Spatial and temporal organization of the genome: Current state and future aims of the 4D nucleome project.

The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.

RAD51AP1 regulates ALT-HDR through chromatin-directed homeostasis of TERRA.

Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in subsets of aggressive cancer. Recent studies have revealed that telomere repeat-containing RNA (TERRA) promotes ALT-associated HDR (ALT-HDR). Here, we report that RAD51AP1, a crucial ALT factor, interacts with TERRA and utilizes it to generate D- and R-loop HR intermediates. We also show that RAD51AP1 binds to and might stabilize TERRA-containing R-loops as RAD51AP1 depletion reduces R-loop formation at telomere DNA breaks. Proteomic analyses uncover a role for RAD51AP1-mediated TERRA R-loop homeostasis in a mechanism of chromatin-directed suppression of TERRA and prevention of transcription-replication collisions (TRCs) during ALT-HDR. Intriguingly, we find that both TERRA binding and this non-canonical function of RAD51AP1 require its intrinsic SUMO-SIM regulatory axis. These findings provide insights into the multi-contextual functions of RAD51AP1 within the ALT mechanism and regulation of TERRA.

RNA Controls PolyQ Protein Phase Transitions.

Compartmentalization in cells is central to the spatial and temporal control of biochemistry. In addition to membrane-bound organelles, membrane-less compartments form partitions in cells. Increasing evidence suggests that these compartments assemble through liquid-liquid phase separation. However, the spatiotemporal control of their assembly, and how they maintain distinct functional and physical identities, is poorly understood. We have previously shown an RNA-binding protein with a polyQ-expansion called Whi3 is essential for the spatial patterning of cyclin and formin transcripts in cytosol. Here, we show that specific mRNAs that are known physiological targets of Whi3 drive phase separation. mRNA can alter the viscosity of droplets, their propensity to fuse, and the exchange rates of components with bulk solution. Different mRNAs impart distinct biophysical properties of droplets, indicating mRNA can bring individuality to assemblies. Our findings suggest that mRNAs can encode not only genetic information but also the biophysical properties of phase-separated compartments.

A General Strategy for the Design and Evaluation of Heterobifunctional Tools: Applications to Protein Localization and Phase Separation.

To mimic the levels of spatiotemporal control that exist in nature, tools for chemically induced dimerization (CID) are employed to manipulate protein-protein interactions. Although linker composition is known to influence speed and efficiency of heterobifunctional compounds, modeling or in vitro experiments are often insufficient to predict optimal linker structure. This can be attributed to the complexity of ternary complex formation and the overlapping factors that impact the effective concentration of probe within the cell, such as efflux and passive permeability. Herein, we synthesize a library of modular chemical tools with varying linker structures and perform quantitative microscopy in live cells to visualize dimerization in real-time. We use our optimized probe to demonstrate our ability to recruit a protein of interest (POI) to the mitochondria, cell membrane, and nucleus. Finally, we induce and monitor local and global phase separation. We highlight the importance of quantitative approaches to linker optimization for dynamic systems and introduce new, synthetically accessible tools for the rapid control of protein localization.

Optogenetic control of kinetochore function.

Kinetochores act as hubs for multiple activities during cell division, including microtubule interactions and spindle checkpoint signaling. Each kinetochore can act autonomously, and activities change rapidly as proteins are recruited to, or removed from, kinetochores. Understanding this dynamic system requires tools that can manipulate kinetochores on biologically relevant temporal and spatial scales. Optogenetic approaches have the potential to provide temporal and spatial control with molecular specificity. Here we report new chemical inducers of protein dimerization that allow us to both recruit proteins to and release them from kinetochores using light. We use these dimerizers to manipulate checkpoint signaling and molecular motor activity. Our findings demonstrate specialized properties of the CENP-E (kinesin-7) motor for directional chromosome transport to the spindle equator and for maintenance of metaphase alignment. This work establishes a foundation for optogenetic control of kinetochore function, which is broadly applicable to experimental probing of other dynamic cellular processes.

Reversible Control of Protein Localization in Living Cells Using a Photocaged-Photocleavable Chemical Dimerizer.

