Small-molecule-induced epigenetic rejuvenation promotes SREBP condensation and overcomes barriers to CNS myelin regeneration.

Remyelination failure in diseases like multiple sclerosis (MS) was thought to involve suppressed maturation of oligodendrocyte precursors; however, oligodendrocytes are present in MS lesions yet lack myelin production. We found that oligodendrocytes in the lesions are epigenetically silenced. Developing a transgenic reporter labeling differentiated oligodendrocytes for phenotypic screening, we identified a small-molecule epigenetic-silencing-inhibitor (ESI1) that enhances myelin production and ensheathment. ESI1 promotes remyelination in animal models of demyelination and enables de novo myelinogenesis on regenerated CNS axons. ESI1 treatment lengthened myelin sheaths in human iPSC-derived organoids and augmented (re)myelination in aged mice while reversing age-related cognitive decline. Multi-omics revealed that ESI1 induces an active chromatin landscape that activates myelinogenic pathways and reprograms metabolism. Notably, ESI1 triggered nuclear condensate formation of master lipid-metabolic regulators SREBP1/2, concentrating transcriptional co-activators to drive lipid/cholesterol biosynthesis. Our study highlights the potential of targeting epigenetic silencing to enable CNS myelin regeneration in demyelinating diseases and aging.

Macromolecular condensation organizes nucleolar sub-phases to set up a pH gradient.

Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass. In vitro reconstitutions and studies in cells showed how condensation, which combines binding and complex coacervation of nucleolar components, contributes to nucleolar organization. D/E tracts of nucleolar proteins contribute to lowering the pH of co-condensates formed with nucleolar RNAs in vitro. In cells, this sets up a pH gradient between nucleoli and the nucleoplasm. By contrast, juxta-nucleolar bodies, which have different macromolecular compositions, featuring protein IDRs with very different charge profiles, have pH values that are equivalent to or higher than the nucleoplasm. Our findings show that distinct compositional specificities generate distinct physicochemical properties for condensates.

Thiolase: A Versatile Biocatalyst Employing Coenzyme A-Thioester Chemistry for Making and Breaking C-C Bonds.

Thiolases are CoA-dependent enzymes that catalyze the thiolytic cleavage of 3-ketoacyl-CoA, as well as its reverse reaction, which is the thioester-dependent Claisen condensation reaction. Thiolases are dimers or tetramers (dimers of dimers). All thiolases have two reactive cysteines: (a) a nucleophilic cysteine, which forms a covalent intermediate, and (b) an acid/base cysteine. The best characterized thiolase is the Zoogloea ramigera thiolase, which is a bacterial biosynthetic thiolase belonging to the CT-thiolase subfamily. The thiolase active site is also characterized by two oxyanion holes, two active site waters, and four catalytic loops with characteristic amino acid sequence fingerprints. Three thiolase subfamilies can be identified, each characterized by a unique sequence fingerprint for one of their catalytic loops, which causes unique active site properties. Recent insights concerning the thiolase reaction mechanism, as obtained from recent structural studies, as well as from classical and recent enzymological studies, are addressed, and open questions are discussed.

DMA-tudor interaction modules control the specificity of in vivo condensates.

Biomolecular condensation is a widespread mechanism of cellular compartmentalization. Because the "survival of motor neuron protein" (SMN) is implicated in the formation of three different membraneless organelles (MLOs), we hypothesized that SMN promotes condensation. Unexpectedly, we found that SMN's globular tudor domain was sufficient for dimerization-induced condensation in vivo, whereas its two intrinsically disordered regions (IDRs) were not. Binding to dimethylarginine (DMA) modified protein ligands was required for condensate formation by the tudor domains in SMN and at least seven other fly and human proteins. Remarkably, asymmetric versus symmetric DMA determined whether two distinct nuclear MLOs-gems and Cajal bodies-were separate or "docked" to one another. This substructure depended on the presence of either asymmetric or symmetric DMA as visualized with sub-diffraction microscopy. Thus, DMA-tudor interaction modules-combinations of tudor domains bound to their DMA ligand(s)-represent versatile yet specific regulators of MLO assembly, composition, and morphology.

