Biology subjects

Wu, D.

Publications and source records attributed to Wu, D..

At least 145 records · Page 8Linked to original sources

bioRxiv2024

A longitudinal single-cell and spatial multiomic atlas of pediatric high-grade glioma

Pediatric high-grade glioma (pHGG) is an incurable central nervous system malignancy that is a leading cause of pediatric cancer death. While pHGG shares many similarities to adult glioma, it is increasingly recognized as a molecularly distinct, yet highly heterogeneous disease. In this study, we longitudinally profiled a molecularly diverse cohort of 16 pHGG patients before and after standard therapy through single-nucleus RNA and ATAC sequencing, whole-genome sequencing, and CODEX spatial proteomics to capture the evolution of the tumor microenvironment during progression following treatment. We found that the canonical neoplastic cell phenotypes of adult glioblastoma are insufficient to capture the range of tumor cell states in a pediatric cohort and observed differential tumor-myeloid interactions between malignant cell states. We identified key transcriptional regulators of pHGG cell states and did not observe the marked proneural to mesenchymal shift characteristic of adult glioblastoma. We showed that essential neuromodulators and the interferon response are upregulated post-therapy along with an increase in non-neoplastic oligodendrocytes. Through in vitro pharmacological perturbation, we demonstrated novel malignant cell-intrinsic targets. This multiomic atlas of longitudinal pHGG captures the key features of therapy response that support distinction from its adult counterpart and suggests therapeutic strategies which are targeted to pediatric gliomas.

cancer biology↗

bioRxiv2024

Post-death Vesicles of Senescent Bone Marrow Mesenchymal Stromal Polyploids Promote Macrophage Aging and Breast Cancer

Potential systemic factors contributing to aging-associated breast cancer (BC) remain elusive. Here, we reveal that the polyploid giant cells (PGCs) that contain more than two sets of genomes prevailing in aging and cancerous tissues constitute 5-10% of healthy female bone marrow mesenchymal stromal cells (fBMSCs). The PGCs can repair DNA damage and stimulate neighboring cells for clonal expansion. However, dying PGCs in advanced-senescent fBMSCs can form "spikings" which are then separated into membraned mtDNA-containing vesicles (Senescent PGC-Spiking Bodies; SPSBs). SPSB-phagocytosed macrophages accelerate aging with diminished clearance on BC cells and protumor M2 polarization. SPSB-carried mitochondrial OXPHOS components are enriched in BC of elder patients and associated with poor prognosis. SPSB-incorporated breast epithelial cells develop aggressive characteristics and PGCs resembling the polyploid giant cancer cells (PGCCs) in clonogenic BC cells and cancer tissues. These findings highlight an aging BMSC-induced BC risk mediated by SPSB-induced macrophage dysfunction and epithelial cell precancerous transition. SIGNIFICANCEMechanisms underlying aging-associated cancer risk remain unelucidated. This work demonstrates that polyploid giant cells (PGCs) in bone marrow mesenchymal stromal cells (BMSCs) from healthy female bone marrow donors can boost neighboring cell proliferation for clonal expansion. However, the dying-senescent PGCs in the advanced-senescent fBMSCs can form "spikings" which are separated into mitochondrial DNA (mtDNA)-containing spiking bodies (senescent PGC-spiking bodies; SPSBs). The SPSBs promote macrophage aging and breast epithelial cell protumorigenic transition and form polyploid giant cancer cells. These results demonstrate a new form of ghost message from dying-senescent BMSCs, that may serve as a systemic factor contributing to aging-associated immunosuppression and breast cancer risk. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/583755v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@f07b60org.highwire.dtl.DTLVardef@33ef98org.highwire.dtl.DTLVardef@1b720c8org.highwire.dtl.DTLVardef@1c9514d_HPS_FORMAT_FIGEXP M_FIG Xie et al demonstrate that the polyploid giant cells (PGCs) in the juvenile phase expansion of female bone marrow mesenchymal stromal cells (fBMSCs) can boost neighboring cell proliferation for clonal expansion. However, when fBMSCs enter to the advanced-senescent phase, the dying-senescent PGCs form "spikings" which are then separated into membraned vesicles termed Senescent PGC spiking bodies, SPSBs). The SPSBs carrying fragmented mitochondrial elements and OXPHOS proteins can be phagocytosed by macrophage and breast epithelial cells leading to macrophage aging and breast epithelial protumorigenic transition. The SPSBs are demonstrated to be a new form of post-cell death vesicle from aging BMSCs and may serve as a systemic factor contributing to aging-associated immunosuppression and breast cancer risk. C_FIG

