HP1 reshapes nucleosome core to promote phase separation of heterochromatin.

Gene silencing is intimately connected to DNA condensation and the formation of transcriptionally inactive heterochromatin by Heterochromatin Protein 1α (HP1α). Because heterochromatin foci are dynamic and HP1α can promote liquid-liquid phase separation, HP1α-mediated phase separation has been proposed as a mechanism of chromatin compaction. The molecular basis of HP1α-driven phase separation and chromatin compaction and the associated regulation by trimethylation of lysine 9 in histone 3 (H3K9me3), which is the hallmark of constitutive heterochromatin, is however largely unknown. Using a combination of chromatin compaction and phase separation assays, site-directed mutagenesis, and NMR-based interaction analysis, we show that human HP1α can compact chromatin in the absence of liquid-liquid phase separation. We further demonstrate that H3K9-trimethylation promotes compaction of chromatin arrays through multimodal interactions. The results provide molecular insights into HP1α-mediated chromatin compaction and thus into the role of human HP1α in the regulation of gene silencing.

Formation and Maturation of Phase-Separated Liquid Droplets by RNA-Binding Proteins

Drosophila Nurf55 is a component of different chromatin-modifying complexes, including the PRC2 (Polycomb repressive complex 2). Based on the 1.75-Å crystal structure of Nurf55 bound to histone H4 helix 1, we analyzed interactions of Nurf55 (Nurf55 or p55 in fly and RbAp48/46 in human) with the N-terminal tail of histone H3, the first helix of histone H4, and an N-terminal fragment of the PRC2 subunit Su(z)12 using isothermal calorimetry and pulldown experiments. Site-directed mutagenesis identified the binding site of histone H3 at the top of the Nurf55 WD40 propeller. Unmodified or K9me3- or K27me3-containing H3 peptides were bound with similar affinities, whereas the affinity for K4me3-containing H3 peptides was reduced. Helix 1 of histone H4 and Su(z)12 bound to the edge of the β-propeller using overlapping binding sites. Our results show similarities in the recognition of histone H4 and Su(z)12 and identify Nurf55 as a versatile interactor that simultaneously contacts multiple partners.

The swift rise in acceptance of molecular principles defining phase separation by a broad array of scientific disciplines is shadowed by increasing discoveries linking phase separation to pathological aggregations associated with numerous neurodegenerative disorders, including Alzheimer’s disease, that contribute to dementia. Phase separation is powered by multivalent macromolecular interactions. Importantly, the release of water molecules from protein hydration shells into bulk creates entropic gains that promote phase separation and the subsequent generation of insoluble cytotoxic aggregates that drive healthy brain cells into diseased states. Higher viscosity in interfacial waters and limited hydration in interiors of biomolecular condensates facilitate phase separation. Light, water, and melatonin constitute an ancient synergy that ensures adequate protein hydration to prevent aberrant phase separation. The 670 nm visible red wavelength found in sunlight and employed in photobiomodulation reduces interfacial and mitochondrial matrix viscosity to enhance ATP production via increasing ATP synthase motor efficiency. Melatonin is a potent antioxidant that lowers viscosity to increase ATP by scavenging excess reactive oxygen species and free radicals. Reduced viscosity by light and melatonin elevates the availability of free water molecules that allow melatonin to adopt favorable conformations that enhance intrinsic features, including binding interactions with adenosine that reinforces the adenosine moiety effect of ATP responsible for preventing water removal that causes hydrophobic collapse and aggregation in phase separation. Precise recalibration of interspecies melatonin dosages that account for differences in metabolic rates and bioavailability will ensure the efficacious reinstatement of the once-powerful ancient synergy between light, water, and melatonin in a modern world.

