Mapiranje površinski izloženih cisteina u ATP-azi ISWI

In an eukaryotic cell, DNA molecule exists in the form of chromatin. The repetitive unit of chromatin is a nucleosome, 147 bp of DNA wrapped around histone octamer. Nucleosome position along DNA is important for transcription regulation. Chromatin remodeler ISWI (Drosophila melanogaster) slides nucleosomes along DNA. The aim of this work is to map surface-exposed cysteines in the protein ISWI. The surface-exposed cysteines are easily avaliable for chemical reaction. To probe accessibility of cysteines for chemical reaction, commonly used reagents for spectrophotometric detection of thiols were used: 5,5ʹ-dithiobis- (2-nitrobenzoic acid) and 2-nitro-5-thiocyanatobenzoic acid. To unequivocally identify surfaceexposed cysteines, ISWI was treated with primary alkylating reagent (N-ethylmaleimide), denaturated and treated with secondary alkylating reagent (iodoacetic acid). The two reactions add to the cysteine different modification groups which can be discriminated by mass spectrometry. Half of the total cysteines could be clasified either as buried or surface-exposed in the protein structure.U eukariotskoj stanici, molekula DNA nalazi se u obliku kromatina. Ponavljajuća jedinica kromatina je nukleosom, 147 parova baza DNA namotano oko histonskog oktamera. Položaj nukleosoma na DNA ima važnu ulogu u kontroli transkripcije. Remodelirajući protein ISWI (Drosophila melanogaster) pomiče nukleosome duž DNA. Cilj ovog rada je mapirati površinski izložene cisteine proteina ISWI. Rezultati će kasnije biti iskorišteni za analizu postojećih strukturnih modela proteina ISWI. Cisteini koji se nalaze na površini proteina, brže podliježu kemijskim reakcijama. Kako bi se potvrdilo da cisteini reagiraju različitom kinetikom, korišteni su reagensi za spektrofotometrijsko utvrđivanje tiola: 5,5ʹ-ditiobis-(2- nitrobenzojeva kiselina) i 2-nitro-5-tiocianatobenzojeva kiselina. Za točnu identifikaciju površinski izloženih cisteina, ISWI je tretiran s primarnim alkilirajućim reagensom (Netilmaleimidom), denaturiran i tretiran sa sekundarnim alkilirajućim reagensom (jodoctenom kiselinom). Produkt tih dviju reakcija je dodatak različitih modifikacijskih skupina na cisteine, koje se potom mogu razlučiti spektrometrijom masa. Polovica od ukupnih cisteina mogla se klasificirati kao površinski izloženi ili zaklonjeni unutar strukture proteina

A CDK-regulated chromatin segregase promoting chromosome replication

The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood. Here, we report the characterization of a chromatin remodeling enzyme-Yta7-entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+ -ATPase that assembles into \~1 MDa hexameric complexes capable of segregating histones from DNA. The Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that the enzymatic activity of Yta7 is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication. Our results present a mechanism for how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase

