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 in] 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.

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.

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.

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.

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.

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.

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.

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.

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.