cancer biology↗

bioRxiv2024

Enhanced microbiota profiling in patients with quiescent Crohn's disease through comparison with paired healthy first-degree relatives using fecal metagenomics and metabolomics

Prior studies indicate no correlation between gut microbiota of healthy first-degree relatives (HFDRs) of Crohns disease (CD) patients and development of CD. Here, we utilized HFDRs as controls to examine the microbiota and metabolome in individuals with active (CD-A) and quiescent (CD-R) CD, thereby minimizing the influence of genetic and environmental factors. Compared to non-relative controls, the use of HFDR controls identified fewer differential taxa. Faecalibacterium, Dorea, and Fusicatenibacter showed decreased abundances in CD-R, independent of inflammation, and correlated with fecal SCFAs. Validation with a large multi-center cohort confirmed decreased abundances in Faecalibacterium and other SCFA-producing genera in CD-R. Classification models based on these genera distinguished CD-R and CD-A from healthy individuals, in both the discovery and validation cohorts. Thus, the decreased presence of Faecalibacterium, Dorea, and Fusicatenibacter in CD-R likely contributed to disease relapse through reduced SCFA production, highlighting their potential as diagnostic markers and therapeutic targets for CD.

pathology↗

bioRxiv2024

Chromatin regulator Kdm6b is required for the establishment and maintenance of neural stem cells in mouse hippocampus

Neural stem cells (NSCs) in the mouse hippocampal dentate gyrus (DG) - a structure important to learning and memory - generate new neurons postnatally and throughout adult life. However, the regulators that enable this lifelong neurogenesis remain incompletely understood. Here we show that the chromatin regulator KDM6B is required for both the establishment and maintenance of NSCs in the mouse DG. Conditional deletion of Kdm6b in embryonic DG precursors results in an adult hippocampus that is essentially devoid of NSCs, and hippocampal-dependent behaviors are defective. Kdm6b-deletion causes precocious neuronal differentiation, and the NSC population fails to become established in the postnatal DG. Using single cell RNA sequencing (scRNA-seq), we observed that Kdm6b-deletion disrupts the transcriptomic signature of NSC maintenance. Furthermore, deleting Kdm6b in adult DG NSCs induces early neuronal differentiation, and the NSC population is not properly maintained. These data illustrate the critical role that Kdm6b plays in adult DG neurogenesis, which may help understand how mutations in this chromatin regulator result in cognitive disorders in human patients.

neuroscience↗

bioRxiv2024

From Sight to Insight: A Multi-task Approach with the Visual Language Decoding Model

Visual neural decoding aims to unlock the mysteries of how the human brain interprets the visual world. While early studies made some progress in decoding visual activity for singular type of information, they failed to concurrently reveal the multi-level interweaving linguistic information in the brain. Here, we developed a novel Visual Language Decoding Model (VLDM) capable of decoding categories, semantic labels, and textual descriptions from visual perceptual activities simultaneously. We selected the large-scale NSD dataset to ensure the efficiency of the decoding model in joint training and evaluation across multiple tasks. For category decoding, we achieved the effective classification of 12 categories with an accuracy of nearly 70%, significantly surpassing the chance level. For label decoding, we attained the precise prediction of 80 specific semantic labels with a 16-fold improvement over the chance level. For text decoding, the scores of the decoded text surpassed the corresponding baseline levels by remarkable margins on six evaluation metrics. This study contributes significantly to extensive applications in multi-layered brain-computer interfaces, potentially leading to more natural and efficient human-computer interaction experiences.