Many dynamic biological processes are regulated by protein-protein interactions and protein localization. Experimental techniques to probe such processes with temporal and spatial precision include photoactivatable proteins and chemically induced dimerization (CID) of proteins. CID has been used to study several cellular events, especially cell signaling networks, which are often reversible. However, chemical dimerizers that can be both rapidly activated and deactivated with high spatiotemporal resolution are currently limited. Herein, we present a novel chemical inducer of protein dimerization that can be rapidly turned on and off using single pulses of light at two orthogonal wavelengths. We demonstrate the utility of this molecule by controlling peroxisome transport and mitotic checkpoint signaling in living cells. Our system highlights and enhances the spatiotemporal control offered by CID. This tool addresses biological questions on subcellular levels by controlling protein-protein interactions.

Septin assemblies form by diffusion-driven annealing on membranes.

Septins assemble into filaments and higher-order structures that act as scaffolds for diverse cell functions including cytokinesis, cell polarity, and membrane remodeling. Despite their conserved role in cell organization, little is known about how septin filaments elongate and are knitted together into higher-order assemblies. Using fluorescence correlation spectroscopy, we determined that cytosolic septins are in small complexes, suggesting that septin filaments are not formed in the cytosol. When the plasma membrane of live cells is monitored by total internal reflection fluorescence microscopy, we see that septin complexes of variable size diffuse in two dimensions. Diffusing septin complexes collide and make end-on associations to form elongated filaments and higher-order structures, an assembly process we call annealing. Septin assembly by annealing can be reconstituted in vitro on supported lipid bilayers with purified septin complexes. Using the reconstitution assay, we show that septin filaments are highly flexible, grow only from free filament ends, and do not exchange subunits in the middle of filaments. This work shows that annealing is a previously unidentified intrinsic property of septins in the presence of membranes and demonstrates that cells exploit this mechanism to build large septin assemblies.

Biophysical characterization of organelle-based RNA/protein liquid phases using microfluidics.

Living cells contain numerous membrane-less RNA/protein (RNP) bodies that assemble by intracellular liquid-liquid phase separation. The properties of these condensed phase droplets are increasingly recognized as important in their physiological function within living cells, and also through the link to protein aggregation pathologies. However, techniques such as droplet coalescence analysis or standard microrheology do not always enable robust property measurements of model RNA/protein droplets in vitro. Here, we introduce a microfluidic platform that drives protein droplets into a single large phase, which facilitates viscosity measurements using passive microrheology and/or active two-phase flow analysis. We use this technique to study various phase separating proteins from structures including P granules, nucleoli, and Whi3 droplets. In each case, droplets exhibit simple liquid behavior, with shear rate-independent viscosities, over observed timescales. Interestingly, we find that a reported order of magnitude difference between the timescale of Whi3 and LAF-1 droplet coalescence is driven by large differences in surface tension rather than viscosity, with implications for droplet assembly and function. The ability to simultaneously perform active and passive microrheological measurements enables studying the impact of ATP-dependent biological activity on RNP droplets, which is a key area for future research.

Image-based discrimination of the early stages of mesenchymal stem cell differentiation.

Mesenchymal stem cells (MSCs) are self-renewing, multipotent cells, which can be used in cellular and tissue therapeutics. MSCs cell number can be expanded in vitro, but premature differentiation results in reduced cell number and compromised therapeutic efficacies. Current techniques fail to discriminate the "stem-like" population from early stages (12 hours) of differentiated MSC population. Here, we imaged nuclear structure and actin architecture using immunofluorescence and used deep learning-based computer vision technology to discriminate the early stages (6-12 hours) of MSC differentiation. Convolutional neural network (CNN) models trained by nucleus and actin images have high accuracy in reporting MSC differentiation; nuclear images alone can identify early stages of differentiation. Concurrently, we show that chromatin fluidity and heterochromatin levels or localization change during early MSC differentiation. This study quantifies changes in cell architecture during early MSC differentiation and describes a novel image-based diagnostic tool that could be widely used in MSC culture, expansion and utilization.

Interplay of condensate material properties and chromatin heterogeneity governs nuclear condensate ripening.

