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.

Adeno-to-squamous transition drives resistance to KRAS inhibition in LKB1 mutant lung cancer.

KRASG12C inhibitors (adagrasib and sotorasib) have shown clinical promise in targeting KRASG12C-mutated lung cancers; however, most patients eventually develop resistance. In lung patients with adenocarcinoma with KRASG12C and STK11/LKB1 co-mutations, we find an enrichment of the squamous cell carcinoma gene signature in pre-treatment biopsies correlates with a poor response to adagrasib. Studies of Lkb1-deficient KRASG12C and KrasG12D lung cancer mouse models and organoids treated with KRAS inhibitors reveal tumors invoke a lineage plasticity program, adeno-to-squamous transition (AST), that enables resistance to KRAS inhibition. Transcriptomic and epigenomic analyses reveal ΔNp63 drives AST and modulates response to KRAS inhibition. We identify an intermediate high-plastic cell state marked by expression of an AST plasticity signature and Krt6a. Notably, expression of the AST plasticity signature and KRT6A at baseline correlates with poor adagrasib responses. These data indicate the role of AST in KRAS inhibitor resistance and provide predictive biomarkers for KRAS-targeted therapies in lung cancer.

Effectiveness and Safety of Non-Vitamin K Oral Anticoagulants versus Warfarin in Patients with Atrial Fibrillation and Previous Stroke: A Systematic Review and Meta-Analysis.

INTRODUCTION: Current evidence regarding the clinical outcomes of non-vitamin K oral anticoagulants (NOACs) versus warfarin in patients with atrial fibrillation (AF) and previous stroke is inconclusive, especially in patients with previous intracranial haemorrhage (ICrH). We aim to undertake a systematic review and meta-analysis assessing the effectiveness and safety of NOACs versus warfarin in AF patients with a history of stroke. METHODS: We searched studies published up to December 10, 2022, on PubMed, Medline, Embase, and Cochrane Central Register of Controlled Trials. Studies on adults with AF and previous ischaemic stroke (IS) or IrCH receiving either NOACs or warfarin and capturing outcome events (thromboembolic events, ICrH, and all-cause mortality) were eligible for inclusion. RESULTS: Six randomized controlled trials (RCTs) (including 19,489 patients with previous IS) and fifteen observational studies (including 132,575 patients with previous IS and 13,068 patients with previous ICrH) were included. RCT data showed that compared with warfarin, NOACs were associated with a significant reduction in thromboembolic events (odds ratio [OR]: 0.85, 95% confidence interval [CI]: 0.75-0.96), ICrH (OR: 0.57, 95% CI: 0.36-0.90), and all-cause mortality (OR: 0.88, 95% CI: 0.80-0.98). In analysing observational studies, similar results were retrieved. Moreover, patients with previous ICrH had a lower OR on thromboembolic events than those with IS (OR: 0.66, 95% CI: 0.46-0.95 vs. OR: 0.80, 95% CI: 0.70-0.93) in the comparison between NOACs and warfarin. CONCLUSIONS: Observational data showed that in AF patients with previous stroke, NOACs showed better clinical performance compared to warfarin and the benefits of NOACs were more pronounced in patients with previous IrCH versus those with IS. RCT data also showed NOACs are superior to warfarin. However, current RCTs only included AF patients who survived an IS, and further large RCTs focused on patients with previous ICrH are warranted.

Aging-disturbed FUS phase transition impairs hematopoietic stem cells by altering chromatin structure.

ABSTRACT: Aged hematopoietic stem cells (HSCs) exhibit compromised reconstitution capacity. The molecular mechanisms behind this phenomenon are not fully understood. Here, we observed that the expression of FUS is increased in aged HSCs, and enforced FUS recapitulates the phenotype of aged HSCs through arginine-glycine-glycine-mediated aberrant FUS phase transition. By using Fus-gfp mice, we observed that FUShigh HSCs exhibit compromised FUS mobility and resemble aged HSCs both functionally and transcriptionally. The percentage of FUShigh HSCs is increased upon physiological aging and replication stress, and FUSlow HSCs of aged mice exhibit youthful function. Mechanistically, FUShigh HSCs exhibit a different global chromatin organization compared with FUSlow HSCs, which is observed in aged HSCs. Many topologically associating domains (TADs) are merged in aged HSCs because of the compromised binding of CCCTC-binding factor with chromatin, which is invoked by aberrant FUS condensates. It is notable that the transcriptional alteration between FUShigh and FUSlow HSCs originates from the merged TADs and is enriched in HSC aging-related genes. Collectively, this study reveals for the first time that aberrant FUS mobility promotes HSC aging by altering chromatin structure.

