space environment. One such model is available at the Community Coordinated Modeling Center and has been running for several years, allowing for an assessment of the quality of the result. Comparisons are made against globally compiled index of near‐Earth space storm activity, including numerous statistical quantities and tests. The skill of the model is remarkable, especially when a few hours after each of the cold restarts of the model are removed from the comparison. It is also shown that a global model alone is not that good at reproducing this storm index; a regional model for the inner part of geospace is necessary for good data‐model agreement.Key PointsThe SWMF model has been running in experimental real‐time mode at CCMC for several years, and all saved output is availableThe comparison against real‐time Dst is quite good, especially when a few hours after cold restarts are removed from the comparisonIt is necessary to include an inner magnetospheric drift physics model to reproduce Dst; a real‐time run without one does much worsePeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146631/1/swe20766.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146631/2/swe20766_am.pd

H3K14ac is linked to methylation of H3K9 by the triple Tudor domain of SETDB1

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 subnuclear 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

Versatile workflow for cell-type resolved transcriptional and epigenetic profiles from cryopreserved human lung

Complexity of lung microenvironment and changes in cellular composition during disease make it exceptionally hard to understand molecular mechanisms driving development of chronic lung diseases. Although recent advances in cell type–resolved approaches hold great promise for studying complex diseases, their implementation relies on local access to fresh tissue, as traditional tissue storage methods do not allow viable cell isolation. To overcome these hurdles, we developed a versatile workflow that allows storage of lung tissue with high viability, permits thorough sample quality check before cell isolation, and befits sequencing-based profiling. We demonstrate that cryopreservation enables isolation of multiple cell types from both healthy and diseased lungs. Basal cells from cryopreserved airways retain their differentiation ability, indicating that cellular identity is not altered by cryopreservation. Importantly, using RNA sequencing and EPIC Array, we show that gene expression and DNA methylation signatures are preserved upon cryopreservation, emphasizing the suitability of our workflow for omics profiling of lung cells. Moreover, we obtained high-quality single-cell RNA-sequencing data of cells from cryopreserved human lungs, demonstrating that cryopreservation empowers single-cell approaches. Overall, thanks to its simplicity, our workflow is well suited for prospective tissue collection by academic collaborators and biobanks, opening worldwide access to viable human tissue

High-resolution transcriptomic and epigenetic profiling identifies novel regulators of COPD

Patients with chronic obstructive pulmonary disease (COPD) are still waiting for curative treatments. Considering its environmental cause, we hypothesized that COPD will be associated with altered epigenetic signaling in lung cells. We generated genome-wide DNA methylation maps at single CpG resolution of primary human lung fibroblasts (HLFs) across COPD stages. We show that the epigenetic landscape is changed early in COPD, with DNA methylation changes occurring predominantly in regulatory regions. RNA sequencing of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair, and extracellular matrix organization. Data integration identified 110 candidate regulators of disease phenotypes that were linked to fibroblast repair processes using phenotypic screens. Our study provides high-resolution multi-omic maps of HLFs across COPD stages. We reveal novel transcriptomic and epigenetic signatures associated with COPD onset and progression and identify new candidate regulators involved in the pathogenesis of chronic lung diseases. The presence of various epigenetic factors among the candidates demonstrates that epigenetic regulation in COPD is an exciting research field that holds promise for novel therapeutic avenues for patients

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Untersuchung von Proteinen verantwortlich für die Entwicklung und Erkennung von wichtigen Lysin-Modifikationen