Eukaryotic cells partition a wide variety of important materials and processes into biomolecular condensates—phase-separated droplets that lack a membrane. In addition to nonspecific electrostatic or hydrophobic interactions, phase separation also depends on specific binding motifs that link together constituent molecules. Nevertheless, few rules have been established for how these ubiquitous specific, saturating, motif-motif interactions drive phase separation. By integrating Monte Carlo simulations of lattice-polymers with mean-field theory, we show that the sequence of heterotypic binding motifs strongly affects a polymer’s ability to phase separate, influencing both phase boundaries and condensate properties (e.g. viscosity and polymer diffusion). We find that sequences with large blocks of single motifs typically form more inter-polymer bonds, which promotes phase separation. Notably, the sequence of binding motifs influences phase separation primarily by determining the conformational entropy of self-bonding by single polymers. This contrasts with systems where the molecular architecture primarily affects the energy of the dense phase, providing a new entropy-based mechanism for the biological control of phase separation.

Microbial biofilms are shaped by the constant dialogue between biological and physical forces in the extracellular matrix.

Transcriptional pausing is a widely used regulatory mechanism to control precise gene expression from bacteria to humans. In metazoans, transcription initiation requires the recruitment of general transcription factors and RNA polymerase II (Pol II) to gene promoters. After it initiates transcription and synthesizes a short RNA, Pol II pauses at the promoter-proximal region, standing by for further cues to enter the productive elongation stage. Promoter-proximal Pol II pausing is one of the rate-limiting steps during transcription, functioning as an early transcriptional elongation check point, to ensure the correct modification of transcription machinery and the addition of the 7-methylguanosine cap to nascent transcripts. Pol II pausing at promoter-proximal region is stabilized by the negative elongation factor (NELF) and the 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF). In response to developmental signals or environmental stress, the release of paused Pol II can be triggered by the positive transcription elongation factor b (P-TEFb), which is composed of the kinase CDK9 and its regulatory subunit CCNT1/2. The kinase activity of P-TEFb towards Pol II carboxy-terminal domain (CTD), NELF and DSIF is critical for P-TEFb to execute its function in pause release, and is thus tightly controlled. To date, it has been identified that P-TEFb mainly exists in three different complexes: the super elongation complex (SEC), BRD4-P-TEFb, and the inactive 7SK snRNP-P-TEFb complex. SEC, one of the most active P-TEFb containing complexes, is required for rapid gene expression in response to stress or developmental signals. The active and inactive forms of P-TEFb complexes co-exist in vivo , reaching equilibrium to meet the transcriptional needs of cells. The transition of Pol II from pause to productive elongation stage is achieved by dynamic exchange between the pausing factors and the positive elongation factors at the proximal promoter region. Liquid-liquid phase separation (LLPS) in living cells is a physicochemical process that confines biochemical reactions within membrane-less compartments. Low complexity intrinsically disordered regions (IDRs) within proteins can initiate and promote phase separation and formation of liquid droplets, which in turn facilitate the multivalent weak interactions among these IDR-containing proteins. Recent studies using super-resolution live cell imaging technology have revealed that RNA Pol II can form dynamic foci in cells through LLPS and transitions between different phases, which are likely mediated by phosphorylated CTD. Moreover, factors involved in various transcription steps can rapidly and dynamically concentrate and exchange at the hot regulatory genomic loci. For example, SEC components are able to compartmentalize and concentrate P-TEFb from its inactive partners. Upon serum stimulation, SEC assembles promptly into phase-separated droplets at the immediate early genes (IEG), triggering rapid transcription induction. Mutations in transcription regulators can lead to developmental defects and many human diseases, including cancers. For instance, abnormal stabilization of SEC components via MLL fusion plays a contributing role in leukemogenesis. Fusion of the SEC core component ENL with MLL can greatly enhance phase separation of SEC, through which the leukemic genes might be mis-activated. In addition, there is growing evidence that disease related mutations in transcriptional regulators can cause malfunction through aberrant condensation, providing potential targets for designing therapeutic strategies through phase separation interventions.