Biomolecular condensates play pivotal roles in many cellular processes, yet predicting condensate growth dynamics within the complex intracellular environment is challenging. While chromatin mechanics are known to influence condensate coarsening in the nucleus, the effect of condensate properties remains unclear. Our study demonstrates that the interplay between condensate properties and chromatin mechanics dictates condensate growth dynamics. Through chemical dimerization, we induced condensates of various properties in the cell nuclei, revealing distinct growth mechanisms: diffusion-driven or ripening-dominated. To explain experimental observations, we developed a quantitative theory that uncovers the role of chromatin in modulating condensate growth via size-dependent pressure. We find that surface tension is a critical factor in determining whether condensates undergo elastic or Ostwald ripening. Our model predicts that different condensates are affected differently by chromatin heterogeneity, validated by experimentally perturbing chromatin organization. Taken together, our work elucidates how condensate surface tension and chromatin heterogeneity govern nuclear condensate ripening.

SUMO Promotes DNA Repair Protein Collaboration to Support Alterative Telomere Lengthening in the Absence of PML.

Alternative lengthening of telomeres (ALT) pathway maintains telomeres in a significant fraction of cancers associated with poor clinical outcomes. A better understanding of ALT mechanisms can provide a basis for developing new treatment strategies for ALT cancers. SUMO modification of telomere proteins plays a critical role in the formation of ALT telomere-associated PML bodies (APBs), where telomeres are clustered and DNA repair proteins are enriched to promote homology-directed telomere DNA synthesis in ALT. However, whether and how SUMO contributes to ALT beyond APB formation remains elusive. Here, we report that SUMO promotes collaboration among DNA repair proteins to achieve APB-independent telomere maintenance. By using ALT cancer cells with PML protein knocked out and thus devoid of APBs, we show that sumoylation is required for manifesting ALT features, including telomere clustering and telomeric DNA synthesis, independent of PML and APBs. Further, small molecule-induced telomere targeting of SUMO produces signatures of phase separation and ALT features in PML null cells in a manner depending on both sumoylation and SUMO interaction with SUMO interaction motifs (SIMs). Mechanistically, SUMO-induced effects are linked to the enrichment of DNA repair proteins, including Rad52, Rad51AP1, and BLM, to the SUMO-containing telomere foci. Finally, we find that Rad52 can undergo phase separation, enrich SUMO on telomeres, and promote telomere DNA synthesis in collaboration with the BLM helicase in a SUMO-dependent manner. Collectively, our findings suggest that, in addition to forming APBs, SUMO also promotes collaboration among DNA repair proteins to support telomere maintenance in ALT cells. Given the promising effects of sumoylation inhibitors in cancer treatment, our findings suggest their potential use in perturbing telomere maintenance in ALT cancer cells.

TERRA-LSD1 phase separation promotes R-loop formation for telomere maintenance in ALT cancer cells.

The telomere repeat-containing RNA (TERRA) forms R-loops to promote homology-directed DNA synthesis in the alternative lengthening of telomere (ALT) pathway. Here we report that TERRA contributes to ALT via interacting with the lysine-specific demethylase 1A (LSD1 or KDM1A). We show that LSD1 localizes to ALT telomeres in a TERRA dependent manner and LSD1 function in ALT is largely independent of its demethylase activity. Instead, LSD1 promotes TERRA recruitment to ALT telomeres via RNA binding. In addition, LSD1 and TERRA undergo phase separation, driven by interactions between the RNA binding properties of LSD1 and the G-quadruplex structure of TERRA. Importantly, the formation of TERRA-LSD1 condensates enriches the R-loop stimulating protein Rad51AP1 and increases TERRA-containing R-loops at telomeres. Our findings suggest that LSD1-TERRA phase separation enhances the function of R-loop regulatory molecules for ALT telomere maintenance, providing a mechanism for how the biophysical properties of histone modification enzyme-RNA interactions impact chromatin function.

Insight into the In-Situ Encapsulation-Reassembly Strategy To Fabricate PW12@NiCo-LDH Acid-Base Bifunctional Catalysts.