TDP-43 condensation properties specify its RNA-binding and regulatory repertoire.

Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These "binding-region condensates" are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution.

Condensed Chromatin Behaves like a Solid on the Mesoscale In Vitro and in Living Cells.

The association of nuclear DNA with histones to form chromatin is essential for temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of condensed chromatin in vitro and in vivo. Our in vitro studies demonstrate that self-association of nucleosomal arrays under a wide range of solution conditions produces supramolecular condensates in which the chromatin is physically constrained and solid-like. By measuring DNA mobility in living cells, we show that condensed chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, display liquid-like behavior and coalesce around the solid chromatin scaffold. Importantly, euchromatin and heterochromatin show solid-like behavior even under conditions that produce limited interactions between chromatin fibers. Our results reveal that condensed chromatin exists in a solid-like state whose properties resist external forces and create an elastic gel and provides a scaffold that supports liquid-liquid phase separation of chromatin binding proteins.

Nuclear Pores Assemble from Nucleoporin Condensates During Oogenesis.

The molecular events that direct nuclear pore complex (NPC) assembly toward nuclear envelopes have been conceptualized in two pathways that occur during mitosis or interphase, respectively. In gametes and embryonic cells, NPCs also occur within stacked cytoplasmic membrane sheets, termed annulate lamellae (AL), which serve as NPC storage for early development. The mechanism of NPC biogenesis at cytoplasmic membranes remains unknown. Here, we show that during Drosophila oogenesis, Nucleoporins condense into different precursor granules that interact and progress into NPCs. Nup358 is a key player that condenses into NPC assembly platforms while its mRNA localizes to their surface in a translation-dependent manner. In concert, Microtubule-dependent transport, the small GTPase Ran and nuclear transport receptors regulate NPC biogenesis in oocytes. We delineate a non-canonical NPC assembly mechanism that relies on Nucleoporin condensates and occurs away from the nucleus under conditions of cell cycle arrest.

The Cohesin Ring Uses Its Hinge to Organize DNA Using Non-topological as well as Topological Mechanisms.

As predicted by the notion that sister chromatid cohesion is mediated by entrapment of sister DNAs inside cohesin rings, there is perfect correlation between co-entrapment of circular minichromosomes and sister chromatid cohesion. In most cells where cohesin loads without conferring cohesion, it does so by entrapment of individual DNAs. However, cohesin with a hinge domain whose positively charged lumen is neutralized loads and moves along chromatin despite failing to entrap DNAs. Thus, cohesin engages chromatin in non-topological, as well as topological, manners. Since hinge mutations, but not Smc-kleisin fusions, abolish entrapment, DNAs may enter cohesin rings through hinge opening. Mutation of three highly conserved lysine residues inside the Smc1 moiety of Smc1/3 hinges abolishes all loading without affecting cohesin's recruitment to CEN loading sites or its ability to hydrolyze ATP. We suggest that loading and translocation are mediated by conformational changes in cohesin's hinge driven by cycles of ATP hydrolysis.

Phase Transitions in the Assembly and Function of Human miRISC.

miRISC is a multi-protein assembly that uses microRNAs (miRNAs) to identify mRNAs targeted for repression. Dozens of miRISC-associated proteins have been identified, and interactions between many factors have been examined in detail. However, the physical nature of the complex remains unknown. Here, we show that two core protein components of human miRISC, Argonaute2 (Ago2) and TNRC6B, condense into phase-separated droplets in vitro and in live cells. Phase separation is promoted by multivalent interactions between the glycine/tryptophan (GW)-rich domain of TNRC6B and three evenly spaced tryptophan-binding pockets in the Ago2 PIWI domain. miRISC droplets formed in vitro recruit deadenylation factors and sequester target RNAs from the bulk solution. The condensation of miRISC is accompanied by accelerated deadenylation of target RNAs bound to Ago2. The combined results may explain how miRISC silences mRNAs of varying size and structure and provide experimental evidence that protein-mediated phase separation can facilitate an RNA processing reaction.