BRD4-targeting PROTAC as a unique tool to study biomolecular condensates.

Biomolecular condensates play key roles in various biological processes. However, specific condensation modulators are currently lacking. PROTAC is a new technology that can use small molecules to degrade target proteins specifically. PROTAC molecules are expected to regulate biomolecular condensates dynamically by degrading/recovering key molecules in biomolecular condensates. In this study, we employed a BRD4-targeting PROTAC molecule to regulate the super-enhancer (SE) condensate and monitored the changes of SE condensate under PROTAC treatment using live-cell imaging and high-throughput sequencing technologies. As a result, we found that BRD4-targeting PROTACs can significantly reduce the BRD4 condensates, and we established a quantitative method for tracking BRD4 condensates by PROTAC and cellular imaging. Surprisingly and encouragingly, BRD4 condensates were observed to preferentially form and play specialized roles in biological process regulation for the first time. Additionally, BRD4 PROTAC makes it possible to observe the dynamics of other condensate components under the continued disruption of BRD4 condensates. Together, these results shed new light on research methods for liquid-liquid phase separation (LLPS), and specifically demonstrate that PROTAC presents a powerful and distinctive tool for the study of biomolecular condensates.

Aging-induced pseudouridine synthase 10 impairs hematopoietic stem cells.

Aged hematopoietic stem cells (HSC) exhibit compromised reconstitution capacity and differentiation-bias towards myeloid lineage, however, the molecular mechanism behind it remains not fully understood. In this study, we observed that the expression of pseudouridine (Ψ) synthase 10 is increased in aged hematopoietic stem and progenitor cells (HSPC) and enforced protein of Ψ synthase 10 (PUS10) recapitulates the phenotype of aged HSC, which is not achieved by its Ψ synthase activity. Consistently, we observed no difference of transcribed RNA pseudouridylation profile between young and aged HSPC. No significant alteration of hematopoietic homeostasis and HSC function is observed in young Pus10-/- mice, while aged Pus10-/- mice exhibit mild alteration of hematopoietic homeostasis and HSC function. Moreover, we observed that PUS10 is ubiquitinated by E3 ubiquitin ligase CRL4DCAF1 complex and the increase of PUS10 in aged HSPC is due to aging-declined CRL4DCAF1- mediated ubiquitination degradation signaling. Taken together, this study for the first time evaluated the role of PUS10 in HSC aging and function, and provided a novel insight into HSC rejuvenation and its clinical application.

Development and Experimental Validation of a Novel Prognostic Signature for Gastric Cancer.

BACKGROUND: Gastric cancer is a malignant tumor with high morbidity and mortality. Therefore, the accurate recognition of prognostic molecular markers is the key to improving treatment efficacy and prognosis. METHODS: In this study, we developed a stable and robust signature through a series of processes using machine-learning approaches. This PRGS was further experimentally validated in clinical samples and a gastric cancer cell line. RESULTS: The PRGS is an independent risk factor for overall survival that performs reliably and has a robust utility. Notably, PRGS proteins promote cancer cell proliferation by regulating the cell cycle. Besides, the high-risk group displayed a lower tumor purity, higher immune cell infiltration, and lower oncogenic mutation than the low-PRGS group. CONCLUSIONS: This PRGS could be a powerful and robust tool to improve clinical outcomes for individual gastric cancer patients.

SOTIP is a versatile method for microenvironment modeling with spatial omics data.

The rapidly developing spatial omics generated datasets with diverse scales and modalities. However, most existing methods focus on modeling dynamics of single cells while ignore microenvironments (MEs). Here we present SOTIP (Spatial Omics mulTIPle-task analysis), a versatile method incorporating MEs and their interrelationships into a unified graph. Based on this graph, spatial heterogeneity quantification, spatial domain identification, differential microenvironment analysis, and other downstream tasks can be performed. We validate each module's accuracy, robustness, scalability and interpretability on various spatial omics datasets. In two independent mouse cerebral cortex spatial transcriptomics datasets, we reveal a gradient spatial heterogeneity pattern strongly correlated with the cortical depth. In human triple-negative breast cancer spatial proteomics datasets, we identify molecular polarizations and MEs associated with different patient survivals. Overall, by modeling biologically explainable MEs, SOTIP outperforms state-of-art methods and provides some perspectives for spatial omics data exploration and interpretation.