Acid-base bifunctional catalysts have attracted increasing attention due to the improved overall efficiency of synthetic reactions. Herein, we reported the successful fabrication of a PW12@NiCo-LDH acid-base bifunctional catalyst by using the in-situ encapsulation-reassembly strategy. The evolution process of morphology and structure was monitored carefully by various time-dependent characterizations. X-ray absorption fine structure (XAFS) and density functional theory (DFT) calculations demonstrated that the terminal oxygen of PW12 in PW12@NiCo-LDH preferred to assemble with the oxygen vacancies on NiCo-LDH. When applied for deacetalization-Knoevenagel condensation, the PW12@NiCo-LDH displayed >99% conversion of benzaldehyde dimethyl acetal (BDMA) and >99% yield of ethyl α-cyanocinnamate (ECC). Moreover, PW12@NiCo-LDH can be recycled at least 10 cycles without obvious structural change, which can be attributed to the confinement of PW12 into the NiCo-LDH nanocage. Such excellent catalytic activity of PW12@NiCo-LDH was benefited from the short mass transfer pathway between acid sites and base sites, which was caused by the stable assembly between PW12 and NiCo-LDH.

Network pharmacology: a bright guiding light on the way to explore the personalized precise medication of traditional Chinese medicine.

Network pharmacology can ascertain the therapeutic mechanism of drugs for treating diseases at the level of biological targets and pathways. The effective mechanism study of traditional Chinese medicine (TCM) characterized by multi-component, multi-targeted, and integrative efficacy, perfectly corresponds to the application of network pharmacology. Currently, network pharmacology has been widely utilized to clarify the mechanism of the physiological activity of TCM. In this review, we comprehensively summarize the application of network pharmacology in TCM to reveal its potential of verifying the phenotype and underlying causes of diseases, realizing the personalized and accurate application of TCM. We searched the literature using "TCM network pharmacology" and "network pharmacology" as keywords from Web of Science, PubMed, Google Scholar, as well as Chinese National Knowledge Infrastructure in the last decade. The origins, development, and application of network pharmacology are closely correlated with the study of TCM which has been applied in China for thousands of years. Network pharmacology and TCM have the same core idea and promote each other. A well-defined research strategy for network pharmacology has been utilized in several aspects of TCM research, including the elucidation of the biological basis of diseases and syndromes, the prediction of TCM targets, the screening of TCM active compounds, and the decipherment of mechanisms of TCM in treating diseases. However, several factors limit its application, such as the selection of databases and algorithms, the unstable quality of the research results, and the lack of standardization. This review aims to provide references and ideas for the research of TCM and to encourage the personalized and precise use of Chinese medicine.

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.

Inhibitory ability of children with developmental dyscalculia.

Inhibitory ability of children with developmental dyscalculia (DD) was investigated to explore the cognitive mechanism underlying DD. According to the definition of developmental dyscalculia, 19 children with DD-only and 10 children with DD&RD (DD combined with reading disability) were selected step by step, children in two control groups were matched with children in case groups by gender and age, and the match ratio was 1:1. Psychological testing software named DMDX was used to measure inhibitory ability of the subjects. The differences of reaction time in number Stroop tasks and differences of accuracy in incongruent condition of color-word Stroop tasks and object inhibition tasks between DD-only children and their controls reached significant levels (P<0.05), and the differences of reaction time in number Stroop tasks between dyscalculic and normal children did not disappear after controlling the non-executive components. The difference of accuracy in color-word incongruent tasks between children with DD&RD and normal children reached significant levels (P<0.05). Children with DD-only confronted with general inhibitory deficits, while children with DD&RD confronted with word inhibitory deficits only.

Chemical Dimerization-Induced Protein Condensates on Telomeres.

Chromatin-associated condensates are implicated in many nuclear processes, but the underlying mechanisms remain elusive. This protocol describes a chemically-induced protein dimerization system to create condensates on telomeres. The chemical dimerizer consists of two linked ligands that can each bind to a protein: Halo ligand to Halo-enzyme and trimethoprim (TMP) to E. coli dihydrofolate reductase (eDHFR), respectively. Fusion of Halo enzyme to a telomere protein anchors dimerizers to telomeres through covalent Halo ligand-enzyme binding. Binding of TMP to eDHFR recruits eDHFR-fused phase separating proteins to telomeres and induces condensate formation. Because TMP-eDHFR interaction is non-covalent, condensation can be reversed by using excess free TMP to compete with the dimerizer for eDHFR binding. An example of inducing promyelocytic leukemia (PML) nuclear body formation on telomeres and determining condensate growth, dissolution, localization and composition is shown. This method can be easily adapted to induce condensates at other genomic locations by fusing Halo to a protein that directly binds to the local chromatin or to dCas9 that is targeted to the genomic locus with a guide RNA. By offering the temporal resolution required for single cell live imaging while maintaining phase separation in a population of cells for biochemical assays, this method is suitable for probing both the formation and function of chromatin-associated condensates.

