69 research outputs found
Integrative modeling of the in-cell architecture of the yeast Nuclear Pore Complex
No full textRepository with source code files and input files utilized for integrative modeling of the in-cell architecture of the yeast Nuclear Pore Complex (ScNPC). All output integrative models of ScNPCs associated with the following work are included:
"In-cell architecture of the nuclear pore and snapshots of its turnover. Matteo Allegretti, Christian E. Zimmerli, Vasileios Rantos, Florian Wilfling, Paolo Ronchi, Herman K.H. Fung, Chia-Wei Lee, Wim Hagen, Beata Turonova, Kai Karius, Mandy Börmel, Xiaojie Zhang, Christoph Müller, Yannick Schwab, Julia Mahamid, Boris Pfander, Jan Kosinski, Martin Beck. Nature, 2020"
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The role of cell cycle, growth, and metabolism in species-specific differentiation timing
No full textDifferent mammalian species progress through similar stages during embryonic development and adult life but the pace of these transitions is species-specific. While classically, developmental timing was viewed merely as a consequence of varying body sizes between species, the use of pluripotent stem cells (PSCs) has shown that species-specific developmental timing is maintained during in vitro differentiation. Since then, uncovering cell-intrinsic mechanisms that regulate the timescales of development has become a rising topic of interest.
This project aimed to identify such cell-intrinsic mechanisms using in vitro neural progenitor differentiation of mouse, monkey and human PSCs. To facilitate inter-species comparisons, all cells were cultured and differentiated under harmonized conditions. Under these circumstances, mouse cells differentiated more than twice as fast as human cells, recapitulating species-specific differentiation timing. As differentiation and growth need to be tightly coordinated during development, I compared cell cycle durations across the species. Cell cycle durations followed a species-specific trend, with the human cell cycle being 1.47-fold and the monkey cell cycle 1.42- fold longer than the mouse cell cycle. To test if differentiation depends on proliferation, I performed cell cycle and growth manipulations by either a retinoblastoma knock-out line or inhibiting the mammalian target of rapamycin (mTOR). Strikingly, mTOR inhibition caused a drastic extension of cell cycle durations, yet single-cell transcriptomics revealed no systematic delay during early neural differentiation. This showed that differentiation can be uncoupled from growth and proliferation, suggesting alternative mechanisms. One candidate identified was the UDP-glucose pyrophosphorylase 2 (UGP2), required for glycogen synthesis. High UGP2 correlated with slow differentiation. Consequently, glycogen content was highest in human cells, intermediate in monkey and lowest in mouse. Thus, glycogen content is a species-specific cellular property that was unknown to this date. Neural differentiation of UGP2 knock-out cells revealed premature expression of the forebrain marker FOXG1, indicating that UGP2 could contribute to setting differentiation timing. It remains to be tested, how loss of UGP2 globally affects differentiation and by which mechanisms UGP2 acts.
Taken together, these findings show that differentiation can be uncoupled from growth and cell cycling and implicate UGP2 and glycogen metabolism in the regulation of timing. This constitutes a novel mechanism by which cells could determine their differentiation speed
System-wide investigation of hydroxymethylcytosine recognition and distribution in the mammalian genome
No full textThe epigenetic cytosine modifications 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) are key regulatory elements of mammalian genomes, occurring within CpG dinucleotides in either strand- symmetric or strand-asymmetric combinations. While reader proteins for symmetrically and hemi- modified CpG dyads have been identified, the recognition of asymmetrically modified CpGs and the potential regulatory implications of their symmetry remain unexplored. In this work, mammalian nu- clear proteins binding to hmC-containing CpG dyads were identified and characterized. A protocol for generating asymmetrically modified DNA probes was established and applied in pull-down assays coupled with mass spectrometry-based proteomics. Comparative enrichment studies were performed using promoter probes bearing symmetric or asymmetric C, mC, and hmC modifications, allowing for direct assessment of reader profiles in the same sequence, tissue and experimental contexts. In hu- man and mouse nuclear extracts, numerous tissue-specific readers of hmC-modified sequences were identified, falling into distinct, probe-specific subgroups. These include transcription factors and chro- matin regulators such as MYC and MAX, which read hmC in a sequence-dependent manner, and RFX5, which selectively discriminated between hmC symmetry states in CpG dyads. The presumably preva- lent asymmetric CpG dyad hmC/mC exhibited a distinct reader protein profile, supporting the hy- pothesis that hmC symmetry information provides unique regulatory outputs. To complement these proteomics analyses, selective enrichment and sequencing of native double-stranded DNA