Profiling and characterization of constitutive chromatin-enriched RNAs.

RNA species act as architectural scaffolds for nuclear structures including chromatin in eukaryotic cells. However, the composition and dynamics of tightly bound chromatin-associated RNAs during mitosis remains elusive. Here we report the identification of chromatin-enriched RNA (cheRNAs) by biochemical nuclear fractionation coupled with RNA sequencing in both interphase and mitotic phase of A549 and HeLa-S3 cell lines. We show that highly abundant cheRNAs, mostly small noncoding RNAs, are largely maintained in mitotic chromatin, and constitute a substantial part of chromatin RNA throughout cell cycle. We also show that the mitotic retained cheRNAs tend to be cell type nonspecific and might be involved in chromatin accessibility and epigenetic memory of gene expression control. Therefore, we reveal an unexpected set of cell type-nonspecific mitotic retained chromatin-enriched RNAs. We anticipate that the landscape of RNA composition of chromatin both in interphase and mitotic phase would help understanding structure and function of chromatin.

MyoD is a 3D genome structure organizer for muscle cell identity.

The genome exists as an organized, three-dimensional (3D) dynamic architecture, and each cell type has a unique 3D genome organization that determines its cell identity. An unresolved question is how cell type-specific 3D genome structures are established during development. Here, we analyzed 3D genome structures in muscle cells from mice lacking the muscle lineage transcription factor (TF), MyoD, versus wild-type mice. We show that MyoD functions as a "genome organizer" that specifies 3D genome architecture unique to muscle cell development, and that H3K27ac is insufficient for the establishment of MyoD-induced chromatin loops in muscle cells. Moreover, we present evidence that other cell lineage-specific TFs might also exert functional roles in orchestrating lineage-specific 3D genome organization during development.

Integration of single cell data by disentangled representation learning.

Recent developments of single cell RNA-sequencing technologies lead to the exponential growth of single cell sequencing datasets across different conditions. Combining these datasets helps to better understand cellular identity and function. However, it is challenging to integrate different datasets from different laboratories or technologies due to batch effect, which are interspersed with biological variances. To overcome this problem, we have proposed Single Cell Integration by Disentangled Representation Learning (SCIDRL), a domain adaption-based method, to learn low-dimensional representations invariant to batch effect. This method can efficiently remove batch effect while retaining cell type purity. We applied it to thirteen diverse simulated and real datasets. Benchmark results show that SCIDRL outperforms other methods in most cases and exhibits excellent performances in two common situations: (i) effective integration of batch-shared rare cell types and preservation of batch-specific rare cell types; (ii) reliable integration of datasets with different cell compositions. This demonstrates SCIDRL will offer a valuable tool for researchers to decode the enigma of cell heterogeneity.

MarkovHC: Markov hierarchical clustering for the topological structure of high-dimensional single-cell omics data with transition pathway and critical point detection.

Clustering cells and depicting the lineage relationship among cell subpopulations are fundamental tasks in single-cell omics studies. However, existing analytical methods face challenges in stratifying cells, tracking cellular trajectories, and identifying critical points of cell transitions. To overcome these, we proposed a novel Markov hierarchical clustering algorithm (MarkovHC), a topological clustering method that leverages the metastability of exponentially perturbed Markov chains for systematically reconstructing the cellular landscape. Briefly, MarkovHC starts with local connectivity and density derived from the input and outputs a hierarchical structure for the data. We firstly benchmarked MarkovHC on five simulated datasets and ten public single-cell datasets with known labels. Then, we used MarkovHC to investigate the multi-level architectures and transition processes during human embryo preimplantation development and gastric cancer procession. MarkovHC found heterogeneous cell states and sub-cell types in lineage-specific progenitor cells and revealed the most possible transition paths and critical points in the cellular processes. These results demonstrated MarkovHC's effectiveness in facilitating the stratification of cells, identification of cell populations, and characterization of cellular trajectories and critical points.

Hardening Mechanism of Coral Aggregates in Cement-Based Systems.