Remodeling of higher order chromatin structures

In fast allen Eukaryoten ist die DNA in einem dynamischen Polymer namens Chromatin organisiert. Das Nukleosom, der elementare Baustein des Chromatins, besteht aus einem Histonoktamer, um das 146 Basenpaare der DNA gewickelt werden. Zwischen den Nukleosomen befindet sich ein Stück freie DNA, die sogenannte Linker-DNA. Die Nukleosomen sind meist auf der DNA in sehr regelmäßigen Abstand angeordnet. Dieser regelmäßige Abstand ist wichtig, um kryptische Transkription zu verhindern und das Genom vor Doppelstrangbrüchen zu schützen. Die Position der Nukleosomen auf der DNA wird durch ATP-abhängige Nukleosomen-Remodeling-Enzyme beeinflusst. Diese Enzyme können die Nukleosomen von der DNA entfernen, sie auf der DNA zusammenbauen, umstrukturieren und entlang der DNA verschieben. Chromatin faltet sich sowohl in vitro als auch in vivo zu Strukturen höherer Ordnung. Nukleosomen-Arrays unterliegen auch einer Phasentrennung und bilden dadurch dichte Chromatin-kondensate. Die Chromatinfaltung und die Phasentrennung stellen eine Herausforderung für Nukleosomen-Remodeling-Enzyme dar, da sie das Chromatin binden und darauf einwirken können müssen. In dieser Arbeit habe ich die Nukleosom Verschiebereaktion von Remodeling-Enzymen in unterschiedlich gefalteten Chromatinsubstraten charakterisiert. Im ersten Kapitel verwendete ich die Spalthefe als Modellsystem, um das nucleosome sliding im Euchromatin und im kompakteren Heterochromatin zu vergleichen. Dafür überexprimierte ich Nukleosomen-Remodeling-Enzyme, deren Targeting zu Heterochromatin durch die Fusion mit einer Heterochromatin-bindenden Domäne erreicht wurde. Allerdings stellte sich heraus, dass die Überexpression des Remodelers Hrp3 für S. pombe toxisch war, unabhängig vom Targeting. Hrp3 Überexpression unterdrückte die Expression eines in Heterochromatin platzierten Reportergens und verursachte Defekte bei der Positionierung von Nukleosomen an den Genkörpern. Obwohl die Informationen, die durch Short-Read-Sequenzierung für Heterochromatin-Regionen erhalten wurden, spärlich waren, ließ sich eine ATP –Hydrolyse-abhängige Zunahme der Regelmäßigkeit der Nukleosomenpositionierung über subtelomerischen Regionen feststellen. Darüber hinaus führten wir erfolgreich eine gene-by-gene Analyse durch, um die Regelmäßigkeit und Wiederholungslängen, die sog. Nucleosome Repeat Length (NRL) von Nukleosomen-Arrays in Wildtyp- und Remodeler-Deletionsstämmen zu messen. Die häufigste NRL beträgt 150 bp; sie ist damit um ein paar Basenpaare noch geringer als bislang angenommen. Im zweiten Kapitel testete ich in vitro, ob die Chromatinfaltung und die Phasentrennung das nucleosome sliding behindern. Diese Studie wurde mit der D. melanogaster ATPase ISWI durchgeführt, die Nukleosomen verschieben kann. Nach der Rekonstitution von Nukleosomen-Arrays induzierte ich die intramolekulare Faltung und Phasentrennung durch Zugabe unterschiedlicher Salzmengen. Die gebildeten Chromatinkondensate enthielten Nukleosomenkonzentrationen wie sie auch im Zellkern zu finden sind. Erstaunlicherweise blieben die Kondensate für sehr voluminöse Komplexe zugänglich, was sie zu einem nützlichen Modellsubstrat macht, um die Herausforderungen zu untersuchen, denen Remodeler in einer dichten Chromatinumge-bung begegnen. ISWI reicherte sich in Chromatinkondensaten an und verlangsamte die Fusion der Kondensate in einer konzentrationsabhängigen Weise. Mit Hilfe eines neuartigen, bildgebenden nucleosome sliding Assays konnten wir die Remodeling-Raten innerhalb und außerhalb von Chromatinkondensaten vergleichen. Wir konnten bestätigen, dass das nucleosome sliding innerhalb von Chromatinkondensaten stattfindet. Die Anfangsgeschwindigkeit für das nucleosome sliding innerhalb der Kondensate war nur um das Zweifache niedriger als in Lösung. Zusammenfassend stellen die Kondensate keine starke Barriere für nucleosome sliding dar. Um die viskoelastischen Eigenschaften von Chromatinkondensaten zu charakterisieren, setzten wir optische Pinzetten ein, um die Kondensate kontrolliert fusionieren zu lassen. Der Verlust der ATP-Hydrolyse führte zu einer Verhärtung der Chromatinkondensate und einer verringerten Dynamik von ISWI. Wir erklären unsere Ergebnisse mit Hilfe eines monkey-bar Modells, in dem die beiden DNA-Bindungsdomänen von ISWI zwischen starken und schwachen Bindungsmodi wechseln. So stellt ISWI sicher, dass es auch im Zellkern, wo die hohe Nukleosomenkonzentration die Dissoziationskonstanten deutlich übersteigt, mobil bleibt. Unsere Ergebnisse deuten darauf hin, dass Pathologie-assoziierte Phänotypen auch zum Teil durch Veränderungen der Chromatindynamik und nicht ausschließlich durch eine Störung der kanonischen Remodeling-Funktionen verursacht werden könnten. Im dritten Kapitel untersuchte ich die Wechselwirkung zwischen ISWI und dem acidic patch, der für seine Aktivierung wichtig ist. ISWI durchläuft während der Katalyse globale Konformationsänderungen, was die Strukturanalyse schwierig macht. Ich verwendete