An integrated view of the essential eukaryotic chaperone FACT in complex with histones H2A-H2B

Summary: Structure of the FACT chaperone domain in complex with histones H2A-H2B, and a model for FACT-mediated nucleosome reorganization Nucleosomes are the smalles unit of chromatin: two coils of DNA are wrapped around a histone octamer core, which neutralizes its charge and `packs' the lengthy molecule. Nucleosomes confer a barrier to processes that require access to the eukaryotic genome such as transcription, DNA replication and repair. A variety of nucleosome remodeling machines and histone chaperones facilitate nucleosome dynamics by depositing or evicting histones and unwrapping the DNA. The eukaryotic FACT complex (composed of the subunits Spt16 and Pob3) is an essential and highly conserved chaperone. It assists the progression of DNA and RNA polymerases, for example by facilitating transcriptional initiation and elongation. Further, it promotes the genome-wide integrity of chromatin structure, including the suppression of cryptic transcription. Genetic and biochemical assays have shown that FACT's chaperone activity is crucially mediated by a direct interaction with histones H2A-H2B. However, the structural basis for how H2A-H2B are recognized and how this integrates with FACT’s other functions, including the recognition of histones H3-H4 and of other nuclear factors, is unknown. In my PhD research project, I was able to reveal the structure of the yeast chaperone domain in complex with the H2A-H2B heterodimer and show that the Spt16M module in FACT’s Spt16 subunit establishes the evolutionarily conserved H2A-H2B binding and chaperoning function. The structure shows how an alpha-helical `U-turn' motif in Spt16M interacts with the alpha-1-helix of H2B. The U-turn motif scaffolds onto a tandem pleckstrin-homology-like (PHL) module, which is structurally and functionally related to the H3-H4 chaperone Rtt106 and the Pob3M domain of FACT. Biochemical and in vivo assays validate the crystal structure and dissect the contribution of histone tails and H3-H4 toward FACT binding. My results show that Spt16M makes multiple interactions with histones, which I suggest allow the module to gradually invade the nucleosome and ultimately block the strongest interaction surface of H2B with nucleosomal DNA by binding the H2B alpha-1-helix. Together, these multiple contact points establish an extended surface that could reorganize the first 30 base-pairs of nucleosomal histone–DNA contacts. Further, I report a brief biochemical analysis of FACT’s heterodimerization domain. Its PHL fold indicates shared evolutionary origin with the H3-H4-binding Spt16M, Pob3M and Rtt106 tandem PHL modules. However, the Spt16D–Pob3N heterodimer does not bind histones, rather it connects FACT to replicative DNA polymerases. The snapshots of FACT’s engagement with H2A-H2B and structure-function analysis of all its domains lay the foundation for the systematic analysis of FACT’s vital chaperoning functions and how the complex promotes the activity of enzymes that require nucleosome reorganization.Zusammenfassung: Struktur der FACT Chaperon-Domäne im Komplex mit Histonen H2A-H2B, und ein Modell für die FACT-vermittelte Restrukturierung des Nukleosoms Nukleosomen sind die kleinsten Bausteine des Chromatin: das DNA Molekül wickelt sich in zwei Windungen um einen Oktamer aus Histon-Proteinen, die seine Ladung neutralisieren und es ordentlich `verpacken'. Deshalb sind Nukleosomen ein Hindernis für alle nukleären Prozesse, die Zugang zur