Biology subjects

Chen, Z.

Publications and source records attributed to Chen, Z..

At least 613 records · Page 34Linked to original sources

bioRxiv2022

Structure of nucleosome-bound human PBAF complex

BAF and PBAF are mammalian SWI/SNF family chromatin remodeling complexes that possess multiple histone/DNA-binding subunits and create nucleosome-depleted/free regions for transcription activation. Despite structural studies of nucleosome-bound human BAF and yeast SWI/SNF family complexes, it remains elusive how PBAF-nucleosome complex is organized. Here we determined structure of 13-subunit human PBAF in complex with acetylated nucleosome in ADP-BeF3-bound state. Four PBAF-specific subunits work together with nine BAF/PBAF-shared subunits to generate PBAF-specific modular organization, distinct from that of BAF at various regions. PBAF-nucleosome structure reveals six histone-binding domains and four DNA-binding domains/modules, the majority of which directly bind histone/DNA. This multivalent nucleosome-binding pattern, not observed in previous studies, suggests that PBAF may integrate comprehensive chromatin information to target genomic loci for function. Our study reveals molecular organization of subunits and histone/DNA-binding domains/modules in PBAF-nucleosome complex and provides a framework to understand chromatin targeting of SWI/SNF family complexes.

molecular biology↗

bioRxiv2022

Selenocysteine metabolism is a targetable vulnerability in MYCN-amplified cancers

Understanding the operational molecular, and metabolic networks that determine the balance between pro- and anti-ferroptotic regulatory pathways could unravel unique vulnerabilities to be exploited for cancer therapy. Here we identify the selenoprotein P (SELENOP) receptor, LRP8, as a key determinant protecting MYCN-amplified neuroblastoma cells from ferroptosis in vitro and in orthotopic neuroblastoma mouse models. Specifically, the exquisite dependency on LRP8-mediated selenocysteine import is caused by the failure of MYCN-amplified cells to efficiently utilize alternative forms of selenium/selenocysteine based uptake necessary for selenoprotein biosynthesis. Increased activity of one of such transporters, SLC7A11, in MYCN-amplified cells leads to cysteine overload, progressive mitochondrial decline and impaired proliferation. These data reveal in LRP8 a targetable, and specific vulnerability of MYCN-amplified neuroblastoma cells and disclose a yet-unaccounted mechanism for selective ferroptosis induction that has the potential to become an important therapeutic entry point for MYCN-amplified neuroblastoma. Statement of significanceGiven the largely unsuccessful repurposing of adult oncology drugs for the treatment of neuroblastoma, our discoveries pave the way for novel ferroptosis based strategies for this entity. Specifically, targeting of LRP8 may offer novel therapeutic and safer opportunities for a number of pediatric malignancies and MYCN driven cancers.

cancer biology↗

bioRxiv2022

In situ cryo-electron tomography reveals the asymmetric architecture of mammalian sperm axonemes

The flagella of mammalian sperm display non-planar, asymmetric beating, in contrast to the planar, symmetric beating of flagella from sea urchin sperm and unicellular organisms. The molecular basis of this difference is unclear. Here, we perform in situ cryo-electron tomography of mouse and human sperm axonemes, providing the highest resolution structural information to date. Our subtomogram averages reveal mammalian sperm- specific protein complexes within the outer microtubule doublets, the radial spokes and nexin-dynein regulatory complexes. The locations and structures of these complexes suggest potential roles in enhancing the mechanical strength of mammalian sperm axonemes and regulating dynein-based axonemal bending. Intriguingly, we find that each of the nine outer microtubule doublets is decorated with a distinct combination of sperm- specific complexes. We propose that this asymmetric distribution of proteins differentially regulates the sliding of each microtubule doublet and may underlie the asymmetric beating of mammalian sperm.

biophysics↗

bioRxiv2022

Multifunctional flexible electro-optical arrays for simultaneous spatiotemporal cardiac mapping and modulation

Bioelectronic devices that allow simultaneous accurate monitoring and control of the spatiotemporal patterns of cardiac activity provide an effective means to understand the mechanisms and optimize therapeutic strategies for heart disease. Optogenetics is a promising technology for cardiac research due to its advantages such as cell-type selectivity and high space-time resolution, but its efficacy is limited by the insufficient number of modulation channels and lack of simultaneous spatiotemporal mapping capabilities in current cardiac optogenetics tools. Here we present soft implantable electro-optical cardiac devices integrating multilayered highly uniform arrays of transparent microelectrodes and multicolor micro-light-emitting-diodes in thin, flexible platforms for mechanically compliant high-content electrical mapping and single-/multi-site optogenetics and electrical stimulation without light-induced artifacts. Systematic benchtop characterizations, together with ex vivo and in vivo evaluations on healthy and diseased small animal and human hearts demonstrate their functionalities in real-time spatiotemporal mapping and control of cardiac rhythm and function, with broad applications in basic and ultimately clinical cardiology.