Mononukleosomen mit einem UV-aktivierten Crosslinker, der in acidic patch Nähe angebracht war, mit dem Ziel ISWI in einer seltenen Konformation anzureichern. In dieser vorläufigen Studie zeigen wir, dass die Affinität von ISWI zum acidic patch mit der Länge der Linker-DNA und im ADPBeFx-gebundenen Zustand zunimmt. Die im Rahmen dieser Dissertation entwickelten Assays und diskutierten Konzepte könnten in Zukunft dazu dienen, neue Wege für Therapeutika eröffnen.In almost all eukaryotes, the DNA is organized in a dynamic polymer called chromatin. The nucleosome, the smallest unit a building monomer of chromatin, is formed by wrapping 146 bp of DNA around an octamer composed of histone proteins. Nucleosomes are interspaced with a piece of free DNA, called linker DNA. Nucleosomes tend to be evenly spaced, and this regular spacing is important for preventing cryptic transcription and protecting the genome from double-strand breaks. Nucleosome positions on DNA are influenced by ATP-dependent chromatin remodeling complexes. These remodelers can evict or assemble nucleosomes, incorporate his-tone variants and slide nucleosomes along DNA. Chromatin can fold into higher order structures, both in vitro and in vivo. Nucleosome arrays also undergo phase separation and form chromatin condensates. Chromatin folding and phase separation put challenges on nucleosome remodelers that must act on it. In this thesis, I characterized nucleosome sliding in differently folded chromatin substrates. In the first chapter, I used fission yeast as a model system to compare nucleosome sliding in euchromatin and the generally more compact heterochromatin using overexpression and heterochromatin targeting approaches. Targeting was achieved by fusion of chromatin remodelers with a heterochromatin-binding domain. Overexpression of the remodeler Hrp3 was toxic to fission yeast, independent of targeting. Hrp3 overexpression derepressed expression of a reporter gene placed into heterochromatin and caused defects in nucleosome positioning over gene bodies. Although the information obtained by short read sequencing for heterochromatin regions was sparse, we have identified an ATP-dependent increase in a regularity over subtelomeric regions. Moreover, we have successfully performed a gene-by-gene analysis to measure the regularity and repeat lengths of nucleosome arrays in wild type and remodeler-deletion strains. The most prevalent NRL turned out to be 150 bp, even tighter than published before. In the second chapter, I tested if nucleosome array folding and phase separation impede nucleosome sliding in vitro. This study was performed with D. melanogaster ATPase ISWI, that slides nucleosomes on its own. I have reconstituted nucleosome arrays and induced intramolecular folding and phase separation by addition of varying amounts of salt. The chromatin condensates that formed contained nucleosome concentrations in the same range as the nucleus and were accessible to large complexes, making them a useful model substrate to study challenges encountered by remodelers in a crowded chromatin environment. ISWI was enriched inside chromatin condensates and it slowed down condensate fusion in a concentration-dependent manner. We have developed a novel, imaging-based nucleosome sliding assay, which allowed us to compare remodeling rates in- and outside of chromatin condensates. We confirmed that nucleosome sliding takes place inside chromatin condensates. The initial velocity for nucleosome sliding inside the condensates was only two-fold lower than in solution. Taken together, ISWI slides nucleosomes inside chromatin condensates and condensates do not pose a strong barrier for sliding. To characterize viscoelastic properties of chromatin condensates, we employed optical tweezers to fuse them in a controlled manner. Loss of ATP hydrolysis led to hardening of chromatin condensates and decreased dynamics of the remodeler. We rationalize our results with the help of a ‘monkey-bar’ model in which ISWI’s two DNA binding domains cycle between strong and weak binding modes, thereby ensuring mobility through the nucleus, where the high nucleosomes concentration well exceeds the dissociation constants. Our findings suggest that pathologies-associated phenotypes might be caused in part by changes in chromatin dynamics, and not exclusively by disruption of canonical remodeler functions. In the third chapter, I investigated the interaction of ISWI and the acidic patch, which is important for its activation. Nucleosome remodelers are going through global conformational changes during nucleosome sliding, making structural analysis challenging. I used mononuclesomes with a UV-activating crosslinker close to the acidic patch that will covalently bind molecules nearby. In this preliminary study, we show that the affinity of ISWI towards the acidic patch increases with linker DNA length and in ADPBeFx bound state. Developed assays and discussed concepts in this dissertation might open new avenues for therapeutics

