Evolutional heterochromatin condensation delineates chromocenter formation and retrotransposon silencing in plants.

Heterochromatic condensates (chromocenters) are critical for maintaining the silencing of heterochromatin. It is therefore puzzling that the presence of chromocenters is variable across plant species. Here we reveal that variations in the plant heterochromatin protein ADCP1 confer a diversity in chromocenter formation via phase separation. ADCP1 physically interacts with the high mobility group protein HMGA to form a complex and mediates heterochromatin condensation by multivalent interactions. The loss of intrinsically disordered regions (IDRs) in ADCP1 homologues during evolution has led to the absence of prominent chromocenter formation in various plant species, and introduction of IDR-containing ADCP1 with HMGA promotes heterochromatin condensation and retrotransposon silencing. Moreover, plants in the Cucurbitaceae group have evolved an IDR-containing chimaera of ADCP1 and HMGA, which remarkably enables formation of chromocenters. Together, our work uncovers a coevolved mechanism of phase separation in packing heterochromatin and silencing retrotransposons.

Arginine methylation-enabled FUS phase separation with SMN contributes to neuronal granule formation.

Various ribonucleoprotein complexes (RNPs) often function in the form of membraneless organelles derived from multivalence-driven liquid-liquid phase separation (LLPS). Post-translational modifications, such as phosphorylation and arginine methylation, govern the assembly and disassembly of membraneless organelles. This study reveals that asymmetric dimethylation of arginine can create extra binding sites for multivalent Tudor domain-containing proteins like survival of motor neuron (SMN) protein, thereby lowering the threshold for LLPS of RNPs, such as fused in sarcoma (FUS). Accordingly, FUS hypomethylation or knockdown of SMN disrupts the formation and transport of neuronal granules in axons. Wild-type SMN, but not the spinal muscular atrophy-associated form of SMN, SMN-Δ7, rescues neuronal defects due to SMN knockdown. Importantly, a fusion of SMN-Δ7 to an exogenous oligomeric protein is sufficient to rescue axon length defects caused by SMN knockdown. Our findings highlight the significant role of arginine methylation-enabled multivalent interactions in LLPS and suggest their potential impact on various aspects of neuronal activities in neurodegenerative diseases.

Verteporfin inhibits TGF-ß signaling by disrupting the Smad2/3-Smad4 interaction.

Transforming growth factor-ß (TGF-ß) signaling plays a crucial role in pathogenesis, such as accelerating tissue fibrosis and promoting tumor development at the later stages of tumorigenesis by promoting epithelial-mesenchymal transition (EMT), cancer cell migration, and invasion. Targeting TGF-ß signaling is a promising therapeutic approach, but nonspecific inhibition may result in adverse effects. In this study, we focus on the Smad2/3-Smad4 complex, a key component in TGF-ß signaling transduction, as a potential target for cancer therapy. Through a phase-separated condensate-aided biomolecular interaction system, we identified verteporfin (VP) as a small-molecule inhibitor that specifically targets the Smad2/3-Smad4 interaction. VP effectively disrupted the interaction between Smad2/3 and Smad4 and thereby inhibited canonical TGF-ß signaling, but not the interaction between Smad1 and Smad4 in bone morphogenetic protein (BMP) signaling. Furthermore, VP exhibited inhibitory effects on TGF-ß-induced EMT and cell migration. Our findings indicate a novel approach to develop protein-protein interaction inhibitors of the canonical TGF-ß signaling pathway for treatments of related diseases.

SnapShot: Condensates in plant biology.

Plant cells share a number of biological condensates with cells from other eukaryotes. There are, however, a growing number of plant-specific condensates that support different cellular functions. Condensates operating in different plant tissues contribute to aspects of development and stress responses. To view this SnapShot, open or download the PDF.

SAFB restricts contact domain boundaries associated with L1 chimeric transcription.

Long interspersed element-1 (LINE-1 or L1) comprises 17% of the human genome, continuously generates genetic variations, and causes disease in certain cases. However, the regulation and function of L1 remain poorly understood. Here, we uncover that L1 can enrich RNA polymerase IIs (RNA Pol IIs), express L1 chimeric transcripts, and create contact domain boundaries in human cells. This impact of L1 is restricted by a nuclear matrix protein scaffold attachment factor B (SAFB) that recognizes transcriptionally active L1s by binding L1 transcripts to inhibit RNA Pol II enrichment. Acute inhibition of RNA Pol II transcription abolishes the domain boundaries associated with L1 chimeric transcripts, indicating a transcription-dependent mechanism. Deleting L1 impairs domain boundary formation, and L1 insertions during evolution have introduced species-specific domain boundaries. Our data show that L1 can create RNA Pol II-enriched regions that alter genome organization and that SAFB regulates L1 and RNA Pol II activity to preserve gene regulation.