Global DNA Compaction in Stationary-Phase Bacteria Does Not Affect Transcription.

In stationary-phase Escherichia coli, Dps (DNA-binding protein from starved cells) is the most abundant protein component of the nucleoid. Dps compacts DNA into a dense complex and protects it from damage. Dps has also been proposed to act as a global regulator of transcription. Here, we directly examine the impact of Dps-induced compaction of DNA on the activity of RNA polymerase (RNAP). Strikingly, deleting the dps gene decompacted the nucleoid but did not significantly alter the transcriptome and only mildly altered the proteome during stationary phase. Complementary in vitro assays demonstrated that Dps blocks restriction endonucleases but not RNAP from binding DNA. Single-molecule assays demonstrated that Dps dynamically condenses DNA around elongating RNAP without impeding its progress. We conclude that Dps forms a dynamic structure that excludes some DNA-binding proteins yet allows RNAP free access to the buried genes, a behavior characteristic of phase-separated organelles.

Centromeres License the Mitotic Condensation of Yeast Chromosome Arms.

During mitosis, chromatin condensation shapes chromosomes as separate, rigid, and compact sister chromatids to facilitate their segregation. Here, we show that, unlike wild-type yeast chromosomes, non-chromosomal DNA circles and chromosomes lacking a centromere fail to condense during mitosis. The centromere promotes chromosome condensation strictly in cis through recruiting the kinases Aurora B and Bub1, which trigger the autonomous condensation of the entire chromosome. Shugoshin and the deacetylase Hst2 facilitated spreading the condensation signal to the chromosome arms. Targeting Aurora B to DNA circles or centromere-ablated chromosomes or releasing Shugoshin from PP2A-dependent inhibition bypassed the centromere requirement for condensation and enhanced the mitotic stability of DNA circles. Our data indicate that yeast cells license the chromosome-autonomous condensation of their chromatin in a centromere-dependent manner, excluding from this process non-centromeric DNA and thereby inhibiting their propagation.

Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds.

Liquid-liquid phase separation plays a key role in the assembly of diverse intracellular structures. However, the biophysical principles by which phase separation can be precisely localized within subregions of the cell are still largely unclear, particularly for low-abundance proteins. Here, we introduce an oligomerizing biomimetic system, "Corelets," and utilize its rapid and quantitative light-controlled tunability to map full intracellular phase diagrams, which dictate the concentrations at which phase separation occurs and the transition mechanism, in a protein sequence dependent manner. Surprisingly, both experiments and simulations show that while intracellular concentrations may be insufficient for global phase separation, sequestering protein ligands to slowly diffusing nucleation centers can move the cell into a different region of the phase diagram, resulting in localized phase separation. This diffusive capture mechanism liberates the cell from the constraints of global protein abundance and is likely exploited to pattern condensates associated with diverse biological processes. VIDEO ABSTRACT.

Spermine enhances antiviral and anticancer responses by stabilizing DNA binding with the DNA sensor cGAS.

Self-nonself discrimination is vital for the immune system to mount responses against pathogens while maintaining tolerance toward the host and innocuous commensals during homeostasis. Here, we investigated how indiscriminate DNA sensors, such as cyclic GMP-AMP synthase (cGAS), make this self-nonself distinction. Screening of a small-molecule library revealed that spermine, a well-known DNA condenser associated with viral DNA, markedly elevates cGAS activation. Mechanistically, spermine condenses DNA to enhance and stabilize cGAS-DNA binding, optimizing cGAS and downstream antiviral signaling. Spermine promotes condensation of viral, but not host nucleosome, DNA. Deletion of viral DNA-associated spermine, by propagating virus in spermine-deficient cells, reduced cGAS activation. Spermine depletion subsequently attenuated cGAS-mediated antiviral and anticancer immunity. Collectively, our results reveal a pathogenic DNA-associated molecular pattern that facilitates nonself recognition, linking metabolism and pathogen recognition.