bioengineering↗

bioRxiv2022

Intrinsic RNA targeting constrains the utility of CRISPR-Cas13 systems

CRISPR-Cas13 systems have been adapted as versatile toolkits for RNA-related applications. Here we systematically evaluate the performance of several popular Cas13 family effectors (Cas13a, Cas13b and Cas13d) under lentiviral vectors and reveal surprisingly differential defects and characteristics of these systems. Using RNA immunoprecipitation sequencing, transcriptome profiling, biochemistry analysis, high-throughput CRISPR-Cas13 screening and machine learning approaches, we determine that each Cas13 system has its intrinsic RNA targets in mammalian cells. Viral process-related host genes can be targeted by Cas13 and affect production of fertile lentiviral particles, thereby restricting the utility of lentiviral Cas13 systems. Multiple RNase activities of Cas13 are involved in endogenous RNA targeting. Unlike target-induced nonspecific collateral effect, intrinsic RNA cleavage can be specific, target-independent and dynamically tuned by varied states of Cas13 nucleases. Our work provides guidance on appropriate use of lentiviral Cas13 systems and further raises cautions about intrinsic RNA targeting during Cas13-based basic and therapeutic applications.

molecular biology↗

bioRxiv2022

Three-dose vaccination-induced immune responses protect against SARS-CoV-2 Omicron-BA.2

BackgroundThe ongoing outbreak of SARS-CoV-2 Omicron BA.2 infections in Hong Kong, the model city of universal masking of the world, has resulted in a major public health crisis. Although the third vaccination resulted in strong boosting of neutralization antibody, vaccine efficacy and corelates of immune protection against the major circulating Omicron BA.2 remains to be investigated. MethodsWe investigated the vaccine efficacy against the Omicron BA.2 breakthrough infection among 470 public servants who had received different SARS-CoV-2 vaccine regimens including two-dose BNT162b2 (2xBNT, n=169), three-dose BNT162b2 (3xBNT, n=170), two-dose CoronaVac (2xCorV, n=34), three-dose CoronaVac (3xCorV, n=67) and third-dose BNT162b2 following 2xCorV (2xCorV+1BNT, n=32). Humoral and cellular immune responses after three-dose vaccination were further characterized and correlated with clinical characteristics of BA.2 infection. FindingsDuring the BA.2 outbreak, 27.7% vaccinees were infected. The timely third-dose vaccination provided significant protection with lower incidence rates of breakthrough infections (2xBNT 49.2% vs 3xBNT 13.1%, p <0.0001; 2xCorV 44.1% vs 3xCoV 19.4%, p=0.003). Investigation of immune response on blood samples derived from 92 subjects in three-dose vaccination cohorts collected before the BA.2 outbreak revealed that the third-dose vaccination activated spike (S)-specific memory B cells and Omicron cross-reactive T cell responses, which correlated with reduced frequencies of breakthrough infections and disease severity rather than with types of vaccines. Moreover, the frequency of S-specific activated memory B cells was significantly lower in infected vaccinees than uninfected vaccinees before vaccine-breakthrough infection whereas IFN-{gamma}+ CD4 T cells were negatively associated with age and viral clearance time. Critically, BA.2 breakthrough infection boosted cross-reactive memory B cells with enhanced cross-neutralizing antibodies to Omicron sublineages, including BA.2.12.1 and BA.4/5, in all vaccinees tested. InterpretationOur results imply that the timely third vaccination and immune responses are likely required for vaccine-mediated protection against Omicron BA.2 pandemic. Although BA.2 conferred the highest neutralization resistance compared with variants of concern tested before the emergence of BA.2.12.1 and BA.4/5, the third dose vaccination-activated S-specific memory B cells and Omicron cross-reactive T cell responses contributed to reduced frequencies of breakthrough infection and disease severity. Neutralizing antibody potency enhanced by BA. 2 breakthrough infection with previous 3 doses of vaccines (CoronaVac or BNT162b2) may reduce the risk for infection of ongoing BA.2.12.1 and BA.4/5. FundingHong Kong Research Grants Council Collaborative Research Fund, Health and Medical Research Fund, Wellcome Trust, Shenzhen Science and Technology Program, the Health@InnoHK, Innovation and Technology Commission of Hong Kong, China, National Program on Key Research Project, Emergency Key Program of Guangzhou Laboratory, donations from the Friends of Hope Education Fund and the Hong Kong Theme-Based Research Scheme.