Adenosine triphosphate (ATP) has been recognized as a hydrotrope in the phase separation process of intrinsically disordered proteins (IDPs). Surprisingly, when using the disordered Arg–Gly/Arg–Gly–Gly (RG/RGG) rich motif from the HNRNPG protein as a model system, we discover a biphasic relationship between the ATP concentration and IDP phase separation. We show that, at a relatively low ATP concentration, ATP dynamically interacts with the IDP, which neutralizes protein surface charges, promotes intermolecular interactions, and consequently promotes phase separation. We further demonstrate that ATP induces a compact conformation of the IDP, accounting for the reduced solvent exchange rate and lower compression ratio during phase separation. As ATP concentration increases, its hydrotropic properties emerge, leading to the dissolution of the phase-separated droplets. Our finding uncovers a complex mechanism by which ATP molecules modulate the structure, interaction, and phase separation of IDPs and accounts for the distinct phase separation behaviors of the charge-rich RGG motif and other low-complexity IDPs.

Phase separation has emerged as an important mechanism explaining the formation of certain biomolecular condensates. Biological phase separation is often driven by the multivalent interactions of modular protein domains. Beyond valency, the physical features of folded domains that promote phase separation are poorly understood. We used a model system─the small ubiquitin modifier (SUMO) and its peptide ligand, the SUMO interaction motif (SIM)─to examine how domain surface charge influences multivalency-driven phase separation. Phase separation of polySUMO and polySIM was altered by pH via a change in the protonation state of SUMO surface histidines. These effects were recapitulated by histidine mutations, which modulated SUMO solubility and polySUMO-polySIM phase separation in parallel and were quantitatively explained by atomistic modeling of weak interactions among proteins in the system. Thus, surface charge can tune the phase separation of multivalent proteins, suggesting a means of controlling phase separation biologically, evolutionarily, and therapeutically.

The eight mammalian Cbx proteins are chromodomain-containing proteins involved in regulation of heterochromatin, gene expression, and developmental programs. They are evolutionarily related to the Drosophila HP1 (dHP1) and Pc (dPc) proteins that are key components of chromatin-associated complexes capable of recognizing repressive marks such as trimethylated Lys-9 and Lys-27, respectively, on histone H3. However, the binding specificity and function of the human homologs, Cbx1–8, remain unclear. To this end we employed structural, biophysical, and mutagenic approaches to characterize the molecular determinants of sequence contextual methyllysine binding to human Cbx1–8 proteins. Although all three human HP1 homologs (Cbx1, -3, -5) replicate the structural and binding features of their dHP counterparts, the five Pc homologs (Cbx2, -4, -6, -7, -8) bind with lower affinity to H3K9me3 or H3K27me3 peptides and are unable to distinguish between these two marks. Additionally, peptide permutation arrays revealed a greater sequence tolerance within the Pc family and suggest alternative nonhistone sequences as potential binding targets for this class of chromodomains. Our structures explain the divergence of peptide binding selectivity in the Pc subfamily and highlight previously unrecognized features of the chromodomain that influence binding and specificity.

Phase separation plays an important role in the formation of membraneless compartments within the cell and intrinsically disordered proteins with low-complexity sequences can drive this compartmentalisation. Various intermolecular forces, such as aromatic-aromatic and cation-aromatic interactions, promote phase separation. However, little is known about how the ability of proteins to phase separate under physiological conditions is encoded in their energy landscapes and this is the focus of the present investigation. Our results provide a first glimpse into how the energy landscapes of minimal peptides that contain - and cation- interactions differ from the peptides that lack amino acids with such interactions. The peaks in the heat capacity () as a function of temperature report on alternative low-lying conformations that differ significantly in terms of their enthalpic and entropic contributions. The analysis and subsequent quantification of frustration of the energy landscape suggest that the interactions that promote phase separation lead to features (peaks or inflection points) at low temperatures in . More features may occur for peptides containing residues with better phase separation propensity and the energy landscape is more frustrated for such peptides. Overall, this work links the features in the underlying single-molecule potential energy landscapes to their collective phase separation behaviour and identifies quantities ( and frustration metric) that can be utilised in soft material design.