Structural characterization of histone binding proteins

Histoni su osnovna proteinska komponenta kromatina. Histonski šaperon Nap1, zajedno s karioferinom Kap114, transportira H2A-H2B iz citoplazme u jezgru. Djelovanje metiltransferaze Nop1, u kompleksu s proteinom Nop56, ključno je za nastanak metilacije H2AQ105, prve epigenetičke oznake posvećene specifičnoj RNA-polimerazi. Cilj ovog rada bio je strukturno karakterizirati komplekse histona s tim histon-vezujućim proteinima. Korištene su standardne metode molekularnog kloniranja. Proteini su pročišćavani afinitetnom kromatografijom, ionizmjenjivačkom kromatografijom i kromatografijom isključenjem. Proteini su analizirani SDS-poliakrilamidnom gel-elektroforezom i masenom spektrometrijom. Pokazali smo da Nap1 (H. sapiens) tvori kompleks s dimerom H2A-H2B (X. laevis). Terminalne domene Nap1 nisu nužne za nastanak kompleksa. Dijelovi kompleksa koji su važni za njegovu stabilnost, zaštićeni su od djelovanja proteaze. Potvrđene su interakcije Nap1-H2A-H2B s Kap114 (S. pombe). Kompleksi Nap1-H2A-H2B i Nap1-H2A-H2B-Kap114 su pročišćeni i postavljene su pretkristalizacijske probe na kristalizacijskom robotu. Kompleks Nop1-Nop56 (K. lactis) dobiven je in vitro. N-terminalna domena Nop1 nije potrebna za interakciju s Nop56. Kompleks tvori topljive agregate što otežava njegovo pročišćavanje. Na temelju ovih rezultata, razvit će se nove strategije za pročišćavanje ciljnih proteina. Daljnja istraživanja doprinjet će razumijevanju biologije kromatina i bolesti povezanih s histonskim šaperonima.Histones are the chief protein components of chromatin. Histone chaperone Nap1, together with karyopherin Kap114, shuttles H2A-H2B from cytosol to the nucleus. Nop1 methyltransferase in complex with Nop56 protein is responsible for H2AQ105 methylation, the first histone epigenetic mark dedicated to a specific RNA polymerase. The aim of this project was to structurally characterize histones in complex with these histone binding proteins. Standard molecular cloning techniques were used. Proteins were purified by affinity, ion exchange and size exclusion chromatography. Proteins were analyzed by SDS-poliacrylamide gel-electrophoresis and mass sprectrometry. We have shown that Nap1 (H. Sapiens) forms a complex with H2A-H2B (X. laevis) dimer. Nap1 terminal domains are not necessary for complex formation. Regions important for complex stability are protected from protease cleavage. Interaction between Nap1-H2A-H2B and Kap114 (S. Pombe) was confirmed. Complexes Nap1-H2A-H2B and Nap1-H2A-H2B-Kap114 were purified and precrystallization screenings were set up on crystallization robot. Nop1-Nop56 (K. lactis) complex was formed in vitro. Nop1 N-terminal domain is dispensable for interaction with Nop56. Complex forms soluble aggregates which has made purification difficult. Based on obtained results, new purification strategies will be developed. Further research will contribute to understanding of chromatin biology and the human diseases associated with histone chaperones