neuroscience↗

bioRxiv2024

Elevated Activity in Left Homologous Music Circuits is Maladaptive for Music Perception but Mediated by Decoupled Structure and Function

Music is inherent in human life and is a significant topic of cognitive neuroscience. Previous studies focused on amusia suggested that two frontotemporal circuits engage in music processing. Structure-function coupling is an important feature of human brain, which is associated with cognition and allows for a more sensitive investigation of brain-behavior association. However, we still have limited knowledge about the relation between structure-function coupling, music processing and other regional neural profiles. We recruited 106 participants (43 subjects were diagnosed with congenital amusia) and measured their music perception by Montreal Battery of Evaluation of Amusia (MBEA). Then we utilized support vector regression algorithm and mediation analysis, and employed amplitude of low frequency fluctuation (ALFF), structural/functional degree centrality (DC) and structure-function coupling to explore their relation with global averaged MBEA score. We found structure-function coupling of widespread brain regions in both hemispheres, rather than ALFF or DC, contributed to predict MBEA score. Left middle frontal gyrus, bilateral inferior temporal gyrus and right insula were most predictive regions, and these regions were involved in memory and cognitive control according to meta-analysis. Further, coupling of left middle frontal gyrus, a region that is homologous to and is connected with typical music circuits, fully mediated the negative relation between ALFF and MBEA score. Our findings provide further understanding for the neural basis of music, and have implications for neural plasticity, neuromodulation therapy and cognitive causes of amusia. HighlightsO_LIOur study first applies structure-function coupling to investigate the neural correlates of music perception, and predicting modeling indicate structure-function coupling is more effective than regional activity and connectivity. C_LIO_LIElevated activity of contralateral homologous of music circuits might be maladaptive rather than compensatory. C_LIO_LIElevated spontaneous regional activity disrupts their connections, which might be a specific expression of neural plasticity for certain regions. C_LIO_LIOur findings have implications for cognitive causes (abnormal memory and/or cognitive control to music salients) of amusia. C_LI

neuroscience↗

bioRxiv2024

The interplay between dormant mutated cells and tumor promotion by chronic tissue damage in determining cancer risk.

While the causal role of mutagenic carcinogens in tumor development is well established, the relative contribution of environmental tumor promoting factors, wounding, and chronic inflammation is still unclear. Recent sequencing studies have suggested that most environmental carcinogens act as promoters rather than through mechanisms that involve direct induction of point mutations, but whether cancer risk factors such as obesity, chronic inflammation, wounding, or tumor promoters contribute directly or indirectly to mutation burden, or induce novel signatures, has not been investigated. Here, we present WGS analysis of over 100 mouse skin tumors to compare the effects of exposure to mutagens, the tumor promoter TPA, chronic wounding, obesity, or chemotherapy, on mutational burden and cancer risk. All tumors initiated by the carcinogen Dimethylbenzanthracene (DMBA) show a very strong A>T mutational signature (SBS.DMBA) attributable to a single exposure to this carcinogen. The number of SBS.DMBA mutations also showed a strong correlation with the "clock" signature SBS5, suggesting that one treatment with this mutagen can induce mutational signatures attributed to endogenous processes. No specific signatures could be attributed to obesity, high fat diet, wounding, or TPA. Cells carrying thousands of mutations persist over very long periods without inducing tumors or causing pathological changes but can give rise to tumors after short term exposure to TPA. Furthermore, normal cell turnover and proliferation during fetal and adult growth, is not sufficient for promotion, but tissue damage followed by regenerative proliferation seems to be required for tumor development. We conclude that tumor promoters, chronic inflammation, wounding, and obesity do not contribute significantly to tumor mutational burden, and that the rate-limiting determinant of tumor growth is exposure to a tumor promoter rather than the nature or number of genomic point mutations. These data are highly relevant to the recent demonstration of persistent oncogenic mutations in histologically normal human tissues during ageing.