fragments containing hmC/mC CpG dyads was implemented, based on an evolved methyl-CpG-binding domain protein with enhanced binding specificity. This approach enabled genome-wide mapping of hmC/mC sites, offering a valuable tool for investigating their roles in chromatin biology. Together, these find- ings provide a comprehensive framework for the study of symmetric and asymmetric hmC-containing CpG dyads and lay the groundwork for elucidating their functional contributions to transcriptional regulation, development and disease.Die epigenetischen Cytosin-Modifikationen 5-Methylcytosin (mC) und 5-Hydroxymethylcytosin (hmC) sind wichtige regulatorische Elemente des Säugergenoms und kommen innerhalb von CpG-Dinukleo- tiden entweder in strangsymmetrischen oder strangasymmetrischen Kombinationen vor. Während Le- serproteine für symmetrisch- und halb-modifizierte CpG-Dyaden identifiziert wurden, sind die Erken- nung asymmetrisch-modifizierter CpGs und damit die potenziellen regulatorischen Auswirkungen ih- rer Symmetrie noch unerforscht. In dieser Arbeit wurden nukleare Säugetierproteine identifiziert und charakterisiert, die an hmC-haltige CpG-Dyaden binden. Es wurde ein Protokoll zur Erzeugung asym- metrisch modifizierter DNA-Sonden erstellt, welches in Pull-down-Analysen in Verbindung mit mas- senspektrometriebasierter Proteomik angewendet wurde. Vergleichende Anreicherungsstudien wur- den mit Promotor-Sonden durchgeführt, die symmetrische oder asymmetrische C-, mC- und hmC- Modifikationen aufwiesen, wodurch eine direkte Bewertung der Leserprofile in derselben Sequenz, demselben Gewebe und denselben experimentellen Kontexten möglich war. In nuklearen Extrakten von Mensch- und Mausgeweben wurden zahlreiche gewebespezifische Leser von hmC-modifizierten Sequenzen identifiziert, die in unterschiedliche, sondenspezifische Untergruppen fallen. Dazu gehören Transkriptionsfaktoren und Chromatin-Regulatoren wie MYC und MAX, die hmC sequenzabhängig le- sen, sowie RFX5, das selektiv zwischen hmC-Symmetriezuständen in CpG-Dyaden unterscheidet. Die vermutlich vorherrschende asymmetrische CpG-Dyade hmC/mC wies ein inividuelles Leserprotein- profil auf, was die Hypothese stützt, dass die Symmetrieinformation von hmC einzigartige regulatori- sche Ergebnisse zur Folge hat. Zur Ergänzung dieser Proteomanalysen wurde eine selektive Anreiche- rung und Sequenzierung von nativen doppelsträngigen DNA-Fragmenten, die hmC/mC-CpG-Dyaden enthalten, durchgeführt, basierend auf einem weiterentwickelten Methyl-CpG-bindenden Domänen- protein mit verbesserter Bindungsspezifität. Dieser Ansatz ermöglichte eine genomweite Kartierung von hmC/mC-Stellen und bietet ein wertvolles Werkzeug zur Untersuchung ihrer Rolle in der Chro- matinbiologie. Zusammen bieten diese Ergebnisse einen umfassenden Rahmen für die Untersuchung symmetrischer und asymmetrischer hmC-haltiger CpG-Dyaden und legen den Grundstein für die Auf- klärung ihrer funktionellen Beiträge zur Transkriptionsregulation, Entwicklung und Krankheit
Engineering cellular systems to study replication stress
No full textThe process of DNA replication is unique in cell biology since all errors will irreversibly change the
nature of the cellular progeny. Failure during DNA replication – so-called DNA replication stress has
been shown to be critical for genome stability and a key driver of cancer. A key limitation in our
understanding of replication stress comes from the fact that it is a poorly defined phenomenon that
encompasses several distinct molecular mechanisms, including replication-transcription collisions,
over- and under-replication and replication stalling. Currently, we lack a molecular marker to
differentiate between different forms of replication stress and utilize this knowledge in cancer
therapy.
Here, we engineered genetic systems in budding yeast to induce unscheduled replication in the G1-
phase of the cell cycle. We characterize the mechanism of G1 replication as well as factors that restrain
DNA replication outside of S phase. G1 replication is highly toxic and induces DNA damage in
subsequent cell cycle phases. We show that - mechanistically - subsequent replication during S-phase
results in over-replication and chromosome breaks via replication collisions. Notably, single-stranded
DNA accumulate with a unique chromosome-wide and strand-biased pattern, which allowed to
deduce a mechanism of head-to-tail fork collisions upon over-replication. The signature of ssDNA
accumulation is therefore an excellent marker for replication stress by unscheduled replication.
The utilization of ssDNA signatures to discriminate different forms of replication stress, for example
after replication-transcription conflicts and accumulation of RNA-DNA hybrids will be discussed
Regulation der Genomstabilität durch SUMO- und Ubiquitin-Modifikation von PCNA
No full textEine akkurate DNA-Replikation ist notwendig, um die Stabilität der genetischen Information zu gewährleisten. Dieser Prozess wird durch DNA-Läsionen erschwert, die durch eine Vielzahl von Ursachen entstehen und häufig nicht vor dem Erreichen der S-Phase repariert werden können. Nicht nur kann durch Läsionen geschädigte DNA häufig nicht dupliziert werden, angehaltene Replikationsgabeln können auch zusammenbrechen und so zu DNA-Strangbrüchen führen.