immunology↗

bioRxiv2022

Identification of transcriptional network disruptions in drug-resistant prostate cancer with TraRe

Metastatic castration-resistant prostate cancer (mCRPC) presents very low survival rates due to lack of response or acquired resistance to the available therapies. To date no molecular mechanisms of resistance have been identified, pointing out their complex dynamics. To identify key genes and processes associated with phenotypically-driven regulatory differences, we developed TraRe, a computational method that provides a three-tier analysis: i) at the network level, inferring differentially regulated modules; ii) at the regulon level, identifying regulatory relationships linked to phenotypic differences; and iii) at the single gene level, identifying TFs consistently linked to rewired modules. We applied TraRe (available in Bioconductor with full documentation) to transcriptomic data from 46 mCRPC patients with Abiraterone-response clinical data and uncovered abrogated immune response regulatory modules that showed strong differential regulation in Abi-resistant patients. These modules were replicated in an independent mCRPC study. Further, we experimentally validated key rewiring predictions and their associated transcription factors. Among them, ELK3, MXD1, and MYB were found to have a differential role in cell survival for Abi-response-specific settings. Moreover, we identified the role of ELK3 in cell migration capacity, which could have direct impact on mCRPC. Collectively, these findings shed light on the underlying regulatory mechanisms driving abiraterone response.

bioinformatics↗

bioRxiv2022

Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain

Capturing mitochondrias intricate and dynamic structure poses a daunting challenge for optical nanoscopy. Different labeling strategies have been demonstrated for live-cell stimulated emission depletion (STED) microscopy of mitochondria, but orthogonal strategies are yet to be established, and image acquisition has suffered either from photodamage to the organelles or from rapid photobleaching. Therefore, live-cell nanoscopy of mitochondria has been largely restricted to 2D single-color recordings of cancer cells. Here, by conjugation of cyclooctatetraene to a benzo-fused cyanine dye, we report a mitochondrial inner-membrane (IM) fluorescent marker, PK Mito Orange (PKMO), featuring efficient STED at 775 nm, strong photostability and markedly reduced phototoxicity. PKMO enables super-resolution recordings of inner-membrane dynamics for extended periods in immortalized mammalian cell lines, primary cells, and organoids. Photostability and reduced phototoxicity of PKMO open the door to live-cell 3D STED nanoscopy of mitochondria for three-dimensional analysis of the convoluted IM. PKMO is optically orthogonal with green and far-red markers allowing multiplexed recordings of mitochondria using commercial STED microscopes. Using multi-color STED, we demonstrate that imaging with PKMO can capture the sub-mitochondrial localization of proteins, or interactions of mitochondria with different cellular components, such as the ER or the cytoskeleton at sub-100 nm resolution. Thereby, this work offers a versatile tool for studying mitochondrial inner-membrane architecture and dynamics in a multiplexed manner.

cell biology↗

bioRxiv2022

Comparative genomic and crystal structure analyses identify a collagen glucosyltransferase from Acanthamoeba Polyphaga Mimivirus

Humans and Acanthamoeba Polyphaga Mimivirus share numerous homologous genes, including collagens and collagen-modifying enzymes. To explore the homology, we performed a genome-wide comparison between human and mimivirus using DELTA-BLAST (Domain Enhanced Lookup Time Accelerated BLAST) and identified 52 new mimiviral proteins that are homologous with human proteins. To gain functional insights into mimiviral proteins, their human protein homologs were organized into Gene Ontology (GO) and REACTOME pathways to build a functional network. Collagen and collagen-modifying enzymes form the largest subnetwork with most nodes. Further analysis of this subnetwork identified a putative collagen glycosyltransferase R699. Protein expression test suggested that R699 is highly expressed in E coli, unlike the human collagen-modifying enzymes. Enzymatic activity assays showed that R699 catalyzes the conversion of unique galactosylhydroxylysine within the GXXXUG motif (U=galactosylhydroxylysine) to glucosylgalactosylhydroxylysine on collagen using uridine diphosphate glucose (UDP-Glc) as a sugar donor, suggesting R699 is a mimiviral collagen galactosylhydroxylysyl glucosyltransferase (GGT) with defined substrate specificity. Structural study of R699 produced the first crystal structure of a collagen GGT with a visible UDP-Glc. Sugar moiety of the UDP-Glc resides in a previously unrecognized pocket. Mn2+ coordination and nucleoside-diphosphate binding site are conserved among GGT family members and critical for R699s collagen GGT activity. To facilitate further analysis of human and mimiviral homologous proteins, we presented an interactive and searchable genome-wide comparison website for quickly browsing human and Acanthamoeba Polyphaga Mimivirus homologs, which is available at RRID Resource ID: SCR_022140 or https://guolab.shinyapps.io/app-mimivirus-publication/.