cancer biology↗

bioRxiv2024

SnapFISH-IMPUTE: an imputation method for multiplexed DNA FISH data

Chromatin spatial organization plays a crucial role in gene regulation. Recently developed and prospering multiplexed DNA FISH technologies enable direct visualization of chromatin conformation in nucleus. However, incomplete data caused by limited detection efficiency can substantially complicate and impair downstream analysis. Here, we present SnapFISH-IMPUTE that imputes missing values in multiplexed DNA FISH data. Analysis on multiple published datasets shows that the proposed method preserves the distribution of pairwise distances between imaging loci, and the imputed chromatin conformations are indistinguishable from the observed conformations. Additionally, imputation greatly improves downstream analyses such as identifying enhancer-promoter loops and clustering cells into distinct cell types. SnapFISH-IMPUTE is freely available at https://github.com/hyuyu104/SnapFISH-IMPUTE.

bioinformatics↗

bioRxiv2024

Development of deaminase-free T-to-S base editor and C-to-G base editor by engineered human uracil DNA glycosylase

DNA base editors could enable direct editing of adenine (A), cytosine (C), or guanine (G), but there is no base editor for direct thymine (T) editing currently. Here, by fusing Cas9 nickase (nCas9) with engineered human uracil DNA glycosylase (UNG) variants, we developed a deaminase-free glycosylase-based thymine base editor (gTBE) with the ability of direct T editing. By several rounds of UNG mutagenesis via rational screening, we demonstrated that gTBE with engineered UNG variants could achieve T editing efficiency by up to 81.5%. Furthermore, the gTBE exhibited high T-to-S (i.e., T-to-C or T-to-G) conversion ratio with up to 0.97 in cultured human cells. Using similar strategy, we developed a deaminase-free cytosine base editor (gCBE) facilitating specifically direct C editing by engineered UNG with mutations different from gTBE. Thus, we provide two novel base editors, gTBE and gCBE, with corresponding engineered UNG variants, broadening the targeting scope of base editors.

molecular biology↗

bioRxiv2023

Efficacy of Drpitor1a, a Dynamin-Related Protein 1 inhibitor, in Pulmonary Arterial Hypertension

RationaleDynamin-related protein 1 (Drp1), a large GTPase, mediates mitochondrial fission. Increased Drp1-mediated fission permits accelerated mitosis, contributing to hyperproliferation of pulmonary artery smooth muscle cells (PASMC), which characterizes pulmonary arterial hypertension (PAH). We developed a Drp1 inhibitor, Drpitor1a, and tested its ability to regress PAH. ObjectivesAssess Drpitor1as efficacy and toxicity in: a)normal and PAH human PASMC (hPASMC); b)normal rats versus rats with established monocrotaline (MCT)-induced PAH. MethodsDrpitor1as effects on recombinant and endogenous Drp1-GTPase activity, mitochondrial fission, and cell proliferation were studied in hPASMCs (normal=3; PAH=5). Drpitor1as pharmacokinetics and tissue concentrations were measured (n=3 rats/sex). In a pilot study (n=3-4/sex/dose), Drpitor1a (1mg/kg/48-hours, intravenous) reduced adverse PA remodeling only in females. Consequently, we compared Drpitor1a to vehicle in normal (n=6 versus 8) and MCT-PAH (n=9 and 11) females, respectively. Drpitor1a treatment began 17-days post-MCT with echocardiography and cardiac catheterization performed 28-29 days post-MCT. ResultsDrpitor1a inhibited recombinant and endogenous Drp1 GTPase activity, which was increased in PAH hPASMC. Drpitor1a inhibited mitochondrial fission and proliferation and induced apoptosis, in PAH hPASMC but not normal hPASMC. Drpitor1a tissue levels were higher in female versus male RVs. In MCT-PAH females, Drpitor1a regressed PA obstruction, lowered pulmonary vascular resistance, and improved RV function, without hematologic, renal, or hepatic toxicity. ConclusionsDrpitor1a inhibits Drp1 GTPase, reduces mitochondrial fission, and inhibits cell proliferation in PAH hPASMC. Drpitor1a caused no toxicity in MCT-PAH and had no significant effect on normal rats or hPASMCs. Drpitor1a is a potential PAH therapeutic which displays an interesting therapeutic sexual dimorphism.