SAMHD1 (SAM domain and HD domain-containing protein 1) is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) with functions in viral restriction, R-loop resolution, DNA repair, telomere maintenance, ssRNA homeostasis, and regulation of self-nucleic acids. As a dNTPase, SAMHD1 functions as an allosterically activated tetramer, where binding of GTP to the A1 activator site of each monomer initiates dNTP-dependent tetramerization. cEM structures reveal that the nucleic-acid-related functions of SAMHD1 involve binding of guanine residues to the A1 site, leading to oligomeric forms that appear as beads-on-a-string on single-stranded RNA and DNA. SAMHD1's cellular activities and known protein interactions involve liquid-liquid phase separation (LLPS), although there are no reports that SAMHD1 itself exhibits phase separation properties. The protein phase separation prediction algorithm MolPhase indicated an overall phase separation probability score of 0.65 and suggested that the amino terminal SAM domain and the disordered carboxyl terminus (CT) may promote phase separation. Although no phase separation behavior was observed in physiological buffer, in the presence of 9% PEG 2000 and ssDNA or ssRNA, SAMHD1 condensed into liquid-like droplets. These droplets were disrupted by deletion of the SAM or CT domains, showed fusion behavior, and were rapidly disrupted by the addition of A1 site ligands GTP, dGTP, and small-molecule inhibitors. We also observed that SAMHD1-ssDNA condensates within the nuclei of human cells in microinjection experiments, supporting a biological relevance for such complexes. LLPS by SAMHD1 could serve a regulatory role in cells and provide a new therapeutic target for the treatment of cancer and viral infections.

Fluorescent protein (FP) tags are extensively used to visualize and characterize the properties of biomolecular condensates despite a lack of investigation into the effects of these tags on phase separation. Here, we characterized the dynamic properties of µNS, a viral protein hypothesized to undergo phase separation and the main component of mammalian orthoreovirus viral factories. Our interest in the sequence determinants and nucleation process of µNS phase separation led us to compare the size and density of condensates formed by FP::µNS to the untagged protein. We found an FP-dependent increase in droplet size and density, which suggests that FP tags can promote µNS condensation. To further assess the effect of FP tags on µNS droplet formation, we fused FP tags to µNS mutants to show that the tags could variably induce phase separation of otherwise noncondensing proteins. By comparing fluorescent constructs with untagged µNS, we identified mNeonGreen as the least artifactual FP tag that minimally perturbed µNS condensation. These results show that FP tags can promote phase separation and that some tags are more suitable for visualizing and characterizing biomolecular condensates with minimal experimental artifacts.

Histone H3 and H4 tails play an important role in nucleosome phase separation

Most loci that are regulated by genomic imprinting have differentially methylated regions (DMRs). Previously, we showed that the DMRs of the mouse Snrpn and U2af1-rs1 genes have paternal allele-specific patterns of acetylation on histones H3 and H4. To investigate the maintenance of acetylation at these DMRs, we performed chromatin immunoprecipitation on trichostatin-A (TSA)-treated and control cells. In embryonic stem (ES) cells and fibroblasts, brief (6-h) TSA treatment induces global hyperacetylation of H3 and H4. In ES cells only, TSA led to a selective increase in maternal acetylation at U2af1-rs1, at lysine 5 of H4 and at lysine 14 of H3. TSA treatment of ES cells did not affect DNA methylation or expression of U2af1-rs1, but was sufficient to increase DNase I sensitivity along the maternal allele to a level comparable with that of the paternal allele. In fibroblasts, TSA did not alter U2af1-rs1 acetylation, and the parental alleles retained their differential DNase I sensitivity. At Snrpn, no changes in acetylation were observed in the TSA-treated cells. Our data suggest that the mechanisms regulating histone acetylation at DMRs are locus and developmental stage-specific and are distinct from those effecting global levels of acetylation. Furthermore, it seems that the allelic U2af1-rs1 acetylation determines DNase I sensitivity/chromatin conformation.