Phase Separation in Biology and Disease; Current Perspectives and Open Questions.

In the past almost 15 years, we witnessed the birth of a new scientific field focused on the existence, formation, biological functions, and disease associations of membraneless bodies in cells, now referred to as biomolecular condensates. Pioneering studies from several laboratories [reviewed in1-3] supported a model wherein biomolecular condensates associated with diverse biological processes form through the process of phase separation. These and other findings that followed have revolutionized our understanding of how biomolecules are organized in space and time within cells to perform myriad biological functions, including cell fate determination, signal transduction, endocytosis, regulation of gene expression and protein translation, and regulation of RNA metabolism. Further, condensates formed through aberrant phase transitions have been associated with numerous human diseases, prominently including neurodegeneration and cancer. While in some cases, rigorous evidence supports links between formation of biomolecular condensates through phase separation and biological functions, in many others such links are less robustly supported, which has led to rightful scrutiny of the generality of the roles of phase separation in biology and disease.4-7 During a week-long workshop in March 2022 at the Telluride Science Research Center (TSRC) in Telluride, Colorado, ∼25 scientists addressed key questions surrounding the biomolecular condensates field. Herein, we present insights gained through these discussions, addressing topics including, roles of condensates in diverse biological processes and systems, and normal and disease cell states, their applications to synthetic biology, and the potential for therapeutically targeting biomolecular condensates.

Generation and Biochemical Characterization of Phase-Separated Droplets Formed by Nucleic Acid Binding Proteins: Using HP1 as a Model System.

Liquid-liquid phase separation (LLPS) has been invoked as an underlying mechanism involved in the formation and function of several cellular membrane-less compartments. Given the explosion of studies in this field in recent years, it has become essential to converge on clear guidelines and methods to rigorously investigate LLPS and advance our understanding of this phenomenon. Here, we describe basic methods to (1) visualize droplets formed by nucleic acid binding proteins and (2) characterize the liquid-like nature of these droplets under controlled in vitro experimental conditions. We discuss the rationale behind these methods, as well as caveats and limitations. Our ultimate goal is to guide scientists interested in learning how to test for LLPS, while appreciating that the field is evolving rapidly and adjusting constantly to the growing knowledge. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Observing phase-separated condensates by microscopy. Support Protocol: Coating of glass-bottom plates. Basic Protocol 2: Assessing condensate reversibility by changing ionic strength. Alternate Protocol 1: Assessing condensate reversibility by dilution. Alternate Protocol 2: Assessing condensate reversibility by altering temperature. Basic Protocol 3: Quantifying phase separation by centrifugation assay. Basic Protocol 4: Quantifying phase separation by turbidity assay.

Liquid-like interactions in heterochromatin: Implications for mechanism and regulation.

A large portion of the eukaryotic genome is packed into heterochromatin, a versatile platform that is essential to maintain genome stability. Often associated with a compact and transcriptionally repressed chromatin state, heterochromatin was earlier considered a static and locked compartment. However, cumulative findings over the last 17 years have suggested that heterochromatin displays dynamics at different timescales and size scales. These dynamics are thought to be essential for the regulation of heterochromatin. This review illustrates how the key principles underlying heterochromatin structure and function have evolved along the years and summarizes the discoveries that have led to the continuous revision of these principles. Using heterochromatin protein 1-mediated heterochromatin as a context, we discuss a novel paradigm for heterochromatin organization based on two emerging concepts, phase separation and nucleosome structural plasticity. We also examine the broader implications of this paradigm for chromatin organization and regulation beyond heterochromatin.

Biophysical Properties of HP1-Mediated Heterochromatin.

Heterochromatin is a classic context for studying the mechanisms of chromatin organization. At the core of a highly conserved type of heterochromatin is the complex formed between chromatin methylated on histone H3 lysine 9 and HP1 proteins. This type of heterochromatin plays central roles in gene repression, genome stability, and nuclear mechanics. Systematic studies over the last several decades have provided insight into the biophysical mechanisms by which the HP1-chromatin complex is formed. Here, we discuss these studies together with recent findings indicating a role for phase separation in heterochromatin organization and function. We suggest that the different functions of HP1-mediated heterochromatin may rely on the increasing diversity being uncovered in the biophysical properties of HP1-chromatin complexes.

Biochemical Basis for Distinct Roles of the Heterochromatin Proteins Swi6 and Chp2.