A liquid-like coat mediates chromosome clustering during mitotic exit.

The individualization of chromosomes during early mitosis and their clustering upon exit from cell division are two key transitions that ensure efficient segregation of eukaryotic chromosomes. Both processes are regulated by the surfactant-like protein Ki-67, but how Ki-67 achieves these diametric functions has remained unknown. Here, we report that Ki-67 radically switches from a chromosome repellent to a chromosome attractant during anaphase in human cells. We show that Ki-67 dephosphorylation during mitotic exit and the simultaneous exposure of a conserved basic patch induce the RNA-dependent formation of a liquid-like condensed phase on the chromosome surface. Experiments and coarse-grained simulations support a model in which the coalescence of chromosome surfaces, driven by co-condensation of Ki-67 and RNA, promotes clustering of chromosomes. Our study reveals how the switch of Ki-67 from a surfactant to a liquid-like condensed phase can generate mechanical forces during genome segregation that are required for re-establishing nuclear-cytoplasmic compartmentalization after mitosis.

Hormone-induced enhancer assembly requires an optimal level of hormone receptor multivalent interactions.

Transcription factors (TFs) activate enhancers to drive cell-specific gene programs in response to signals, but our understanding of enhancer assembly during signaling events is incomplete. Here, we show that androgen receptor (AR) forms condensates through multivalent interactions mediated by its N-terminal intrinsically disordered region (IDR) to orchestrate enhancer assembly in response to androgen signaling. AR IDR can be substituted by IDRs from selective proteins for AR condensation capacity and its function on enhancers. Expansion of the poly(Q) track within AR IDR results in a higher AR condensation propensity as measured by multiple methods, including live-cell single-molecule microscopy. Either weakening or strengthening AR condensation propensity impairs its heterotypic multivalent interactions with other enhancer components and diminishes its transcriptional activity. Our work reveals the requirement of an optimal level of AR condensation in mediating enhancer assembly and suggests that alteration of the fine-tuned multivalent IDR-IDR interactions might underlie AR-related human pathologies.

Catalytic and non-catalytic mechanisms of histone H4 lysine 20 methyltransferase SUV420H1.

SUV420H1 di- and tri-methylates histone H4 lysine 20 (H4K20me2/H4K20me3) and plays crucial roles in DNA replication, repair, and heterochromatin formation. It is dysregulated in several cancers. Many of these processes were linked to its catalytic activity. However, deletion and inhibition of SUV420H1 have shown distinct phenotypes, suggesting that the enzyme likely has uncharacterized non-catalytic activities. Our cryoelectron microscopy (cryo-EM), biochemical, biophysical, and cellular analyses reveal how SUV420H1 recognizes its nucleosome substrates, and how histone variant H2A.Z stimulates its catalytic activity. SUV420H1 binding to nucleosomes causes a dramatic detachment of nucleosomal DNA from the histone octamer, which is a non-catalytic activity. We hypothesize that this regulates the accessibility of large macromolecular complexes to chromatin. We show that SUV420H1 can promote chromatin condensation, another non-catalytic activity that we speculate is needed for its heterochromatin functions. Together, our studies uncover and characterize the catalytic and non-catalytic mechanisms of SUV420H1, a key histone methyltransferase that plays an essential role in genomic stability.

Evolutionary conservation of the structure and function of meiotic Rec114-Mei4 and Mer2 complexes.