In terms of the infrastructure construction near coral reefs, native coral aggregates have been widely implemented as alternative efficient building materials to prepare the "coral concrete". This study focused on the mechanical properties and hardening mechanism of coral particles under cement-based systems. Firstly, coral particles were divided into two categories: coral biological debris particles (calcium sand) and coral parent rock particles (limestone). Subsequently, several elementary laboratory tests were conducted to compare the physical and chemical properties of the samples. The results demonstrate that the specific surface area and open pores of coral particles are bigger than those of quartz sand. Moreover, the water absorption rate of debris and parent rock particles reach 9.9% and 22%, respectively. To further examine the hardening mechanism of coral particles, we carried out particle crushing strength tests, compressive strength tests and nanoindentation tests. Regardless of the tested groups and particle categories, the results show that the wrapped cement slurry universally demonstrated the successful enhancement of crushing strength σ0,d. Particularly, in the size range from 1.18-2.36 mm, wrapped particles of debris and parent rock both reached unusually high σ0,d values: 10.14 MPa and 8.74 MPa, respectively. However, in the size range from 9.5 mm to 16 mm, the σ0,d values of wrapped debris and parent rock reached 4.75 MPa and 3.18 MPa, respectively. According to the nanoindentation tests, the sub-microscopic strength of ITZs was enhanced to more than 1 GPa, which is higher than that of conventional concrete, in terms of the sample with 28-day maintenance. In conclusion, this study has provided a further basis for studying coral concrete material and its hardening mechanism.

SEAM is a spatial single nuclear metabolomics method for dissecting tissue microenvironment.

Spatial metabolomics can reveal intercellular heterogeneity and tissue organization. Here we report on the spatial single nuclear metabolomics (SEAM) method, a flexible platform combining high-spatial-resolution imaging mass spectrometry and a set of computational algorithms that can display multiscale and multicolor tissue tomography together with identification and clustering of single nuclei by their in situ metabolic fingerprints. We first applied SEAM to a range of wild-type mouse tissues, then delineated a consistent pattern of metabolic zonation in mouse liver. We further studied the spatial metabolic profile in the human fibrotic liver. We discovered subpopulations of hepatocytes with special metabolic features associated with their proximity to the fibrotic niche, and validated this finding by spatial transcriptomics with Geo-seq. These demonstrations highlighted SEAM's ability to explore the spatial metabolic profile and tissue histology at the single-cell level, leading to a deeper understanding of tissue metabolic organization.

Quantifying the phase separation property of chromatin-associated proteins under physiological conditions using an anti-1,6-hexanediol index.

BACKGROUND: Liquid-liquid phase separation (LLPS) is an important organizing principle for biomolecular condensation and chromosome compartmentalization. However, while many proteins have been reported to undergo LLPS, quantitative and global analysis of chromatin LLPS property remains absent. RESULTS: Here, by combining chromatin-associated protein pull-down, quantitative proteomics and 1,6-hexanediol (1,6-HD) treatment, we develop Hi-MS and define an anti-1,6-HD index of chromatin-associated proteins (AICAP) to quantify 1,6-HD sensitivity of chromatin-associated proteins under physiological conditions. Compared with known physicochemical properties involved in phase separation, we find that proteins with lower AICAP are associated with higher content of disordered regions, higher hydrophobic residue preference, higher mobility and higher predicted LLPS potential. We also construct BL-Hi-C libraries following 1,6-HD treatment to study the sensitivity of chromatin conformation to 1,6-HD treatment. We find that the active chromatin and high-order structures, as well as the proteins enriched in corresponding regions, are more sensitive to 1,6-HD treatment. CONCLUSIONS: Our work provides a global quantitative measurement of LLPS properties of chromatin-associated proteins and higher-order chromatin structure. Hi-MS and AICAP data provide an experimental tool and quantitative resources valuable for future studies of biomolecular condensates.

3D genome alterations associated with dysregulated HOXA13 expression in high-risk T-lineage acute lymphoblastic leukemia.

3D genome alternations can dysregulate gene expression by rewiring enhancer-promoter interactions and lead to diseases. We report integrated analyses of 3D genome alterations and differential gene expressions in 18 newly diagnosed T-lineage acute lymphoblastic leukemia (T-ALL) patients and 4 healthy controls. 3D genome organizations at the levels of compartment, topologically associated domains and loop could hierarchically classify different subtypes of T-ALL according to T cell differentiation trajectory, similar to gene expressions-based classification. Thirty-four previously unrecognized translocations and 44 translocation-mediated neo-loops are mapped by Hi-C analysis. We find that neo-loops formed in the non-coding region of the genome could potentially regulate ectopic expressions of TLX3, TAL2 and HOXA transcription factors via enhancer hijacking. Importantly, both translocation-mediated neo-loops and NUP98-related fusions are associated with HOXA13 ectopic expressions. Patients with HOXA11-A13 expressions, but not other genes in the HOXA cluster, have immature immunophenotype and poor outcomes. Here, we highlight the potentially important roles of 3D genome alterations in the etiology and prognosis of T-ALL.