pharmacology and toxicology↗

bioRxiv2023

Biodiversity in mountain soils above the treeline

Despite the importance of healthy soils for human livelihood, wellbeing, and safety, current gaps in our knowledge and understanding of biodiversity in soil are numerous, undermining conservation efforts. These gaps are particularly wide in mountain regions where healthy soils are especially important for human safety and yet evidence is accumulating of ongoing degradation, posing significant threats to ecosystem functioning and human settlements. To analyse these gaps in detail, we synthesise current research on the global diversity of microorganisms, cryptogams, and invertebrates in mountain soils above the treeline. This synthesis is based on a semi-quantitative survey of the literature and an expert-based analysis. Our work reveals not only deficiencies in geographic cover but also significant gaps in taxonomic coverage, particularly among soil protists and invertebrates, and a lack of (functional and ecological) description of the uncultivated majority of prokaryotes, fungi, and protists. We subsequently build on this overview to highlight opportunities for research on mountain soils as systems of co-occurring species that interact in complex environmental matrices to fulfil critical functions and make essential contributions to life on land. Closing gaps in biodiversity research in mountain soil is crucial to enhance our understanding and to promote laws and guidelines advancing international soil biodiversity conservation targets in mountains. Addressing sparse and biased data, recognizing the impact of environmental changes on mountain ecosystems, and advocating dedicated policies are essential strategies to safeguard mountain soils and their biodiversity. GLOSSARY O_TBL View this table: org.highwire.dtl.DTLVardef@16682fdorg.highwire.dtl.DTLVardef@121b9feorg.highwire.dtl.DTLVardef@139eadeorg.highwire.dtl.DTLVardef@16abd74org.highwire.dtl.DTLVardef@bc3da9_HPS_FORMAT_FIGEXP M_TBL C_TBL

ecology↗

bioRxiv2023

Unraveling the Impact of W215A/E217A Mutations on Thrombin Dynamics and Thrombomodulin Binding through Molecular Dynamics Simulations

Thrombin, a central serine protease in hemostasis, exhibits dual functionality in coagulation processes--favoring fibrinogen cleavage in its native form while shifting towards protein C activation when complexed with thrombomodulin (TM). Thrombin also plays roles in cancer-associated thrombosis and may be involved in metastasis and tumorigenesis. The W215A/E217A (WE) double mutant of thrombin presents a unique case, with its fibrinogen cleavage activity diminished by 19,000-fold, contrasting a modest 7-fold reduction in protein C activation in the presence of TM. The differential substrate specificity of this mutant raises fundamental questions about the underlying molecular mechanisms. In this study, we employed all-atom microsecond-scale molecular dynamics (MD) simulations, complemented by Root Mean Square Fluctuation (RMSF) analysis, clustering algorithms, PCA-based free-energy surfaces, and logistic regression modeling, to dissect the structural and allosteric changes driving thrombins substrate specificity. Our results unveil distinct conformational states within the catalytic triad, each optimized for specific substrate interactions. We demonstrate that the WE mutations synergize with TM456 binding, resulting in altered hydrogen bond networks and distinct free energy landscapes. A key finding of our research is the identification of ARG125 as a pivotal element in these interactions, consistently forming critical hydrogen bonds across different thrombin variants. The persistent role of ARG125 not only elucidates aspects of thrombins functional plasticity but also positions it as a promising target for novel therapies. This comprehensive analysis enhances our understanding of thrombins structural dynamics, paving the way for more effective and targeted therapeutics.

biophysics↗

bioRxiv2023

SntB triggers the antioxidant pathways to regulate development and aflatoxin biosynthesis in Aspergillus flavus