Heterochromatin protein 1 (HP1) family proteins are conserved chromatin binding proteins involved in gene silencing, chromosome packaging, and chromosome segregation. These proteins recognize histone H3 lysine 9 methylated tails via their chromodomain and recruit additional ligand proteins with diverse activities through their dimerization domain, the chromoshadow domain. Species that have HP1 proteins possess multiple paralogs that perform non-overlapping roles in vivo. How different HP1 proteins, which are highly conserved, perform different functions is not well understood. Here, we use the two Schizosaccharomyces pombe HP1 paralogs, Swi6 and Chp2, as model systems to compare and contrast their biophysical properties. We find that Swi6 and Chp2 have similar dimerization and oligomerization equilibria, and that Swi6 binds slightly (~3-fold) more strongly to nucleosomes than Chp2. Furthermore, while Swi6 binding to the H3K9me3 mark is regulated by a previously described auto-inhibition mechanism, the binding of Chp2 to the H3K9me3 mark is not analogously regulated. In the context of chromoshadow domain interactions, we show using a newly identified peptide sequence from the Clr3 histone deacetylase and a previously identified sequence from the protein Shugoshin that the Swi6 chromoshadow domain binds both ligands more strongly than the Chp2. Overall, our findings uncover quantitative differences in how Swi6 and Chp2 interact with nucleosomal and non-nucleosomal ligands and qualitative differences in how their assembly on nucleosomes is regulated. These findings provide a biochemical framework to explain the varied functions of Chp2 and Swi6 in vivo.

Jarid2 Methylation via the PRC2 Complex Regulates H3K27me3 Deposition during Cell Differentiation.

Polycomb Group (PcG) proteins maintain transcriptional repression throughout development, mostly by regulating chromatin structure. Polycomb Repressive Complex 2 (PRC2), a component of the Polycomb machinery, is responsible for the methylation of histone H3 lysine 27 (H3K27me2/3). Jarid2 was previously identified as a cofactor of PRC2, regulating PRC2 targeting to chromatin and its enzymatic activity. Deletion of Jarid2 leads to impaired orchestration of gene expression during cell lineage commitment. Here, we reveal an unexpected crosstalk between Jarid2 and PRC2, with Jarid2 being methylated by PRC2. This modification is recognized by the Eed core component of PRC2 and triggers an allosteric activation of PRC2's enzymatic activity. We show that Jarid2 methylation is important to promote PRC2 activity at a locus devoid of H3K27me3 and for the correct deposition of this mark during cell differentiation. Our results uncover a regulation loop where Jarid2 methylation fine-tunes PRC2 activity depending on the chromatin context.

Jarid2 Is Implicated in the Initial Xist-Induced Targeting of PRC2 to the Inactive X Chromosome.

During X chromosome inactivation (XCI), the Polycomb Repressive Complex 2 (PRC2) is thought to participate in the early maintenance of the inactive state. Although Xist RNA is essential for the recruitment of PRC2 to the X chromosome, the precise mechanism remains unclear. Here, we demonstrate that the PRC2 cofactor Jarid2 is an important mediator of Xist-induced PRC2 targeting. The region containing the conserved B and F repeats of Xist is critical for Jarid2 recruitment via its unique N-terminal domain. Xist-induced Jarid2 recruitment occurs chromosome-wide independently of a functional PRC2 complex, unlike at other parts of the genome, such as CG-rich regions, where Jarid2 and PRC2 binding are interdependent. Conversely, we show that Jarid2 loss prevents efficient PRC2 and H3K27me3 enrichment to Xist-coated chromatin. Jarid2 thus represents an important intermediate between PRC2 and Xist RNA for the initial targeting of the PRC2 complex to the X chromosome during onset of XCI.

Legionella pneumophila effector RomA uniquely modifies host chromatin to repress gene expression and promote intracellular bacterial replication.

Histone posttranslational modifications control eukaryotic gene expression and regulate many biological processes including immunity. Pathogens alter host epigenetic control to aid pathogenesis. We find that the intracellular bacterial pathogen Legionella pneumophila uses a Dot/Icm type IV secreted effector, RomA, to uniquely modify the host chromatin landscape. RomA, a SET domain-containing methyltransferase, trimethylates K14 of histone H3, a histone mark not previously described in mammals. RomA localizes to the infected cell nucleus where it promotes a burst of H3K14 methylation and consequently decreases H3K14 acetylation, an activating histone mark, to repress host gene expression. ChIP-seq analysis identified 4,870 H3K14 methylated promoter regions, including innate immune genes. Significantly reduced replication of a RomA-deleted strain in host cells was trans-complemented by wild-type, but not by catalytically inactive, RomA. Thus, a secreted L. pneumophila effector targets the host cell nucleus and modifies histones to repress gene expression and promote efficient intracellular replication.

Skeletal muscle stem cells adopt a dormant cell state post mortem and retain regenerative capacity.

The accessibility to stem cells from healthy or diseased individuals, and the maintenance of their potency are challenging issues for stem cell biology. Here we report the isolation of viable and functional skeletal myogenic cells from humans up to 17 days, and mice up to 14 days post mortem, much longer beyond previous reports. Muscle stem cells are enriched in post mortem tissue, suggesting a selective survival advantage compared with other cell types. Transplantation of mouse muscle and haematopoietic stem cells regenerates tissues robustly. Cellular quiescence contributes to this cell viability where cells adopt a reversible dormant state characterized by reduced metabolic activity, a prolonged lag phase before the first cell division, elevated levels of reactive oxygen species and a transcriptional status less primed for commitment. Finally, severe hypoxia, or anoxia is critical for maintaining stem cell viability and regenerative capacity. Thus, these cells provide a useful resource for studying stem cell biology.