Proteome-scale analysis of phase-separated proteins in immunofluorescence images.

Phase separation is an important mechanism that mediates the spatial distribution of proteins in different cellular compartments. While phase-separated proteins share certain sequence characteristics, including intrinsically disordered regions (IDRs) and prion-like domains, such characteristics are insufficient for making accurate predictions; thus, a proteome-wide understanding of phase separation is currently lacking. Here, we define phase-separated proteomes based on the systematic analysis of immunofluorescence images of 12 073 proteins in the Human Protein Atlas. The analysis of these proteins reveals that phase-separated candidate proteins exhibit higher IDR contents, higher mean net charge and lower hydropathy and prefer to bind to RNA. Kinases and transcription factors are also enriched among these candidate proteins. Strikingly, both phase-separated kinases and phase-separated transcription factors display significantly reduced substrate specificity. Our work provides the first global view of the phase-separated proteome and suggests that the spatial proximity resulting from phase separation reduces the requirement for motif specificity and expands the repertoire of substrates. The source code and data are available at https://github.com/cheneyyu/deepphase.

Computational Screening of Phase-separating Proteins.

Phase separation is an important mechanism that mediates the compartmentalization of proteins in cells. Proteins that can undergo phase separation in cells share certain typical sequence features, like intrinsically disordered regions (IDRs) and multiple modular domains. Sequence-based analysis tools are commonly used in the screening of these proteins. However, current phase separation predictors are mostly designed for IDR-containing proteins, thus inevitably overlook the phase-separating proteins with relatively low IDR content. Features other than amino acid sequence could provide crucial information for identifying possible phase-separating proteins: protein-protein interaction (PPI) networks show multivalent interactions that underlie phase separation process; post-translational modifications (PTMs) are crucial in the regulation of phase separation behavior; spherical structures revealed in immunofluorescence (IF)images indicate condensed droplets formed by phase-separating proteins, distinguishing these proteins from non-phase-separating proteins. Here, we summarize the sequence-based tools for predicting phase-separating proteins and highlight the importance of incorporating PPIs, PTMs, and IF images into phase separation prediction in future studies.

PhaSepDB: a database of liquid-liquid phase separation related proteins.

It's widely appreciated that liquid-liquid phase separation (LLPS) underlies the formation of membraneless organelles, which function to concentrate proteins and nucleic acids. In the past few decades, major efforts have been devoted to identify the phase separation associated proteins and elucidate their functions. To better utilize the knowledge dispersed in published literature, we developed PhaSepDB (http://db.phasep.pro/), a manually curated database of phase separation associated proteins. Currently, PhaSepDB includes 2914 non-redundant proteins localized in different organelles curated from published literature and database. PhaSepDB provides protein summary, publication reference and sequence features of phase separation associated proteins. The sequence features which reflect the LLPS behavior are also available for other human protein candidates. The online database provides a convenient interface for the research community to easily browse, search and download phase separation associated proteins. As a centralized resource, we believe PhaSepDB will facilitate the future study of phase separation.

Intein-mediated backbone cyclization of entolimod confers enhanced radioprotective activity in mouse models.

BACKGROUND: Entolimod is a Salmonella enterica flagellin derivate. Previous work has demonstrated that entolimod effectively protects mice and non-human primates from ionizing radiation. However, it caused a "flu-like" syndrome after radioprotective and anticancer clinical application, indicating some type of immunogenicity and toxicity. Cyclization is commonly used to improve the in vivo stability and activity of peptides and proteins. METHODS: We designed and constructed cyclic entolimod using split Nostoc punctiforme DnaE intein with almost 100% cyclization efficiency. We adopted different strategies to purify the linear and circular entolimod due to their different topologies. Both of linear and circular entolimod were first purified by Ni-chelating affinity chromatography, and then the linear and circular entolimod were purified by size-exclusion and ion-exchange chromatography, respectively. RESULTS: The circular entolimod showed significantly increased both the in vitro NF-κB signaling and in vivo radioprotective activity in mice. CONCLUSION: Our data indicates that circular entolimod might be a good candidate for further clinical investigation.