Dual-role transcription factors stabilize intermediate expression levels.

Precise control of gene expression levels is essential for normal cell functions, yet how they are defined and tightly maintained, particularly at intermediate levels, remains elusive. Here, using a series of newly developed sequencing, imaging, and functional assays, we uncover a class of transcription factors with dual roles as activators and repressors, referred to as condensate-forming level-regulating dual-action transcription factors (TFs). They reduce high expression but increase low expression to achieve stable intermediate levels. Dual-action TFs directly exert activating and repressing functions via condensate-forming domains that compartmentalize core transcriptional unit selectively. Clinically relevant mutations in these domains, which are linked to a range of developmental disorders, impair condensate selectivity and dual-action TF activity. These results collectively address a fundamental question in expression regulation and demonstrate the potential of level-regulating dual-action TFs as powerful effectors for engineering controlled expression levels.

A phase separation-fortified bi-specific adaptor for conditional tumor killing.

A common approach in therapeutic protein development involves employing synthetic ligands with multivalency, enabling sophisticated control of signal transduction. Leveraging the emerging concept of liquid-liquid phase separation (LLPS) and its ability to organize cell surface receptors into functional compartments, we herein have designed modular ligands with phase-separation modalities to engineer programmable interreceptor communications and precise control of signal pathways, thus inducing the rapid, potent, and specific apoptosis of tumor cells. Despite their simplicity, these "triggers", named phase-separated Tumor Killers (hereafter referred to as psTK), are sufficient to yield interreceptor clustering of death receptors (represented by DR5) and tumor-associated receptors, with notable features: LLPS-mediated robust high-order organization, well-choreographed conditional activation, and broad-spectrum capacity to potently induce apoptosis in tumor cells. The development of novel therapeutic proteins with phase-separation modalities showcases the power of spatially reorganizing signal transduction. This approach facilitates the diversification of cell fate and holds promising potential for targeted therapies against challenging tumors.

Substrate-induced condensation activates plant TIR domain proteins.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR-NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.

Linker histone H1 drives heterochromatin condensation via phase separation in Arabidopsis.

In the eukaryotic nucleus, heterochromatin forms highly condensed, visible foci known as heterochromatin foci (HF). These HF are enriched with linker histone H1, a key player in heterochromatin condensation and silencing. However, it is unknown how H1 aggregates HF and condenses heterochromatin. In this study, we established that H1 facilitates heterochromatin condensation by enhancing inter- and intrachromosomal interactions between and within heterochromatic regions of the Arabidopsis (Arabidopsis thaliana) genome. We demonstrated that H1 drives HF formation via phase separation, which requires its C-terminal intrinsically disordered region (C-IDR). A truncated H1 lacking the C-IDR fails to form foci or recover HF in the h1 mutant background, whereas C-IDR with a short stretch of the globular domain (18 out of 71 amino acids) is sufficient to rescue both defects. In addition, C-IDR is essential for H1's roles in regulating nucleosome repeat length and DNA methylation in Arabidopsis, indicating that phase separation capability is required for chromatin functions of H1. Our data suggest that bacterial H1-like proteins, which have been shown to condense DNA, are intrinsically disordered and capable of mediating phase separation. Therefore, we propose that phase separation mediated by H1 or H1-like proteins may represent an ancient mechanism for condensing chromatin and DNA.

A bivalent inhibitor against TDRD3 to suppress phase separation of methylated G3BP1.

The formation of membrane-less organelles is driven by multivalent weak interactions while mediation of such interactions by small molecules remains an unparalleled challenge. Here, we uncovered a bivalent inhibitor that blocked the recruitment of TDRD3 by the two methylated arginines of G3BP1. Relative to the monovalent inhibitor, this bivalent inhibitor demonstrated an enhanced binding affinity to TDRD3 and capability to suppress the phase separation of methylated G3BP1, TDRD3, and RNAs, and in turn inhibit the stress granule growth in cells. Our result paves a new path to mediate multivalent interactions involved in SG assembly for potential combinational chemotherapy by bivalent inhibitors.