Histone post-translational modifications influence chromatin architecture, either by direct effects on the interaction between histones and DNA, or indirectly, by serving as docking places for regulatory proteins, which bind through conserved functional domains termed “reading” domains. Different combinations of histone modifications define various chromatin states, each of which being associated with a particular set of regulatory enzymes. Lysine methylation is an important histone post-translational modification, which can occur at various positions in histones, with different roles in epigenetic regulation. This mark is generally established by SET domain Protein Lysine Methyltransferases (PKMTs). Recently, PKMTs have been reported to also methylate numerous non-histone substrates, which subsequently recruit so called “reading” domains. These domains specifically interact with the methylated lysine in a sequence context-dependent manner. In this work, I tried to establish a Yeast-3-Hybrid method for the identification of methylation-dependent interactors of methylated non-histone proteins. For validation, I attempted to test the interaction between reported PKMT substrates fused to the Gal4-DNA-Binding Domain and methyl-“readers” fused to the Gal4-Activation Domain in yeast, either in the presence or absence of the corresponding PKMTs. Later in the project the known “reading” domains would be replaced by a library of human cDNA, in order to search for novel “readers” of protein lysine methylation marks. Additionally, this work presents the study of the substrate specificities of two SET domain methyltransferases responsible for the methylation of histone 3 lysine 4 (H3K4), which are mutually exclusive members of the same coactivator complex, the human COMPASS. In this study, SET1A, an H3K4 trimethylase, was shown to be active only as part of the core COMPASS complex. This PKMT proved to have a higher preference for some sequences other than histone 3, justifying a search for novel non-histone substrates. MLL2, a member of the mixed lineage leukemia (MLL) family, responsible for H3K4 monomethylation, revealed stimulation of activity when part of the core COMPASS complex, and showed some differences in the substrate specificity when acting alone, compared to the complex. The search for non-histone protein substrates is in progress for SET1A/COMPASS, and also MLL2 alone and within the complex. The targeting of most PKMTs is achieved with the help of histone modification “reading” or DNA-binding domains. The binding specificity of the PHD finger “reading” domains of MLL2, and its paralog MLL3, was investigated during this doctoral study. Although most of the PHD fingers did not bind to histone tails, the MLL2 PHD 3-5 group of domains and the MLL3 PHD 4-6 group of domains bound specifically to modified histone tail peptides. Preference towards both histone 3 (H3) and histone 4 (H4) was identified and the strongest binding was seen on H4 peptides containing acetylation at lysine 16 together with multiple acetylations or methylations. This finding suggested recruitment to active chromatin, which is enriched in acetylation marks, but the specificity needs to be further confirmed and characterized in more detail. I also investigated the histone binding specificity of PHF1, a member of the Polycomb Repressive Complex 2. This complex is responsible for developmental gene repression by the trimethylation of histone 3 lysine 27 (H3K27me3). The tudor domain of PHF1 showed preferred binding to its target, H3K27me3 in the sequence context of testis-identified histone variant H3T, in comparison to the canonical histone H3.1. The specificity for the same mark and histone variant was also identified for the chromodomain of the Polycomb Repressive Complex 1 member, CBX7, while the chromodomain of its paralog, CBX2, did not show discrimination between the histone variants, although it presented the same specificity towards the H3K27me3 mark. We propose that the discrimination between histone variants is a unique feature of some “reading” domains, and the role of this particular function needs to be elucidated. Moreover, the H3K27me3-specific CBX7 chromodomain was used as a tool in the validation of new methods developed by Kungulovski et al., 2014, with the purpose of replacing antibodies raised against specific histone modifications in adaptations of several antibody-based assays. Finally, this PhD work also presents the binding specificity of the chromodomain of the SUV39H1 methyltransferase. SUV39H1 is responsible for histone 3 lysine 9 trimethylation (H3K9me3), and the consequent gene repression and silencing of heterochromatin. I showed that the chromodomain of SUV39H1 bound specifically to H3K9me3, and binding of the chromodomain to its target peptide seemed to inhibit the catalytic activity of the enzyme in our in vitro conditions.Posttranslationale Modifikationen an Histonen beeinflussen die Chromatin Struktur. Dies erfolgt entweder direkt durch die Interaktion zwischen Histonen und DNA, oder indirekt, durch Bindung von regulatorischen