HP1 reshapes nucleosome core to promote phase separation of heterochromatin
TL;DR: The results indicate that Swi6 couples its oligomerization to the phase separation of chromatin by a counterintuitive mechanism, namely the dynamic exposure of buried nucleosomal regions, which increases opportunities for multivalent interactions between nucleosomes, thereby promoting phase separation.
Abstract: Heterochromatin affects genome function at many levels. It enables heritable gene repression, maintains chromosome integrity and provides mechanical rigidity to the nucleus1,2. These diverse functions are proposed to arise in part from compaction of the underlying chromatin2. A major type of heterochromatin contains at its core the complex formed between HP1 proteins and chromatin that is methylated on histone H3, lysine 9 (H3K9me). HP1 is proposed to use oligomerization to compact chromatin into phase-separated condensates3-6. Yet, how HP1-mediated phase separation relates to chromatin compaction remains unclear. Here we show that chromatin compaction by the Schizosaccharomyces pombe HP1 protein Swi6 results in phase-separated liquid condensates. Unexpectedly, we find that Swi6 substantially increases the accessibility and dynamics of buried histone residues within a nucleosome. Restraining these dynamics impairs compaction of chromatin into liquid droplets by Swi6. Our results indicate that Swi6 couples its oligomerization to the phase separation of chromatin by a counterintuitive mechanism, namely the dynamic exposure of buried nucleosomal regions. We propose that such reshaping of the octamer core by Swi6 increases opportunities for multivalent interactions between nucleosomes, thereby promoting phase separation. This mechanism may more generally drive chromatin organization beyond heterochromatin.
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TL;DR: A review of the role of biomolecular condensates in ageing and disease can be found in this paper, where the authors discuss how cellular stress, ageing-related loss of homeostasis and a decline in protein quality control may contribute to the formation of aberrant, disease-causing condensate.
Abstract: Biomolecular condensates are membraneless intracellular assemblies that often form via liquid-liquid phase separation and have the ability to concentrate biopolymers. Research over the past 10 years has revealed that condensates play fundamental roles in cellular organization and physiology, and our understanding of the molecular principles, components and forces underlying their formation has substantially increased. Condensate assembly is tightly regulated in the intracellular environment, and failure to control condensate properties, formation and dissolution can lead to protein misfolding and aggregation, which are often the cause of ageing-associated diseases. In this Review, we describe the mechanisms and regulation of condensate assembly and dissolution, highlight recent advances in understanding the role of biomolecular condensates in ageing and disease, and discuss how cellular stress, ageing-related loss of homeostasis and a decline in protein quality control may contribute to the formation of aberrant, disease-causing condensates. Our improved understanding of condensate pathology provides a promising path for the treatment of protein aggregation diseases.
376 citations
TL;DR: A framework for the study of condensate functions across these cellular length scales is provided, offering to bring new understanding of biological processes.
Abstract: Biomolecular condensates are found throughout eukaryotic cells, including in the nucleus, in the cytoplasm and on membranes. They are also implicated in a wide range of cellular functions, organizing molecules that act in processes ranging from RNA metabolism to signalling to gene regulation. Early work in the field focused on identifying condensates and understanding how their physical properties and regulation arise from molecular constituents. Recent years have brought a focus on understanding condensate functions. Studies have revealed functions that span different length scales: from molecular (modulating the rates of chemical reactions) to mesoscale (organizing large structures within cells) to cellular (facilitating localization of cellular materials and homeostatic responses). In this Roadmap, we discuss representative examples of biochemical and cellular functions of biomolecular condensates from the recent literature and organize these functions into a series of non-exclusive classes across the different length scales. We conclude with a discussion of areas of current interest and challenges in the field, and thoughts about how progress may be made to further our understanding of the widespread roles of condensates in cell biology.
355 citations
TL;DR: The interplay of genetic, biochemical, biophysical, and cell biological methods can account for the emergence of structural chromatin features, the diversity and single-cell heterogeneity of nuclear architecture in cell types and tissues, and explains evolutionarily conserved functional features of genomes, including plasticity and robustness.
260 citations
TL;DR: The shared features of nuclear condensate, the components that produce locus-specific condensates, elements of specificity, and the emerging understanding of mechanisms regulating these compartments are described.
217 citations
TL;DR: The current understanding of the mechanisms by which pioneer factors initiate gene network changes and will ultimately contribute to the ability to control cell fates at will are focused on.
Abstract: Pioneer transcription factors have the intrinsic biochemical ability to scan partial DNA sequence motifs that are exposed on the surface of a nucleosome and thus access silent genes that are inaccessible to other transcription factors. Pioneer factors subsequently enable other transcription factors, nucleosome remodeling complexes, and histone modifiers to engage chromatin, thereby initiating the formation of an activating or repressive regulatory sequence. Thus, pioneer factors endow the competence for fate changes in embryonic development, are essential for cellular reprogramming, and rewire gene networks in cancer cells. Recent studies with reconstituted nucleosomes in vitro and chromatin binding in vivo reveal that pioneer factors can directly perturb nucleosome structure and chromatin accessibility in different ways. This review focuses on our current understanding of the mechanisms by which pioneer factors initiate gene network changes and will ultimately contribute to our ability to control cell fates at will.
