RESUMEN
Biomolecular condensates assemble in living cells through phase separation and related phase transitions. An underappreciated feature of these dynamic molecular assemblies is that they form interfaces with other cellular structures, including membranes, cytoskeleton, DNA and RNA, and other membraneless compartments. These interfaces are expected to give rise to capillary forces, but there are few ways of quantifying and harnessing these forces in living cells. Here, we introduce viscoelastic chromatin tethering and organization (VECTOR), which uses light-inducible biomolecular condensates to generate capillary forces at targeted DNA loci. VECTOR can be utilized to programmably reposition genomic loci on a timescale of seconds to minutes, quantitatively revealing local heterogeneity in the viscoelastic material properties of chromatin. These synthetic condensates are built from components that naturally form liquid-like structures in living cells, highlighting the potential role for native condensates to generate forces and do work to reorganize the genome and impact chromatin architecture.
RESUMEN
Nuclear compartments form via biomolecular phase separation, mediated through multivalent properties of biomolecules concentrated within condensates. Certain compartments are associated with specific chromatin regions, including transcriptional initiation condensates, which are composed of transcription factors and transcriptional machinery, and form at acetylated regions including enhancer and promoter loci. While protein self-interactions, especially within low-complexity and intrinsically disordered regions, are known to mediate condensation, the role of substrate-binding interactions in regulating the formation and function of biomolecular condensates is underexplored. Here, utilizing live-cell experiments in parallel with coarse-grained simulations, we investigate how chromatin interaction of the transcriptional activator BRD4 modulates its condensate formation. We find that both kinetic and thermodynamic properties of BRD4 condensation are affected by chromatin binding: nucleation rate is sensitive to BRD4-chromatin interactions, providing an explanation for the selective formation of BRD4 condensates at acetylated chromatin regions, and thermodynamically, multivalent acetylated chromatin sites provide a platform for BRD4 clustering below the concentration required for off-chromatin condensation. This provides a molecular and physical explanation of the relationship between nuclear condensates and epigenetically modified chromatin that results in their mutual spatiotemporal regulation, suggesting that epigenetic modulation is an important mechanism by which the cell targets transcriptional condensates to specific chromatin loci.
Asunto(s)
Proteínas que Contienen Bromodominio , Proteínas de Ciclo Celular , Cromatina , Proteínas Nucleares , Factores de Transcripción , Humanos , Acetilación , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica , Termodinámica , Factores de Transcripción/metabolismoRESUMEN
Intrinsically disordered regions (IDRs) represent a large percentage of overall nuclear protein content. The prevailing dogma is that IDRs engage in non-specific interactions because they are poorly constrained by evolutionary selection. Here, we demonstrate that condensate formation and heterotypic interactions are distinct and separable features of an IDR within the ARID1A/B subunits of the mSWI/SNF chromatin remodeler, cBAF, and establish distinct "sequence grammars" underlying each contribution. Condensation is driven by uniformly distributed tyrosine residues, and partner interactions are mediated by non-random blocks rich in alanine, glycine, and glutamine residues. These features concentrate a specific cBAF protein-protein interaction network and are essential for chromatin localization and activity. Importantly, human disease-associated perturbations in ARID1B IDR sequence grammars disrupt cBAF function in cells. Together, these data identify IDR contributions to chromatin remodeling and explain how phase separation provides a mechanism through which both genomic localization and functional partner recruitment are achieved.
Asunto(s)
Ensamble y Desensamble de Cromatina , Complejos Multiproteicos , Proteínas Nucleares , Humanos , Cromatina , Proteínas de Unión al ADN/química , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismoRESUMEN
The nucleolus is the largest biomolecular condensate and facilitates transcription, processing, and assembly of ribosomal RNA (rRNA). Although nucleolar function is thought to require multiphase liquid-like properties, nucleolar fluidity and its connection to the highly coordinated transport and biogenesis of ribosomal subunits are poorly understood. Here, we use quantitative imaging, mathematical modeling, and pulse-chase nucleotide labeling to examine nucleolar material properties and rRNA dynamics. The mobility of rRNA is several orders of magnitude slower than that of nucleolar proteins, with rRNA steadily moving away from the transcriptional sites in a slow (â¼1 Å/s), radially directed fashion. This constrained but directional mobility, together with polymer physics-based calculations, suggests that nascent rRNA forms an entangled gel, whose constant production drives outward flow. We propose a model in which progressive maturation of nascent rRNA reduces its initial entanglement, fluidizing the nucleolar periphery to facilitate the release of assembled pre-ribosomal particles.