Meiosis-specific Rec114-Mei4 and Mer2 complexes are thought to enable Spo11-mediated DNA double-strand break (DSB) formation through a mechanism that involves DNA-dependent condensation. However, the structure, molecular properties, and evolutionary conservation of Rec114-Mei4 and Mer2 are unclear. Here, we present AlphaFold models of Rec114-Mei4 and Mer2 complexes supported by nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), and mutagenesis. We show that dimers composed of the Rec114 C terminus form α-helical chains that cup an N-terminal Mei4 α helix, and that Mer2 forms a parallel homotetrameric coiled coil. Both Rec114-Mei4 and Mer2 bind preferentially to branched DNA substrates, indicative of multivalent protein-DNA interactions. Indeed, the Rec114-Mei4 interaction domain contains two DNA-binding sites that point in opposite directions and drive condensation. The Mer2 coiled-coil domain bridges coaligned DNA duplexes, likely through extensive electrostatic interactions along the length of the coiled coil. Finally, we show that the structures of Rec114-Mei4 and Mer2 are conserved across eukaryotes, while DNA-binding properties vary significantly. This work provides insights into the mechanism whereby Rec114-Mei4 and Mer2 complexes promote the assembly of the meiotic DSB machinery and suggests a model in which Mer2 condensation is the essential driver of assembly, with the DNA-binding activity of Rec114-Mei4 playing a supportive role.

Mesenchymal condensation in tooth development and regeneration: a focus on translational aspects of organogenesis.

The teeth are vertebrate-specific, highly specialized organs performing fundamental functions of mastication and speech, the maintenance of which is crucial for orofacial homeostasis and is further linked to systemic health and human psychosocial well-being. However, with limited ability for self-repair, the teeth can often be impaired by traumatic, inflammatory, and progressive insults, leading to high prevalence of tooth loss and defects worldwide. Regenerative medicine holds the promise to achieve physiological restoration of lost or damaged organs, and in particular an evolving framework of developmental engineering has pioneered functional tooth regeneration by harnessing the odontogenic program. As a key event of tooth morphogenesis, mesenchymal condensation dictates dental tissue formation and patterning through cellular self-organization and signaling interaction with the epithelium, which provides a representative to decipher organogenetic mechanisms and can be leveraged for regenerative purposes. In this review, we summarize how mesenchymal condensation spatiotemporally assembles from dental stem cells (DSCs) and sequentially mediates tooth development. We highlight condensation-mimetic engineering efforts and mechanisms based on ex vivo aggregation of DSCs, which have achieved functionally robust and physiologically relevant tooth regeneration after implantation in animals and in humans. The discussion of this aspect will add to the knowledge of development-inspired tissue engineering strategies and will offer benefits to propel clinical organ regeneration.

Stressful steps: Progress and challenges in understanding stress-induced mRNA condensation and accumulation in stress granules.

Stress-induced condensation of mRNA and protein into massive cytosolic clusters is conserved across eukaryotes. Known as stress granules when visible by imaging, these structures remarkably have no broadly accepted biological function, mechanism of formation or dispersal, or even molecular composition. As part of a larger surge of interest in biomolecular condensation, studies of stress granules and related RNA/protein condensates have increasingly probed the biochemical underpinnings of condensation. Here, we review open questions and recent advances, including the stages from initial condensate formation to accumulation in mature stress granules, mechanisms by which stress-induced condensates form and dissolve, and surprising twists in understanding the RNA components of stress granules and their role in condensation. We outline grand challenges in understanding stress-induced RNA condensation, centering on the unique and substantial barriers in the molecular study of cellular structures, such as stress granules, for which no biological function has been firmly established.

A walk through the SMC cycle: From catching DNAs to shaping the genome.

SMC protein complexes are molecular machines that provide structure to chromosomes. These complexes bridge DNA elements and by doing so build DNA loops in cis and hold together the sister chromatids in trans. We discuss how drastic conformational changes allow SMC complexes to build such intricate DNA structures. The tight regulation of these complexes controls fundamental chromosomal processes such as transcription, recombination, repair, and mitosis.