The epigenetic reader SntB was identified as an important transcriptional regulator of growth, development, and secondary metabolite synthesis in Aspergillus flavus. However, the underlying molecular mechanism is still unclear. In this study, sntB gene deletion ({Delta}sntB), complementary (Com-sntB), and HA tag fused to sntB (sntB-HA) strains were constructed by using the homologous recombination method, respectively. Our results revealed that deletion of sntB inhibited the processes of mycelia growth, conidial production, sclerotia formation, aflatoxin synthesis, and ability to colonize host, and the defective phenotype of knockout strain {Delta}sntB could be restored in its complementary strain Com-sntB. Chromatin immunoprecipitation sequencing (ChIP-seq) of sntB-HA and WT, and RNA sequencing (RNA-seq) of {Delta}sntB and WT strains revealed that SntB played key roles in oxidative stress response of A. flavus. The function of catC gene (encode a catalase) was further analyzed based on the integration results of ChIP-seq and RNA-seq. In {Delta}sntB strain, the relative expression level of catC was significantly higher than in WT strain, while a secretory lipase encoding gene (G4B84_008359) was down-regulated. Under the stress of oxidant menadione sodium bisulfite (MSB), the deletion of sntB obvious down-regulated the expression level of catC. After deletion of catC gene, the mycelia growth, conidial production, and sclerotia formation were inhibited, while ROS level and aflatoxin production were increased compared to the WT strain. Results also showed that the inhibition rate of MSB to {Delta}catC strain was significantly lower than that of WT group and AFB1 yield of the {Delta}catC strain was significantly decreased than that of WT strain under the stress of MSB. Our study revealed the potential machinery that SntB regulated fungal morphogenesis, mycotoxin anabolism, and fungal virulence through the axle of from SntB to fungal virulence and mycotoxin bio-synthesis, i.e. H3K36me3 modification-SntB-Peroxisomes-Lipid hydrolysis-fungal virulence and mycotoxin bio-synthesis. The results of this study shed light into the SntB mediated transcript regulation pathways of fungal mycotoxin anabolism and virulence, which provided potential strategy for control the contamination of A. flavus and its aflatoxins.

pharmacology and toxicology↗

bioRxiv2023

Site Effects in Multisite Fetal Brain MRI: Morphological Insights into Early Brain Development

BackgroundMultisite MRI studies have become prevalent given their advantage in revealing reliable biological or clinical findings. Adult and adolescent multisite studies have demonstrated inevitable site-related non-biological effects that introduce bias. However, the site effect on fetal brain MRI remains unknown. PurposeTo identify crucial acquisition factors affecting fetal brain structural measurements and developmental patterns, while assessing the effectiveness of existing harmonization methods in mitigating site effects. Materials and MethodsBetween May 2017 and March 2022, T2-weighted fast spin-echo sequences in-utero MRI were performed in healthy fetuses from prospectively recruited pregnant volunteers on four different scanners at four sites. A generalized additive model (GAM) was used to quantitatively assess site effects, including field strength (FS), manufacture (M), in-plane resolution (R), and slice thickness (ST), on subcortical volume and cortical morphological measurements, including cortical thickness, curvature, and sulcal depth. Growth models were selected to elucidate developmental trajectories of these morphological measurements. Welchs test was performed to evaluate the influence of site effects on developmental trajectories. ComBat-GAM harmonization method was applied to mitigate site-related biases. ResultsThe final analytic sample consisted of 340 MRI scans from 218 fetuses (mean gestational age, 30.1 weeks {+/-} 4.4 [range, 21.7-40 weeks]). GAM results showed that low FS and low spatial resolution led to overestimations in selected brain regions of subcortical volumes and cortical morphological measurements, and cortical measurements were more susceptible to site effects than subcortical volumes. Only the peak cortical thickness in developmental trajectories was significantly influenced by the effects of FS and R. Notably, ComBat-GAM harmonization effectively removed site effects while preserving developmental patterns. ConclusionOur findings pinpointed the key acquisition factors in in-utero fetal brain MRI and underscored the necessity of data harmonization when pooling multisite data for fetal brain morphology investigations.