DNA erfordern, wie zum Beispiel Transkription, Replikation oder Reparatur der DNA. Verschiedene Protein-Komplexe (ATP-abhängige `Remodeler' und ATP-unabhängige Histon-Chaperone) halten Nukleosomen in einem dynamischen und zugänglichen Zustand, indem sie Histone aus- oder ein-bauen, oder die DNA vom Oktamer abwickeln. Der eukaryotische FACT Komplex ist ein hochkonserviertes, heterodimeres Histon-Chaperon (aus den Unterheiten Spt16 und Pob3), das DNA und RNA Polymerasen unterstützt, durch Nukleosomen hindurchzuschreiben. Gleichzeitig stellt es sicher, dass die Chromatin-Integrität erhalten bleibt und unterdrückt dadurch z.B. Transkription von sogenannten kryptischen Promotoren. Genetische und biochemische Experimente haben gezeigt, dass die Interaktion mit Histonen, vor allem mit dem H2A-H2B Histon-Dimer, entscheidend für die Funktionalität von FACT als Histon Chaperon ist. Es fehlten jedoch molekulare oder strukturelle Informationen wie die Histone gebunden werden und wie dies mit den anderen biologischen Funktionen von FACT zusammenspielt, wie zum Beispiel der Interaktion mit Histonen H3-H4 oder anderen nukleären Faktoren, und letztendlich wie das reorganisierte Nukleosom aussehen könnte. In dieser Arbeit habe ich die H2A-H2B bindende Domäne von FACT, Spt16M, identifiziert und ihre Struktur im Komplex mit H2A-H2B gelöst. Die H2A-H2B Bindung habe ich biochemisch verifiziert, verfeinert und den Phänotyp von wichtigen Spt16M-Aminosäuren in vivo in Hefe analysiert. Ein strukturell und funktionell konserviertes, neuartiges `U-turn' (Kehrtwende) Motif interagiert mit der alpha-1-Helix des globulären Kerns von Histon H2B; diese hydrophobe Interaktion mit mikromolarer Affnität ist essentiell für die Komplex-Stabilität. Ein konservierter `acidic patch' (`negativ geladene Partie') interagiert zusätzlich mit dem unstrukturierten N-terminalen Ende von H2B und stabilisiert dadurch den Komplex kinetisch. Das Spt16M U-turn Motif ist auf ein Tandem-PHL (pleckstrin-homology like) Modul aufgebaut, das hohe strukturelle Verwandtschaft zu den Histon-Chaperonen Rtt106 und Pob3M aufweist. Wie Rtt106 und Pob3M bindet auch Spt16M Histone H3-H4. Die Interaktion wurde biochemisch auf die alpha-N-Helix von H3 eingegrenzt. Zusammenfassend bindet Spt16M an drei Stellen auf der Histon-Oktamer Oberfläche des Nukleosoms. Diese bilden eine zusammenhängende Fläche, welche die ersten 30 Basenpaare der nukleosomalen DNA koordiniert. Vermutlich erfolgt die Interaktion von Spt16M mit dem Nukleosom schrittweise: Zunächst bindet Spt16M über das frei zugänglichen N-terminale Ende von H2B an das Nukleosom. Dort `verharrt' das Chaperon bis die beiden stärkeren Interaktions-Stellen (die alpha-N Helix von H3 und die alpha-1 Helix von H2B), welche meist von DNA bedeckt sind, durch spontanes Ablösen der DNA freigelegt werden. Letztendlich würde die vollständige Bindung von FACT an das Nukleosom die ersten 30 Basenpaare DNA verdrängen und dadurch das Nukleosom destablisieren, so dass andere nukläere Prozesse (z.B. Polymerasen) auf die DNA Stränge zugreifen können. Des Weiteren habe ich die Heterodimerisierungs-Domäne von FACT biochemisch analysiert. Spt16D-Pob3N besteht ebenfalls aus PHL Domänen, diese können jedoch keine Histone binden. Stattdessen koppeln sie den Chaperon-Komplex an die DNA Replikations-Maschinerie. Die vorgestellten Ergebnisse legen den Grundstein für strukturelle und mechanistische Studien wie der holo-FACT Komplex mit dem Nukleosom interagiert, und wie sich dies in den Replikations- und Transkriptions-Prozess eingliedert