Aliphatic Quaternary Ammonium Functionalized Nanogels for Gene Delivery.

Gene therapy is a promising treatment for hereditary diseases, as well as acquired genetic diseases, including cancer. Facing the complicated physiological and pathological environment in vivo, developing efficient non-viral gene vectors is needed for their clinical application. Here, poly(N-isopropylacrylamide) (p(NIPAM)) nanogels are presented with either protonatable tertiary amine groups or permanently charged quaternized ammonium groups to achieve DNA complexation ability. In addition, a quaternary ammonium-functionalized nanogel was further provided with an aliphatic moiety using 1-bromododecane to add a membrane-interacting structure to ultimately facilitate intracellular release of the genetic material. The ability of the tertiary amine-, quaternized ammonium-, and aliphatic quaternized ammonium-functionalized p(NIPAM) nanogels (i.e., NGs, NGs-MI, and NGs-BDD, respectively) to mediate gene transfection was evaluated by fluorescence microscopy and flow cytometry. It is observed that NGs-BDD/pDNA complexes exhibit efficient gene loading, gene protection ability, and intracellular uptake similar to that of NGs-MI/pDNA complexes. However, only the NGs-BDD/pDNA complexes show a notable gene transfer efficiency, which can be ascribed to their ability to mediate DNA escape from endosomes. We conclude that NGs-BDD displays a cationic lipid-like behavior that facilitates endosomal escape by perturbing the endosomal/lysosomal membrane. These findings demonstrate that the presence of aliphatic chains within the nanogel is instrumental in accomplishing gene delivery, which provides a rationale for the further development of nanogel-based gene delivery systems.

CRISPR Cas13-Based Tools to Track and Manipulate Endogenous Telomeric Repeat-Containing RNAs in Live Cells.

TERRA, TElomeric Repeat-containing RNA, is a long non-coding RNA transcribed from telomeres. Emerging evidence indicates that TERRA regulates telomere maintenance and chromosome end protection in normal and cancerous cells. However, the mechanism of how TERRA contributes to telomere functions is still unclear, partially owing to the shortage of approaches to track and manipulate endogenous TERRA molecules in live cells. Here, we developed a method to visualize TERRA in live cells via a combination of CRISPR Cas13 RNA labeling and SunTag technology. Single-particle tracking reveals that TERRA foci undergo anomalous diffusion in a manner that depends on the timescale and telomeric localization. Furthermore, we used a chemically-induced protein dimerization system to manipulate TERRA subcellular localization in live cells. Overall, our approaches to monitor and control TERRA locations in live cells provide powerful tools to better understand its roles in telomere maintenance and genomic integrity.

Tension promotes kinetochore-microtubule release by Aurora B kinase.

To ensure accurate chromosome segregation, interactions between kinetochores and microtubules are regulated by a combination of mechanics and biochemistry. Tension provides a signal to discriminate attachment errors from bi-oriented kinetochores with sisters correctly attached to opposite spindle poles. Biochemically, Aurora B kinase phosphorylates kinetochores to destabilize interactions with microtubules. To link mechanics and biochemistry, current models regard tension as an input signal to locally regulate Aurora B activity. Here, we show that the outcome of kinetochore phosphorylation depends on tension. Using optogenetics to manipulate Aurora B at individual kinetochores, we find that kinase activity promotes microtubule release when tension is high. Conversely, when tension is low, Aurora B activity promotes depolymerization of kinetochore-microtubules while maintaining attachment. Thus, phosphorylation converts a catch-bond, in which tension stabilizes attachments, to a slip-bond, which releases microtubules under tension. We propose that tension is a signal inducing distinct error-correction pathways, with release or depolymerization being advantageous for typical errors characterized by high or low tension, respectively.