Proteinen. Die Bindung dieser Proteine erfolgt durch konservierte funktionelle Domänen, genannt „reading“ Domänen. Verschiedene Kombinationen von Histonmodifikationen definieren eine Vielzahl von verschiedenen Chromatinzuständen, von denen jeder Zustand mit einem bestimmten Repertoire an regulatorischen Enzymen assoziiert ist. Lysin-Methylierung ist eine wichtige posttranslationale Modifikation von Histonen. Diese kann an verschiedene Stellen mit unterschiedlichen Funktionen in der epigenetischen Regulation auftreten. Diese Markierung wird generell durch Protein Lysin Methyltransferasen (PKMTs) mit einer SET Domäne eingeführt. Vor kurzem konnte gezeigt werden, dass PKMTs auch zahlreiche Nicht-Histon Substrate methylieren können, welche anschließend mit sogenannten „Lesedomänen“ spezifisch interagieren. Ein Ziel dieser Arbeit war es, eine Yeast-3-Hybrid Methode zu entwickeln, um damit methylierungsabhängige Interaktionspartner von Lysin methylierten Proteinen zu identifizieren. Zur Validierung dieser Methode wurde in Hefe die Wechselwirkung zwischen bekannten PKMT Substraten, welche an die Gal4-DNA-Bindungsdomäne fusioniert waren, und Methyl-„Lesedomänen“, welche an die Gal4-Aktivierungsdomäne fusioniert waren, werden in An- und Abwesenheit der PKMT untersucht. Später in diesen Project sollen die bekannten “Lesedomänen” durch eine Sammlung von humanen cDNAs ersetzt werden, um anschließend nach neuen “Lesedomänen” für Lysin methylierte Proteinen zu suchen. Zusätzlich, wurde in diese Arbeit die Substratspezifität zweier Methyltransferasen untersucht, die eine SET Domäne enthalten und Histon 3 Lysin 4 (H3K4) methylieren. Beide Methyltransferasen binden unabhängig voneinander an denselben Koaktivator Komplex, welcher COMPASS genannt wird. In dieser Arbeit konnte gezeigt werden, dass SET1A, eine H3K4 trimethyl-Transferase, nur im COMPASS Komplex aktiv ist. Es konnte allerdings gezeigt werden, dass diese PKMT einige Sequenzen bevorzugt methyliert, welche nicht in Histon 3 vorkommen, was eine Suche nach neuen Nicht-Histon Substraten nahelegt. MLL2, ein Mitglied der „mixed lineage leukemia“ (MLL) Familie, ist verantwortlich für H3K4 mono-Methylierung. Es wurden unterschiedliche Substratspezifitäten beobachtet, wenn MLL2 alleine oder als Teil des „COMPASS“ Komplexes eingesetzt wurde. Die Identifikation von Nicht- Histon Substraten ist sowohl für SET1A als auch MLL2 allein und innerhalb des Komplex in Arbeit. Das Targeting der meisten PKMTs wird mit Hilfe von Histon „Lesedomänen“ oder DNA-Bindungsdomänen ermöglicht. In dieser Arbeit wurde die Bindungsspezifität der PHD Finger „reading“ Domänen von MLL2 und seinem Paralog MLL3 untersucht. Obwohl die meisten PHD Finger nicht mit dem N-terminalen Ende der Histone interagieren, konnte dennoch gezeigt werden, dass die Domänen MLL2 PHD Gruppe 3-5 und MLL3 PHD 4-6 spezifisch an das modifizierten N-terminale Ende von Histonpeptiden binden. Eine Bindungspräferenz konnte sowohl für Histone 3 (H3) und Histone 4 (H4) identifiziert werden. Die stärkste Bindung konnte für das H4 Peptid gezeigt werden, welches am Lysin 16 eine Acetylierung zusammen mit weiteren Acetylierungen oder Methylierungen aufweist. Der allgemeine Trend zeigt eine Rekrutierung an aktives Chromatin, in welchem hauptsächlich Acetylierungsmarkierungen vorhanden sind. Die Spezifität sollte jedoch weiter untersucht und detaillierter charakterisiert werden. In dieser Arbeit wurde außerdem die Histon Bindungsspezifität von PHF1, einem Mitglied des „Polycom Repressiv Complex 2“, charakterisiert. Durch diesen Komplex erfolgt die tri-Methylierung von Lysin 27 an Histon 3 (H3K27me3), welche zu einer Genrepression führt. Die Tudor Domäne von PHF1 zeigt unterschiedliche Bindungsspezifitäten für H3K27me3, und bevorzugt die in Testis identifizierte Variante H3T im Vergleich zur kanonische Variante H3.1. Ähnliche Ergebnisse wurden für die Chromodomäne von CBX7, einem Mitglied des Polycomb Repressive Complex 1, erzielt. Das Paralog CBX2 weist allerdings keine Unterschiede in der H3K27me3 Bindung zwischen den Histon Varianten auf. Daraus kann gefolgert werden, dass die Unterschiede zwischen den Histon Varianten eine besondere Eigenschaft einiger „Lesedomänen“ darstellt und diese besondere Eigenschaft weitere Untersuchungen erfordert. Außerdem wurde die Chromodomäne von CBX7,welche spezifisch für H3K27me3 ist, als ein Werkzeug für die Validation einer neuen Methode verwendet. Diese Methode,entwickelt von Kungulovski et al., (2014), hat das Ziel, Antikörper zu ersetzen, welche gegen spezifische Histon Modifikationen eingesetzt werden. Schließlich wurde die Bindungsspezifität der Chromodomäne der SUV39H1 Methyltransferase untersucht. SUV39H1 ist verantwortlich für die Histon 3 tri-Methylierung am Lysin 9 (H3K9me3), woraus eine Genrepression und Stilllegung von Heterochromatin resultiert. Es konnte gezeigt werden, dass die Chromodomäne von SUV39H1 spezifisch mit H3K9me3 interagiert und dass die Bindung der Chromodomäne zu seinem Targetpeptid zu einer Inhibiton der katalytischen Aktivität des Enzyms unter den verwendeten in vitro Bedingungen führt