biochemistry↗

bioRxiv2022

Improving the RNA velocity approach using long-read single cell sequencing

The concept of RNA velocity has been recently developed that allowed to look at the otherwise static single-cell RNA sequencing data in a dynamic way, which permitted inferences about cell fates. However, the more precise parameters, such as the number of exons/introns, can also be determined using long-read methods. Comparing the numbers of exons and introns allows including more genes for downstream velocity analysis and resolves the precise cell fate. The recently developed concept of "RNA velocity" concerns with dynamic changes in mRNA expression and complements single-cell RNA sequencing (scRNA-seq) data, which are static snapshots of a certain cell state taken at a given time point1. RNA velocity measures the change in mRNA abundance by differentiating the newly transcribed unspliced pre-mRNAs from mature spliced mRNAs. The rapidly developing long-read sequencing technology lends itself for RNA velocity analysis of scRNA-seq data, which was previously performed primarily using second-generation sequencing.

bioinformatics↗

bioRxiv2022

The contribution of Neanderthal introgression and natural selection to neurodegenerative diseases

Humans are thought to be more susceptible to neurodegeneration than equivalently-aged primates. It is not known whether this vulnerability is specific to anatomically-modern humans or shared with other hominids. The contribution of introgressed Neanderthal DNA to neurodegenerative disorders remains uncertain. It is also unclear how common variants associated with neurodegenerative disease risk are maintained by natural selection in the population despite their deleterious effects. In this study, we aimed to quantify the genome-wide contribution of Neanderthal introgression and positive selection to the heritability of complex neurodegenerative disorders to address these questions. We used stratified-linkage disequilibrium score regression to investigate the relationship between five SNP-based signatures of natural selection, reflecting different timepoints of evolution, and genome-wide associated variants of the three most prevalent neurodegenerative disorders: Alzheimers disease, amyotrophic lateral sclerosis and Parkinsons disease. We found no evidence for enrichment of positively-selected SNPs in the heritability of Alzheimers disease, amyotrophic lateral sclerosis and Parkinsons disease,, suggesting that common deleterious disease variants are unlikely to be maintained by positive selection. There was no enrichment of Neanderthal introgression in the SNP-heritability of these disorders, suggesting that Neanderthal admixture is unlikely to have contributed to disease risk. These findings provide insight into the origins of neurodegenerative disorders within the evolution of Homo sapiens and addresses a long-standing debate, showing that Neanderthal admixture is unlikely to have contributed to common genetic risk of neurodegeneration in anatomically-modern humans.

neuroscience↗

bioRxiv2022

Integrative analysis of the 3D genome and epigenome in mouse embryonic tissues

While a rich set of putative cis-regulatory sequences involved in mouse fetal development has been annotated recently based on chromatin accessibility and histone modification patterns, delineating their role in developmentally regulated gene expression continues to be challenging. To fill this gap, we mapped chromatin contacts between gene promoters and distal sequences genome-wide in seven mouse fetal tissues, and for one of them, across six developmental stages. We identified 248,620 long-range chromatin interactions centered at 14,138 protein-coding genes and characterized their tissue-to-tissue variations as well as developmental dynamics. Integrative analysis of the interactome with previous epigenome and transcriptome datasets from the same tissues revealed a strong correlation between the chromatin contacts and chromatin state at distal enhancers, as well as gene expression patterns at predicted target genes. We predicted target genes of 15,098 candidate enhancers, and used them to annotate target genes of homologous candidate enhancers in the human genome that harbor risk variants of human diseases. We present evidence that schizophrenia and other adult disease risk variants are frequently found in fetal enhancers, providing support for the hypothesis of fetal origins of adult diseases.

developmental biology↗

bioRxiv2022

Spatial chromatin accessibility sequencing resolves next-generation genome architecture