Die Funktion des RAD6-pathways liegt darin, die Umgehung (Bypass) von DNA-Läsionen während der Replikation zu ermöglichen, wodurch eine Toleranz gegenüber Schädigungen der DNA erreicht wird. In dieser Arbeit wurde die Regulation des RAD6-vermittelten Bypass von DNA-Läsionen durch posttranslationale Ubiquitin- und SUMO-Modifikationen des Replikationsfaktors PCNA untersucht.
PCNA bildet einen trimeren Ring um die DNA und verstärkt durch Bindung der replikativen Polymerase deren Assoziation zur DNA und somit die Prozessivität der Replikation. Als DNA gebundener Faktor des Replikations-komplexes ohne katalytische Aktivität ist PCNA ideal geeignet, um durch seine Modifikation Replikations-assoziierte Prozesse zu regulieren.
Die Ubiquitinierung von PCNA durch Enzyme des RAD6-pathways erfolgt als spezifische Antwort auf DNA-Läsionen während der Replikation und ermöglicht deren Bypass. Dabei bewirken unterschiedliche Ubiquitin-Modifikationen verschiedene Arten des Bypass. Die Mono-Ubiquitin-Modifikation führt zum Einsatz von speziellen Transläsions-Polymerasen, die eine größere Toleranz für geschädigte DNA haben, aber auch für die Entstehung von Mutationen verantwortlich sind. Einen mechanistisch anderen Bypass von DNA-Schäden bewirkt die Modifikation von PCNA mit einer Lysin K63-verknüpften Multi-Ubiquitinkette. Für diesen wird wahrscheinlich der neureplizierte, unbeschädigte Schwester-Strang als Vorlage benutzt.
Unabhängig von Schädigungen der DNA wird PCNA während der S-Phase zusätzlich mit dem ubiquitin-ähnlichen Protein SUMO modifiziert. Dies führt zu einer Interaktion mit der Helikase Srs2, die als Antagonist zu dem zentralen Rekombinationsprotein Rad51 wirkt. Dadurch wird spezfisch die homologe Rekombination zwischen Schwesterchromatiden an der Rekombinationsgabel inhibiert, nicht jedoch andere Rekombinationsereignisse, wie. z.B. Rekom-bination zwischen homologen Chromosomen. Deshalb ist es wahrscheinlich, dass spezifisch die Replikationsgabel durch PCNA-SUMO-Srs2 geschützt wird, um schädliche Rekombination oder Rekombinationsstrukturen zu vermeiden, die mit Replikations-assoziierten Prozessen interferieren.
Ubiquitin- und SUMO-Modifikation regulieren demnach unabhängige Prozesse. Interessanterweise haben diese aber eine verwandte Funktion im Bypass von DNA-Läsionen während der Replikation. Die Inhibition der Schwesterchromatid-Rekombination durch PCNA-SUMO-Srs2 lenkt den Bypass von DNA-Läsionen in einen durch PCNA-Ubiquitinierung gesteuerten Mechanismus
Robust Replication Control Is Generated by Temporal Gaps between Licensing and Firing Phases and Depends on Degradation of Firing Factor Sld2
No full textSummaryTemporal separation of DNA replication initiation into licensing and firing phases ensures the precise duplication of the genome during each cell cycle. Cyclin-dependent kinase (CDK) is known to generate this separation by activating firing factors and at the same time inhibiting licensing factors but may not be sufficient to ensure robust separation at transitions between both phases. Here, we show that a temporal gap separates the inactivation of firing factors from the re-activation of licensing factors during mitosis in budding yeast. We find that gap size critically depends on phosphorylation-dependent degradation of the firing factor Sld2 mediated by CDK, DDK, Mck1, and Cdc5 kinases and the ubiquitin-ligases Dma1/2. Stable mutants of Sld2 minimize the gap and cause increased genome instability in an origin-dependent manner when combined with deregulation of other replication regulators or checkpoint mechanisms. Robust separation of licensing and firing phases therefore appears indispensable to safeguard genome stability
Author response
No full textHolliday junctions (HJs) are key DNA intermediates in homologous recombination. They link homologous DNA strands and have to be faithfully removed for proper DNA segregation and genome integrity. Here, we present the crystal structure of human HJ resolvase GEN1 complexed with DNA at 3.0 Å resolution. The GEN1 core is similar to other Rad2/XPG nucleases. However, unlike other members of the superfamily, GEN1 contains a chromodomain as an additional DNA interaction site. Chromodomains are known for their chromatin-targeting function in chromatin remodelers and histone(de)acetylases but they have not previously been found in nucleases. The GEN1 chromodomain directly contacts DNA and its truncation severely hampers GEN1's catalytic activity. Structure-guided mutations in vitroand in vivo in yeast validated our mechanistic findings. Our study provides the missing structure in the Rad2/XPG family and insights how a well-conserved nuclease core acquires versatility in recognizing diverse substrates for DNA repair and maintenance
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