Structural characterization of histone binding proteins

Histoni su osnovna proteinska komponenta kromatina. Histonski šaperon Nap1, zajedno s karioferinom Kap114, transportira H2A-H2B iz citoplazme u jezgru. Djelovanje metiltransferaze Nop1, u kompleksu s proteinom Nop56, ključno je za nastanak metilacije H2AQ105, prve epigenetičke oznake posvećene specifičnoj RNA-polimerazi. Cilj ovog rada bio je strukturno karakterizirati komplekse histona s tim histon-vezujućim proteinima. Korištene su standardne metode molekularnog kloniranja. Proteini su pročišćavani afinitetnom kromatografijom, ionizmjenjivačkom kromatografijom i kromatografijom isključenjem. Proteini su analizirani SDS-poliakrilamidnom gel-elektroforezom i masenom spektrometrijom. Pokazali smo da Nap1 (H. sapiens) tvori kompleks s dimerom H2A-H2B (X. laevis). Terminalne domene Nap1 nisu nužne za nastanak kompleksa. Dijelovi kompleksa koji su važni za njegovu stabilnost, zaštićeni su od djelovanja proteaze. Potvrđene su interakcije Nap1-H2A-H2B s Kap114 (S. pombe). Kompleksi Nap1-H2A-H2B i Nap1-H2A-H2B-Kap114 su pročišćeni i postavljene su pretkristalizacijske probe na kristalizacijskom robotu. Kompleks Nop1-Nop56 (K. lactis) dobiven je in vitro. N-terminalna domena Nop1 nije potrebna za interakciju s Nop56. Kompleks tvori topljive agregate što otežava njegovo pročišćavanje. Na temelju ovih rezultata, razvit će se nove strategije za pročišćavanje ciljnih proteina. Daljnja istraživanja doprinjet će razumijevanju biologije kromatina i bolesti povezanih s histonskim šaperonima.Histones are the chief protein components of chromatin. Histone chaperone Nap1, together with karyopherin Kap114, shuttles H2A-H2B from cytosol to the nucleus. Nop1 methyltransferase in complex with Nop56 protein is responsible for H2AQ105 methylation, the first histone epigenetic mark dedicated to a specific RNA polymerase. The aim of this project was to structurally characterize histones in complex with these histone binding proteins. Standard molecular cloning techniques were used. Proteins were purified by affinity, ion exchange and size exclusion chromatography. Proteins were analyzed by SDS-poliacrylamide gel-electrophoresis and mass sprectrometry. We have shown that Nap1 (H. Sapiens) forms a complex with H2A-H2B (X. laevis) dimer. Nap1 terminal domains are not necessary for complex formation. Regions important for complex stability are protected from protease cleavage. Interaction between Nap1-H2A-H2B and Kap114 (S. Pombe) was confirmed. Complexes Nap1-H2A-H2B and Nap1-H2A-H2B-Kap114 were purified and precrystallization screenings were set up on crystallization robot. Nop1-Nop56 (K. lactis) complex was formed in vitro. Nop1 N-terminal domain is dispensable for interaction with Nop56. Complex forms soluble aggregates which has made purification difficult. Based on obtained results, new purification strategies will be developed. Further research will contribute to understanding of chromatin biology and the human diseases associated with histone chaperones

ChromatoShiny: an interactive R/Shiny App for plotting chromatography profiles [version 1; peer review: 2 approved]

Background: UnicornTM software on Äkta liquid chromatography instruments outputs chromatography profiles of purified biological macromolecules. While the plots generated by the instrument software are very helpful to inspect basic chromatogram properties, they lack a range of useful annotation, customization and export options. Methods: We use the R Shiny framework to build an interactive app that facilitates the interpretation of chromatograms and the generation of figures for publications. Results: The app allows users to fit a baseline, to highlight selected fractions and elution volumes inside or under the plot (e.g. those used for downstream biochemical/biophysical/structural analysis) and to zoom into the plot. The app is freely available at https://ChromatoShiny.bio.ed.ac.uk. Conclusions:  It requires no programming experience, so we anticipate that it will enable chromatography users to create informative, annotated chromatogram plots quickly and simply