Chirurgie des Hyperparathyreoidismus

Diese Langzeitstudie sollte die Wertigkeit des iPTH-Schnelltests und die „50 / 60 % - Regel“ in der Chirurgie des Hyperparathyreoidismus überprüfen. Bei der Bewertung der postoperativen Normokalzämie des pHPT im Mittel nach 2,1 Jahren, zeigte sich eine Erfolgsrate von 95,1 %, bei einer Persistenzen von 3,6 % und Rezidivrate von 1,2 %. Bei der Betrachtung jedoch sowohl des Kalziums als auch des Parathormons kristallisiert sich eine Gruppe heraus, die wir weder den Gesunden noch den eindeutig Kranken zuordnen wollten. Die postoperativ normokalzämischen hyperparathyreoten Patienten, mit einer Häufigkeit von 5,0 % (Gruppe 3). Intraoperativ fiel im Mittel bei dieser Gruppe das Parathormon um 82,74 % ab auf einen Endwert von 96,74 pg/ml. Wir konnten zeigen, dass sich die eindeutig Kranken von den Gesunden sowohl im prozentuellen Abfall (p 0,003) als auch im Absolutwert (p 0,009) signifikant unterschieden. Bezüglich des postoperativen normokalzämischen HPTs´ konnte gezeigt werden, dass diese Gruppe sich signifikant von den Gesunden im Absolutwert (p 0,01) und von den Kranken im prozentuellen Abfall (p 0,043) unterschied. Beim renalen HPT hatten wir eine mittlere Nachbeobachtungszeit von 1,81 Jahren. Dabei waren 93,6 % postoperativ normokalzämisch und 1,6 % persistent. Es zeigte sich eine Rezidivrate von 4,8 %. Vergleicht man die postoperativ normokalzämischen Patienten mit den Kranken, so zeigt sich weder im Endwert (p 0,339) noch im prozentuellen Abfall (p 0,729) ein signifikanter Unterschied. Wir kamen zu dem Schluss, dass der iPTH-Schnelltest für die Chirurgie des pHPT ein probates Mittel darstellt intraoperativ dem Operateur in seiner Entscheidung zu helfen, weiter nach EK-Gewebe zu suchen oder es dabei belassen zu können. Wir gehen davon aus, dass es nicht ausreicht, wie bisher, sich nur den prozentuellen Parathormonabfall anzusehen, sondern es auch notwendig ist den Absolutwert, auf welchen Wert das Parathormon intraoperativ abfällt, im Auge zu behalten. Unsere Empfehlung dabei ist, dass das iPTH mindestens auf einen Endwert von 60 pg/ml (Norm 12-72) abfallen sollte, ist diese Bedingung nicht gegeben, so muss der prozentulle Abfall mindestens über 60 % sein, damit die Operation mit einer Vorhersagewahrscheinlichkeit von 94,67 % als erfolgreich abgeschlossen werden kann. Für die Operation des renalen Hyperparathyreoidismus konnten wir zeigen, dass der iPTH-Abfall keinen Hinweis auf den postoperativen Erfolg bringt

Membraneless organelles: phasing out of equilibrium

Over the past years, liquid-liquid phase separation (LLPS) has emerged as a ubiquitous principle of cellular organization implicated in many biological processes ranging from gene expression to cell division. The formation of biological condensates, like the nucleolus or stress granules, by LLPS is at its core a thermodynamic equilibrium process. However, life does not operate at equilibrium, and cells have evolved multiple strategies to keep condensates in a non-equilibrium state. In this review, we discuss how these non-equilibrium drivers counteract solidification and potentially detrimental aggregation, and at the same time enable biological condensates to perform work and control the flux of substrates and information in a spatial and temporal manner

Dynamic arrest and aging of biomolecular condensates are modulated by low-complexity domains, RNA and biochemical activity

Biomolecular condensates require suitable control of material properties for their function. Here we apply Differential Dynamic Microscopy (DDM) to probe the material properties of an in vitro model of processing bodies consisting of out-of-equilibrium condensates formed by the DEAD-box ATPase Dhh1 in the presence of ATP and RNA. By applying this single-droplet technique we show that condensates within the same population exhibit a distribution of material properties, which are regulated on several levels. Removal of the low-complexity domains (LCDs) of the protein decreases the fluidity of the condensates. Structured RNA leads to a larger fraction of dynamically arrested condensates with respect to unstructured polyuridylic acid (polyU). Promotion of the enzymatic ATPase activity of Dhh1 reduces aging of the condensates and the formation of arrested structures, indicating that biochemical activity and material turnover can maintain fluid-like properties over time

Pat1 promotes processing body assembly by enhancing the phase separation of the DEAD-box ATPase Dhh1 and RNA

Processing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid-liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. Previously, we identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly; in vivo; (Mugler et al., 2016). Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly,; in vivo; PB dynamics can be recapitulated; in vitro; , since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs

Characterization of RNA content in individual phase-separated coacervate microdroplets

Liquid-liquid phase separation or condensation is a form of macromolecular compartmentalization. Condensates formed by complex coacervation were hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of condensation in cells and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and how single condensates differ in their RNA composition. Therefore, we developed an approach to read the RNA content from single condensates using high-throughput sequencing. We find that RNAs which are enriched for specific sequence motifs efficiently accumulate in condensates. These motifs show high sequence similarity to short interspersed elements (SINEs). We observed similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution.Competing Interest StatementThe authors have declared no competing interest

