Connections between cell mechanics and Chromatin condensation in embryonic stem cells

Pluripotenz spielt eine Schlüsselrolle in embryonalen Stammzellen (ES Zellen) sowie im Epiblasten, dem Ursprungsgewebe des Embryos. Die Charakterisierung dieses speziellen Zustands der Pluripotenz und der zugrunde liegenden molekularen Mechanismen hat die Wissenschaft seit Jahrzehnten inspiriert. In dieser Studie haben wir eine Mischung aus bewährten und neuen Methoden verwendet um biophysikalische Veränderungen zu untersuchen, die in Stammzellen, die auf transkriptioneller Ebene heterogen sind, schon vor der Differenzierung passieren. Nanog ist ein Transkriptionsfaktor, der Pluripotenz stabilisiert und dessen Expressionslevel zwei unterschiedliche Zustände innerhalb der Pluripotenz definieren: Zellen mit hohem Nanog Expressionslevel sind stabil pluripotent während Zellen mit niedrigen Levels zur Differenzierung tendieren. Wir haben mittels optischer Zelldehnung herausgefunden, dass letztere Zellen leichter verformbar sind als stabil pluripotente Zellen. Das Zytoskelett spielt eine prominente Rolle für die mechanischen Eigenschaften einer Zelle; deswegen haben wir die Struktur von Zytoskelettfilamenten in ES Zellen untersucht. In immunohistochemischen Analysen von adherenten sowie abgelösten Zellen fanden wir jedoch keine Unterschiede zwischen den beiden Zuständen. Zusätzlich konnten wir ausschließen, dass Veränderungen in der Proliferationsgeschwindigkeit oder in den relativen Volumenverhältnissen zwischen Kern und Zytoplasma für die mechanischen Unterschiede verantwortlich waren. Der Einfluss des Zellkerns auf die mechanischen Eigenschaften der gesamten Zelle sind nicht sehr gut untersucht, allerdings sind Kerne in ES Zellen vergleichsweise groß und daher zogen wir in Betracht, dass Kerne die Zellverformbarkeit mitbestimmen könnten. Um die mechanischen Eigenschaften von Kernen zu messen, haben wir eine neue Methode entwickelt, bei der Kerne in intakten Zellen verformt werden können. Mit dieser Methode fanden wir Unterschiede in der Kernverformung wie wir sie für die unterschiedlichen Zellpopulationen gesehen haben. Um die Substruktur des Kerns zu untersuchen, haben wir Histon-Mobilität eines markierten Histons mit einer Photobleaching Technik untersucht und zusätzlich die Verteilung des Heterochromatin-spezifischen Proteins HP1α analysiert. Die Ergebnisse beider Analysen stimmten darin überein, dass Chromatin im stabil pluripotenten Zustant, in dem Zellen und Kerne steifer waren, stärker kondensiert ist. Mit verschiedenen Chemikalien beeinflussten wir spezifisch die Chromatin-Kondensierung und konnten zeigen, dass sowohl Zell- als auch Kern-Verformbarkeit mit Dekondensierung steigen, ein Hinweis darauf, dass die Kernmechanik die Zellmechanik insgesamt beeinflusst. Wir postulieren daher, dass Chromatin-Kondensation eine zentrale Rolle für die Regulation der Pluripotenz spielt und dass Chromatin erst dekondensiert werden muss bevor Zellen einer Entwicklunslinie folgen können und die Formation von Heterochromatin stattfinden kann. Die Verbindung zwischen den Eigenschaften des Chromatins mit der Zellmechanik hat weitreichende Konsequenzen für die Regulation von frühen embryonalen Entscheidungen und Sortierungsprozessen von Zellen. Zusätzlich zu bekannten biochemischen und positionsabhängigen Signalen könnten biophysikalische Eigenschaften regulatorische Mechanismen in der frühen Embryonalentwicklung darstellen.Pluripotency is a key feature of embryonic stem (ES) cells and the founder tissue of the embryo proper, the epiblast. The characterization of the properties of pluripotent cells and the molecular regulation of this state has inspired much research over the last decades. Here, we used a mix of well established and novel techniques to investigate biophysical changes that occur in the transcriptionally heterogeneous state of pluripotency in mouse ES cells even before differentiation. The expression levels of Nanog, a transcription factor that stabilizes pluripotency, can distinguish two states of pluripotency: Cells with high Nanog expression are in a stable pluripotent state while cells with low levels are prone to differentiate. We found that differentiation-prone cells are softer on average as compared to stable pluripotent cells when we tested global cell mechanics by optical stretching. The cytoskeleton is usually critical in determinining mechanical properties of cells; therefore, we investigated the structure of the cytoskeleton in ES cells but we found no detectable difference between the two states in immunohistochemical stainings of attached and suspended cells. Furthermore, we ruled out that changes in proliferation speed or in the relative amounts of nuclear and cytoplasmatic volume caused the observed effect. The role of nuclear stiffness on overall cell mechanics is not well understood, however, nuclei in ES cells are comparably big and so we considered a role of nuclei in cell compliance. To probe nuclear mechanics, we demonstrated a novel method that allows to stretch nuclei within intact cells. Indeed, we found changes of the response of nuclei to stretching forces that are in line with cell mechanics changes. To assess nuclear substructure, we analyzed chromatin structure by testing histone mobility using fluorescence recovery after photobleaching of a tagged histone and analyzing the distribution of the heterochromatin marker HP1α in ES nuclei. The results of both assays agree well in that chromatin was more condensed in the stable pluripotent state where cells and nuclei were stiffer. We used chemical treatments to change chromatin condensation and showed that both nuclear compliance and cell compliance increase with chromatin decondensation, indicating that nuclear mechanics influence overall cell mechanics in ES cells. We propose that chromatin condensation plays a crucial role in the regulation of pluripotency and that chromatin has to be decondensed before lineage commitment and heterochromatinization can occur. The connection between chromatin state and cell mechanics has far reaching implications for the regulation of early embryonic fate decisions and cell sorting processes. In addition to well-known biochemical and positional cues, biophysical properties could act as a regulatory mechanism in early embryonic development