207 citations
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TL;DR: The asynchronous pipeline scheme provides other substantial advantages, including high flexibility, favorable processing speeds, choice of both all-in-memory and disk-bound processing, easy adaptation to different data formats, simpler software development and maintenance, and the ability to distribute processing tasks on multi-CPU computers and computer networks.
Abstract: The NMRPipe system is a UNIX software environment of processing, graphics, and analysis tools designed to meet current routine and research-oriented multidimensional processing requirements, and to anticipate and accommodate future demands and developments. The system is based on UNIX pipes, which allow programs running simultaneously to exchange streams of data under user control. In an NMRPipe processing scheme, a stream of spectral data flows through a pipeline of processing programs, each of which performs one component of the overall scheme, such as Fourier transformation or linear prediction. Complete multidimensional processing schemes are constructed as simple UNIX shell scripts. The processing modules themselves maintain and exploit accurate records of data sizes, detection modes, and calibration information in all dimensions, so that schemes can be constructed without the need to explicitly define or anticipate data sizes or storage details of real and imaginary channels during processing. The asynchronous pipeline scheme provides other substantial advantages, including high flexibility, favorable processing speeds, choice of both all-in-memory and disk-bound processing, easy adaptation to different data formats, simpler software development and maintenance, and the ability to distribute processing tasks on multi-CPU computers and computer networks.
13,804 citations
TL;DR: The X-ray crystal structure of the nucleosome core particle of chromatin shows in atomic detail how the histone protein octamer is assembled and how 146 base pairs of DNA are organized into a superhelix around it.
Abstract: The X-ray crystal structure of the nucleosome core particle of chromatin shows in atomic detail how the histone protein octamer is assembled and how 146 base pairs of DNA are organized into a superhelix around it. Both histone/histone and histone/DNA interactions depend on the histone fold domains and additional, well ordered structure elements extending from this motif. Histone amino-terminal tails pass over and between the gyres of the DNA superhelix to contact neighbouring particles. The lack of uniformity between multiple histone/DNA-binding sites causes the DNA to deviate from ideal superhelix geometry.
7,841 citations
TL;DR: In this article, the authors propose guidelines for rigorous experimental characterization of liquid-liquid phase separation processes in vitro and in cells, discuss the caveats of common experimental approaches, and point out experimental and theoretical gaps in the field.
1,482 citations
TL;DR: It is proposed 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.
Abstract: HP1a can nucleate into foci that display liquid properties during the early stages of heterochromatin domain formation in Drosophila embryos, suggesting that the repressive action of heterochromatin may be mediated in part by emergent properties of phase separation. The gene-silencing action of heterochromatin is thought to arise from the spread of proteins such as HP1 that compact the underlying chromatin and recruit repressors. Two papers in this issue demonstrate that HP1α has the ability to form phase-separated droplets. Gary Karpen and colleagues show that HP1α can nucleate into foci that display liquid properties during the early stages of heterochromatin domain formation in Drosophila embryos. Geeta Narlikar and colleagues demonstrate that human HP1α protein also forms phase-separated droplets. Phosphorylation or DNA binding promotes the physical partitioning of HP1α out of the soluble aqueous phase into droplets. These related findings suggest that the repressive action of heterochromatin may be in part mediated by the phase separation of HP1, with the droplets being initiated or dissolved by various ligands depending on nuclear context. Constitutive heterochromatin is an important component of eukaryotic genomes that has essential roles in nuclear architecture, DNA repair and genome stability1, and silencing of transposon and gene expression2. 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 DNA3. 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 compartments4. 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.
1,306 citations
TL;DR: It is shown by direct protein delivery into mammalian cells that an HP1 α mutant incapable of phase separation in vitro forms smaller and fewer nuclear puncta than phosphorylated HP1α, suggesting that heterochromatin-mediated gene silencing may occur in part through sequestration of compacted chromatin in phase-separated HP1 droplets, which are dissolved or formed by specific ligands on the basis of nuclear context.
Abstract: Gene silencing by heterochromatin is proposed to occur in part as a result of the ability of heterochromatin protein 1 (HP1) proteins to spread across large regions of the genome, compact the underlying chromatin and recruit diverse ligands. Here we identify a new property of the human HP1α protein: the ability to form phase-separated droplets. While unmodified HP1α is soluble, either phosphorylation of its N-terminal extension or DNA binding promotes the formation of phase-separated droplets. Phosphorylation-driven phase separation can be promoted or reversed by specific HP1α ligands. Known components of heterochromatin such as nucleosomes and DNA preferentially partition into the HP1α droplets, but molecules such as the transcription factor TFIIB show no preference. Using a single-molecule DNA curtain assay, we find that both unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, although with different characteristics. We show by direct protein delivery into mammalian cells that an HP1α mutant incapable of phase separation in vitro forms smaller and fewer nuclear puncta than phosphorylated HP1α. These findings suggest that heterochromatin-mediated gene silencing may occur in part through sequestration of compacted chromatin in phase-separated HP1 droplets, which are dissolved or formed by specific ligands on the basis of nuclear context.
1,247 citations