Structure specific recognition protein-1 (SSRP1) is an elongated homodimer that binds histones

The histone chaperone complex facilitates chromatin transcription (FACT) plays important roles in DNA repair, replication, and transcription. In the formation of this complex, structure-specific recognition protein-1 (SSRP1) heterodimerizes with suppressor of Ty 16 (SPT16). SSRP1 also has SPT16-independent functions, but how SSRP1 functions alone remains elusive. Here, using analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS) techniques, we characterized human SSRP1 and that from the amoeba Dictyostelium discoideum and show that both orthologs form an elongated homodimer in solution. We found that substitutions in the SSRP1 pleckstrin homology domain known to bind SPT16 also disrupt SSRP1 homodimerization. Moreover, AUC and SAXS analyses revealed that SSRP1 homodimerization and heterodimerization with SPT16 (resulting in FACT) involve the same SSRP1 surface, namely, the PH2 region, and that the FACT complex contains only one molecule of SSRP1. These observations suggest that SSRP1 homo- and heterodimerization might be mutually exclusive. Moreover, isothermal titration calorimetry analyses disclosed that SSRP1 binds both histones H2A-H2B and H3-H4 and that disruption of SSRP1 homodimerization decreases its histone-binding affinity. Together, our results provide evidence for regulation of SSRP1 by homodimerization and suggest a potential role for homodimerization in facilitating SPT16-independent functions of SSRP1

Characterization of RNA content in individual phase-separated coacervate microdroplets

Condensates formed by complex coacervation are hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of biological condensates and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and to which extend single condensates differ in their RNA composition. To address this, we developed an approach to read the RNA content from single synthetic and protein-based condensates using high-throughput sequencing. We find that certain RNAs efficiently accumulate in condensates. These RNAs are strongly enriched in sequence motifs which show high sequence similarity to short interspersed elements (SINEs). We observe similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution

Repair or destruction: an intimate liaison between ubiquitin ligases and molecular chaperones in proteostasis

Cellular differentiation, developmental processes, and environmental factors challenge the integrity of the proteome in every eukaryotic cell. The maintenance of protein homeostasis, or proteostasis, involves folding and degradation of damaged proteins, and is essential for cellular function, organismal growth, and viability [1, 2]. Misfolded proteins that cannot be refolded by chaperone machineries are degraded by specialized proteolytic systems. A major degradation pathway regulating cellular proteostasis is the ubiquitin/proteasome-system (UPS), which regulates turnover of damaged proteins that accumulate upon stress and during aging. Despite the large number of structurally unrelated substrates, ubiquitin conjugation is remarkably selective. Substrate selectivity is mainly provided by the group of E3 enzymes. Several observations indicate that numerous E3 ubiquitin ligases intimately collaborate with molecular chaperones to maintain the cellular proteome. In this Review, we provide an overview of specialized quality control E3 ligases playing a critical role in the degradation of damaged proteins. The process of substrate recognition and turnover, the type of chaperones they team up with, and the potential pathogeneses associated with their malfunction will be further discusse

Glutamylation of Nap1 modulates histone H1 dynamics and chromosome condensation in Xenopus

Linker histone H1 is required for mitotic chromosome architecture in Xenopus laevis egg extracts and, unlike core histones, exhibits rapid turnover on chromatin. Mechanisms regulating the recruitment, deposition, and dynamics of linker histones in mitosis are largely unknown. We found that the cytoplasmic histone chaperone nucleosome assembly protein 1 (Nap1) associates with the embryonic isoform of linker histone H1 (H1M) in egg extracts. Immunodepletion of Nap1 decreased H1M binding to mitotic chromosomes by nearly 50%, reduced H1M dynamics as measured by fluorescence recovery after photobleaching and caused chromosome decondensation similar to the effects of H1M depletion. Defects in H1M dynamics and chromosome condensation were rescued by adding back wild-type Nap1 but not a mutant lacking sites subject to posttranslational modification by glutamylation. Nap1 glutamylation increased the deposition of H1M on sperm nuclei and chromatin-coated beads, indicating that charge-shifting posttranslational modification of Nap1 contributes to H1M dynamics that are essential for higher order chromosome architecture