Asunto(s)
ARN Ribosómico , ARN , ARN/genética , ARN Ribosómico/genética , Condensados Biomoleculares , Nucléolo Celular/genética , Proteínas Nucleares/genéticaRESUMEN
The cell nucleus can be thought of as a complex, dynamic, living material, which functions to organize and protect the genome and coordinate gene expression. These functions are achieved via intricate mechanical and biochemical interactions among its myriad components, including the nuclear lamina, nuclear bodies, and the chromatin itself. While the biophysical organization of the nuclear lamina and chromatin have been thoroughly studied, the concept that liquid-liquid phase separation and related phase transitions play a role in establishing nuclear structure has emerged only recently. Phase transitions are likely to be intimately coupled to the mechanobiology of structural elements in the nucleus, but their interplay with one another is still not understood. Here, we review recent developments on the role of phase separation and mechanics in nuclear organization and discuss the functional implications in cell physiology and disease states.
RESUMEN
Telomeres form unique nuclear compartments that prevent degradation and fusion of chromosome ends by recruiting shelterin proteins and regulating access of DNA damage repair factors. To understand how these dynamic components protect chromosome ends, we combine in vivo biophysical interrogation and in vitro reconstitution of human shelterin. We show that shelterin components form multicomponent liquid condensates with selective biomolecular partitioning on telomeric DNA. Tethering and anomalous diffusion prevent multiple telomeres from coalescing into a single condensate in mammalian cells. However, telomeres coalesce when brought into contact via an optogenetic approach. TRF1 and TRF2 subunits of shelterin drive phase separation, and their N-terminal domains specify interactions with telomeric DNA in vitro. Telomeric condensates selectively recruit telomere-associated factors and regulate access of DNA damage repair factors. We propose that shelterin mediates phase separation of telomeric chromatin, which underlies the dynamic yet persistent nature of the end-protection mechanism.
Asunto(s)
Complejo Shelterina/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Línea Celular , Cromatina/genética , ADN/metabolismo , Daño del ADN/fisiología , Reparación del ADN/genética , Reparación del ADN/fisiología , Humanos , Optogenética/métodos , Unión Proteica/genética , Unión Proteica/fisiología , Complejo Shelterina/genética , Complejo Shelterina/fisiología , Telómero/fisiología , Proteínas de Unión a Telómeros/genética , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/genéticaRESUMEN
Chromatin, which consists of DNA and associated proteins, contains genetic information and is a mechanical component of the nucleus. Heterochromatic histone methylation controls nucleus and chromosome stiffness, but the contribution of heterochromatin protein HP1α (CBX5) is unknown. We used a novel HP1α auxin-inducible degron human cell line to rapidly degrade HP1α. Degradation did not alter transcription, local chromatin compaction, or histone methylation, but did decrease chromatin stiffness. Single-nucleus micromanipulation reveals that HP1α is essential to chromatin-based mechanics and maintains nuclear morphology, separate from histone methylation. Further experiments with dimerization-deficient HP1αI165E indicate that chromatin crosslinking via HP1α dimerization is critical, while polymer simulations demonstrate the importance of chromatin-chromatin crosslinkers in mechanics. In mitotic chromosomes, HP1α similarly bolsters stiffness while aiding in mitotic alignment and faithful segregation. HP1α is therefore a critical chromatin-crosslinking protein that provides mechanical strength to chromosomes and the nucleus throughout the cell cycle and supports cellular functions.
Asunto(s)
Núcleo Celular/metabolismo , Cromatina , Proteínas Cromosómicas no Histona , Cromosomas , Mitosis/fisiología , Línea Celular , Núcleo Celular/química , Cromatina/química , Cromatina/metabolismo , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/química , Cromosomas/metabolismo , Humanos , MetilaciónRESUMEN
Membraneless organelles or condensates form through liquid-liquid phase separation1-4, which is thought to underlie gene transcription through condensation of the large-scale nucleolus5-7 or in smaller assemblies known as transcriptional condensates8-11. Transcriptional condensates have been hypothesized to phase separate at particular genomic loci and locally promote the biomolecular interactions underlying gene expression. However, there have been few quantitative biophysical tests of this model in living cells, and phase separation has not yet been directly linked with dynamic transcriptional outputs12,13. Here, we apply an optogenetic approach to show that FET-family transcriptional regulators exhibit a strong tendency to phase separate within living cells, a process that can drive localized RNA transcription. We find that TAF15 has a unique charge distribution among the FET family members that enhances its interactions with the C-terminal domain of RNA polymerase II. Nascent C-terminal domain clusters at primed genomic loci lower the energetic barrier for nucleation of TAF15 condensates, which in turn further recruit RNA polymerase II to drive transcriptional output. These results suggest that positive feedback between interacting transcriptional components drives localized phase separation to amplify gene expression.