Slx5/Slx8-dependent ubiquitin hotspots on chromatin contribute to stress tolerance in saccharomyces cerevisiae

Chromatin is a tightly controlled cellular environment and protein association with chromatin is often regulated by post-translational modifications (PTMs), including modification with SUMO and ubiquitin. In the last decades, both these modifications and their corresponding enzymatic machineries have emerged as pivotal regulators involved in nuclear quality control, DNA repair and transcriptional regulation. More recently, SUMO-targeted ubiquitin ligases (STUbLs) were discovered to provide an important link between those pathways, as they recognize SUMOylated proteins and catalyze their ubiquitylation. However, many of the physiological functions of STUbLs and how exactly they recognize specific substrates, while SUMOylated proteins are highly prevalent on chromatin, remained elusive. In this study, my analysis of the genome-wide distribution of the yeast STUbL Slx5/Slx8 demonstrates a remarkably specific localization of Slx5/Slx8 to seven loci of strong ubiquitin accumulation, so-called “ubiquitin hotspots”. My data show that Slx5/Slx8 is recruited to ubiquitin hotspots by the uncharacterized transcription factor-like protein Ymr111c/Euc1. Slx5/Slx8 recruitment relies on a bipartite interaction between Ymr111c/Euc1 and Slx5, which involves the Slx5 SUMO-interacting motifs and a novel, uncharacterized substrate recognition domain of Slx5 directly interacting with Ymr111c/Euc1. Importantly, the Euc1–ubiquitin hotspot pathway and Slx5/Slx8 are required for the cellular response to various stresses like temperature shifts, in particular when general gene expression control is impaired by mutation of members of the H2A.Z and Rpd3L pathways. Thus, my data suggest that the STUbL-dependent ubiquitin hotspots shape chromatin during stress adaptation, and the bipartite recruitment mechanism exemplifies how specificity can be generated in the STUbL pathway. These findings can guide future research elucidating how different substrate recognition domains control the diverse STUbL functions, which range from the response to DNA damage to early embryonic development

Structural mechanism of extranucleosomal DNA readout by the INO80 complex

The nucleosomal landscape of chromatin depends on the concerted action of chromatin remodelers. The INO80 remodeler specifically places nucleosomes at the boundary of gene regulatory elements, which is proposed to be the result of an ATP-dependent nucleosome sliding activity that is regulated by extranucleosomal DNA features. Here, we use cryo–electron microscopy and functional assays to reveal how INO80 binds and is regulated by extranucleosomal DNA. Structures of the regulatory A-module bound to DNA clarify the mechanism of linker DNA binding. The A-module is connected to the motor unit via an HSA/post-HSA lever element to chemomechanically couple the motor and linker DNA sensing. Two notable sites of curved DNA recognition by coordinated action of the four actin/actin-related proteins and the motor suggest how sliding by INO80 can be regulated by extranucleosomal DNA features. Last, the structures clarify the recruitment of YY1/Ies4 subunits and reveal deep architectural similarities between the regulatory modules of INO80 and SWI/SNF complexes

Mechanism of ribosome-associated mRNA degradation during tubulin autoregulation

Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits affects polymerization dynamics and microtubule function. When cells sense excess free tubulin, they trigger degradation of the encoding mRNAs, which requires recognition of the nascent polypeptide by the tubulin-specific ribosome-binding factor TTC5. How TTC5 initiates the decay of tubulin mRNAs is unknown. Here, our biochemical and structural analysis reveals that TTC5 recruits the poorly studied protein SCAPER to the ribosome. SCAPER, in turn, engages the CCR4-NOT deadenylase complex through its CNOT11 subunit to trigger tubulin mRNA decay. SCAPER mutants that cause intellectual disability and retinitis pigmentosa in humans are impaired in CCR4-NOT recruitment, tubulin mRNA degradation, and microtubule-dependent chromosome segregation. Our findings demonstrate how recognition of a nascent polypeptide on the ribosome is physically linked to mRNA decay factors via a relay of protein-protein interactions, providing a paradigm for specificity in cytoplasmic gene regulation