bioengineering↗

bioRxiv2023

Bioorthogonal labeling enables in situ fluorescence imaging of expressed gas vesicle nanostructures

Gas vesicles (GVs) are proteinaceous nanostructures that, along with virus-like particles, encapsulins, nano-cages, and other macromolecular assemblies are being developed for potential biomedical applications. To facilitate such development, it would be valuable to characterize these nanostructures sub-cellular assembly and localization. However, traditional fluorescent protein fusions are not tolerated by GVs primary constituent protein, making optical microscopy a challenge. Here, we introduce a method for fluorescently visualizing intracellular GVs using the bioorthogonal label FlAsH, which becomes fluorescent upon binding the six-amino acid tetracysteine (TC) tag. We engineered the GV subunit protein, GvpA, to display the TC tag, and showed that GVs bearing TC-tagged GvpA can be successfully assembled and fluorescently visualized in HEK 293T cells. We used fluorescence images of the tagged GVs to study GV size and distance distributions within these cells. This bioorthogonal labeling approach will enable research to provide a greater understanding of GVs and could be adapted to similar proteinaceous nanostructures.

synthetic biology↗

bioRxiv2023

Epiregulon: Inference of single-cell transcription factor activity to dissect mechanisms of lineage plasticity and drug response

Transcription factors (TFs) and transcriptional coregulators represent an emerging class of therapeutic targets in oncology. Gene regulatory networks (GRNs) can be used to evaluate pharmacological agents targeting these factors and to identify drivers of disease and drug resistance. However, GRN methods that rely solely on gene expression often fail to account for post-transcriptional modulation of TF function. We present Epiregulon, a method that constructs GRNs from single-cell ATAC-seq and RNA-seq data for accurate prediction of TF activity. This is achieved by considering the co-occurrence of TF expression and chromatin accessibility at TF binding sites in each cell. We leverage ChIP-seq data to extend inference to transcriptional coregulators lacking defined motifs or TF harboring neomorphic mutations. Epiregulon accurately predicted the effects of AR inhibition across various drug modalities including an AR antagonist and an AR degrader, delineated the mechanisms of a SMARCA4 degrader by identifying context-dependent interaction partners and prioritized known and novel drivers of lineage reprogramming and tumorigenesis. By mapping gene regulation across various cellular contexts, Epiregulon can accelerate the discovery of therapeutics targeting transcriptional regulators.

bioinformatics↗

bioRxiv2023

Assembly reactions of SARS-CoV-2 nucleocapsid protein with nucleic acid

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-) protein into ribonucleoprotein particles (RNPs), 38{+/-}10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining mutant proteins in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multi-valent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N- protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

biophysics↗

bioRxiv2023

Modulation of Biophysical Properties of Nucleocapsid Protein in the Mutant Spectrum of SARS-CoV-2

Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we examine the impact of mutations across the spectrum of viable amino acid sequences on the biophysical phenotypes of the highly expressed and multifunctional nucleocapsid protein. We find variation in the physicochemical parameters of its extended intrinsically disordered regions (IDRs) sufficient to allow local plasticity, but also exhibiting functional constraints that similarly occur in related coronaviruses. In biophysical experiments with several N-protein species carrying mutations associated with major variants, we find that point mutations in the IDRs can have nonlocal impact and modulate thermodynamic stability, secondary structure, protein oligomeric state, particle formation, and liquid-liquid phase separation. In the Omicron variant, distant mutations in different IDRs have compensatory effects in shifting a delicate balance of interactions controlling protein assembly properties, and include the creation of a new protein-protein interaction interface in the N-terminal IDR through the defining P13L mutation. A picture emerges where genetic diversity is accompanied by significant variation in biophysical characteristics of functional N-protein species, in particular in the IDRs.

biophysics↗