Asunto(s)
Nucléolo Celular/metabolismo , Regulación de la Expresión Génica , Proteínas Intrínsecamente Desordenadas/metabolismo , Orgánulos/metabolismo , ARN Polimerasa II/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Animales , Nucléolo Celular/genética , Citoplasma/metabolismo , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Ratones , Orgánulos/genética , Transición de Fase , ARN Polimerasa II/química , ARN Polimerasa II/genética , Factores Asociados con la Proteína de Unión a TATA/química , Factores Asociados con la Proteína de Unión a TATA/genéticaRESUMEN
Liquid-liquid phase separation (LLPS) mediates formation of membraneless condensates such as those associated with RNA processing, but the rules that dictate their assembly, substructure, and coexistence with other liquid-like compartments remain elusive. Here, we address the biophysical mechanism of this multiphase organization using quantitative reconstitution of cytoplasmic stress granules (SGs) with attached P-bodies in human cells. Protein-interaction networks can be viewed as interconnected complexes (nodes) of RNA-binding domains (RBDs), whose integrated RNA-binding capacity determines whether LLPS occurs upon RNA influx. Surprisingly, both RBD-RNA specificity and disordered segments of key proteins are non-essential, but modulate multiphase condensation. Instead, stoichiometry-dependent competition between protein networks for connecting nodes determines SG and P-body composition and miscibility, while competitive binding of unconnected proteins disengages networks and prevents LLPS. Inspired by patchy colloid theory, we propose a general framework by which competing networks give rise to compositionally specific and tunable condensates, while relative linkage between nodes underlies multiphase organization.
Asunto(s)
Gránulos Citoplasmáticos/fisiología , Estructuras Citoplasmáticas/fisiología , Mapas de Interacción de Proteínas/fisiología , Fenómenos Biofísicos , Línea Celular Tumoral , Citoplasma/metabolismo , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Extracción Líquido-Líquido/métodos , Orgánulos/química , ARN/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/fisiologíaRESUMEN
Cells organize membrane-less internal compartments through a process called liquid-liquid phase separation (LLPS) to create chemically distinct compartments, referred to as condensates, which emerge from interactions among biological macromolecules. These condensates include various cytoplasmic structures such as P-granules and stress granules. However, an even wider array of condensates subcompartmentalize the cell nucleus, forming liquid-like structures that range from nucleoli and Cajal bodies to nuclear speckles and gems. Phase separation provides a biophysical assembly mechanism underlying this non-covalent form of fluid compartmentalization and functionalization. In this Cell Science at a Glance article and the accompanying poster, we term these phase-separated liquids that organize the nucleus the liquid nucleome; we discuss examples of biological phase transitions in the nucleus, how the cell utilizes biophysical aspects of phase separation to form and regulate condensates, and suggest interpretations for the role of phase separation in nuclear organization and function.
Asunto(s)
Núcleo Celular/fisiología , Extracción Líquido-Líquido/métodosRESUMEN
Constitutive heterochromatin is an important component of eukaryotic genomes that has essential roles in nuclear architecture, DNA repair and genome stability, and silencing of transposon and gene expression. Heterochromatin is highly enriched for repetitive sequences, and is defined epigenetically by methylation of histone H3 at lysine 9 and recruitment of its binding partner heterochromatin protein 1 (HP1). A prevalent view of heterochromatic silencing is that these and associated factors lead to chromatin compaction, resulting in steric exclusion of regulatory proteins such as RNA polymerase from the underlying DNA. However, compaction alone does not account for the formation of distinct, multi-chromosomal, membrane-less heterochromatin domains within the nucleus, fast diffusion of proteins inside the domain, and other dynamic features of heterochromatin. Here we present data that support an alternative hypothesis: that the formation of heterochromatin domains is mediated by phase separation, a phenomenon that gives rise to diverse non-membrane-bound nuclear, cytoplasmic and extracellular compartments. We show that Drosophila HP1a protein undergoes liquid-liquid demixing in vitro, and nucleates into foci that display liquid properties during the first stages of heterochromatin domain formation in early Drosophila embryos. Furthermore, in both Drosophila and mammalian cells, heterochromatin domains exhibit dynamics that are characteristic of liquid phase-separation, including sensitivity to the disruption of weak hydrophobic interactions, and reduced diffusion, increased coordinated movement and inert probe exclusion at the domain boundary. We conclude that heterochromatic domains form via phase separation, and mature into a structure that includes liquid and stable compartments. We propose that emergent biophysical properties associated with phase-separated systems are critical to understanding the unusual behaviours of heterochromatin, and how chromatin domains in general regulate essential nuclear functions.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Heterocromatina/química , Heterocromatina/metabolismo , Animales , Línea Celular , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/química , ADN/química , ADN/genética , ADN/metabolismo , Difusión , Drosophila melanogaster , Femenino , Silenciador del Gen , Heterocromatina/genética , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Células 3T3 NIH , Transición de Fase , SolubilidadRESUMEN
Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors and regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions.