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

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

Probing Liquid-Liquid Phase Separation of RNA-Binding Proteins In Vitro and In Vivo

Biomolecular condensates and the concept of liquid-liquid phase separation (LLPS) have transformed cell biology in recent years. Condensates organize cellular content and compartmentalize biochemical reactions, in particular many processes involving RNA. This protocol is aimed at readers new to the LLPS field who want to test their protein or cellular structure of interest. We describe the basic principles of liquid-liquid phase separation, and outline initial approaches-both in vitro and in yeast cells-for the characterization of a candidate cellular condensate. First, we focus on strategies to purify phase-separating proteins and to reconstitute condensates from recombinant proteins in vitro for observation by light microscopy. Second, we describe in vivo experiments (including fluorescence recovery after photobleaching (FRAP) microscopy and 1,6-Hexanediol treatment) to test whether a subcellular structure displays liquid-like behavior in cells

The Role of DEAD-Box ATPases in Gene Expression and the Regulation of RNA-Protein Condensates

DEAD-box ATPases constitute a very large protein family present in all cells, often in great abundance. From bacteria to humans, they play critical roles in many aspects of RNA metabolism, and due to their widespread importance in RNA biology, they have been characterized in great detail at both the structural and biochemical levels. DEAD-box proteins function as RNA-dependent ATPases that can unwind short duplexes of RNA, remodel ribonucleoprotein (RNP) complexes, or act as clamps to promote RNP assembly. Yet, it often remains enigmatic how individual DEAD-box proteins mechanistically contribute to specific RNA-processing steps. Here, we review the role of DEAD-box ATPases in the regulation of gene expression and propose that one common function of these enzymes is in the regulation of liquid-liquid phase separation of RNP condensates. Expected final online publication date for the; Annual Review of Biochemistry; , Volume 91 is June 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates

DEAD-box ATPases as regulators of biomolecular condensates and membrane-less organelles

RNA-dependent DEAD-box ATPases (DDXs) are emerging as major regulators of RNA-containing membrane-less organelles (MLOs). On the one hand, oligomerizing DDXs can promote condensate formation 'in cis', often using RNA as a scaffold. On the other hand, DDXs can disrupt RNA-RNA and RNA-protein interactions and thereby 'in trans' remodel the multivalent interactions underlying MLO formation. In this review, we discuss the best studied examples of DDXs modulating MLOs in cis and in trans. Further, we illustrate how this contributes to the dynamic assembly and turnover of MLOs which might help cells to modulate RNA sequestration and processing in a temporal and spatial manner

A mitotic beacon reveals its nucleosome anchor

Mitosis, nuclear envelope formation, and nucleocytoplasmic transport require chromosomes to identify themselves by enriching Ran-GTP around the chromatin fiber. In a recent Nature report, Makde et al. (2010) describe the structure of the Ran activator RCC1 anchored onto nucleosomes

Catch me if you can

Nucleosomes confer a barrier to processes that require access to the eukaryotic genome such as transcription, DNA replication and repair. A variety of ATP-dependent nucleosome remodeling machines and ATP-independent histone chaperones facilitate nucleosome dynamics by depositing or evicting histones and unwrapping the DNA. It is clear that remodeling machines can use the energy from ATP to actively destabilize, translocate or disassemble nucleosomes. But how do ATP-independent histone chaperones, which “merely” bind histones, contribute to this process? Using our recent structural analysis of the conserved and essential eukaryotic histone chaperone FACT in complex with histones H2A-H2B as an example, we suggest that FACT capitalizes on transiently exposed surfaces of the nucleosome. By binding these surfaces, FACT stabilizes thermodynamically unfavorable intermediates of the intrinsically dynamic nucleosome particle. This makes the nucleosome permissive to DNA and RNA polymerases, providing temporary access, passage, and read-out