As the genome is organized into a three-dimensional structure in intracellular space, epigenomic information also has a complex spatial arrangement. However, most epigenetic studies describe locations of methylation marks, chromatin accessibility regions, and histone modifications in the horizontal dimension. Proper spatial epigenomic information has rarely been obtained. In this study, we designed spatial chromatin accessibility sequencing (SCA-seq) to resolve the genome conformation by capturing the epigenetic information in single-molecular resolution while simultaneously resolving the genome conformation. Using SCA-seq, we are able to examine the spatial interaction of chromatin accessibility (e.g. enhancer-promoter contacts), CpG island methylation, and spatial insulating functions of the CCCTC-binding factor. We demonstrate that SCA-seq paves the way to explore the mechanism of epigenetic interactions and extends our knowledge in 3D packaging of DNA in the nucleus.

genomics↗

bioRxiv2022

Quantitative landscapes reveal trajectories of cell-state transitions associated with drug resistance in melanoma

Drug resistance and tumor relapse in melanoma patients is attributed to a combination of genetic and non-genetic mechanisms. Non-genetic mechanisms of drug resistance commonly involve reversible changes in the cell-state or phenotype, i.e., alterations in molecular profiles that can help cells escape being killed by targeted therapeutics. In melanoma, one of the most common mechanisms of non-genetic resistance is dedifferentiation, which is characterized by loss of melanocytic markers. While various molecular attributes of de-differentiation have been identified, the transition dynamics remains poorly understood. Here, we construct cell-state transition landscapes, to quantify the stochastic dynamics driving phenotypic switch in melanoma based on its underlying regulatory network. These landscapes reveal the existence of multiple alternative paths to resistance - de-differentiation and transition to a hyper-pigmented phenotype. Finally, by visualizing the changes in the landscape during in silico molecular perturbations, we identify combinatorial strategies that can lead to the most optimal outcome - a landscape with the minimal occupancy of the two drug-resistant states. Therefore, we present these landscapes as platforms to screen possible therapeutic interventions in terms of their ability to lead to most favourable patient outcomes.

systems biology↗

bioRxiv2022

A landscape of differentiated biological processes involved in the initiation of sex differentiation in zebrafish

Zebrafish (Danio rerio) has been used as a promising animal model to study gonadal development and gametogenesis. Although previous studies have identified critical molecules participating in zebrafish gonad differentiation, a landscape view of the biological processes involved in this process is still lacking. Here we isolated intact zebrafish differentiating gonads, at 25 days post-fertilization (dpf) and 30 dpf, and conducted RNA-seq analysis between the juvenile gonads that tended to develop into ovaries or testes. Our study demonstrates that the juvenile ovary and testis at 25 dpf and 30 dpf are different at the level of biological process. During ovary differentiation, the biological processes related to metabolic activities in production of energy and maternal substances, RNA degradation, and DNA repair were enriched. During testis differentiation, the biological processes related to cell proliferation, differentiation, and morphogenesis were enriched, with a total of 15 signaling pathways. Notably, we reveal that the immune-related processes are extensively involved in the regulation of testis development. Overall, this study provides a landscape of differentiated biological processes and novel insights into the initiation of sex differentiation in zebrafish.

developmental biology↗

bioRxiv2022

EstG is a novel esterase required for cell envelope integrity

Proper regulation of the bacterial cell envelope is critical for cell survival. Identification and characterization of enzymes that maintain cell envelope homeostasis is crucial, as they can be targets for effective antibiotics. In this study, we have identified a novel enzyme, called EstG, whose activity protects cells from a variety of lethal assaults in the -proteobacterium Caulobacter crescentus. Despite homology to transpeptidase family cell wall enzymes and an ability to protect against cell wall-targeting antibiotics, EstG does not demonstrate biochemical activity towards cell wall substrates. Instead, EstG is genetically connected to the periplasmic enzymes OpgH and BglX, responsible for synthesis and hydrolysis of osmoregulated periplasmic glucans (OPGs), respectively. The crystal structure of EstG revealed similarities to esterases and transesterases, and we demonstrated esterase activity of EstG in vitro. Using biochemical fractionation, we identified a cyclic hexamer of glucose as a likely substrate of EstG. This molecule is the first OPG described in Caulobacter and establishes a novel class of OPGs, the regulation and modification of which is important for stress survival and adaptation to fluctuating environments. Our data indicate that EstG, BglX, and OpgH comprise a previously unknown OPG pathway in Caulobacter. Ultimately, we propose that EstG is a novel enzyme that, instead of acting on the cell wall, acts on cyclic OPGs to provide resistance to a variety of cellular stresses.

microbiology↗

bioRxiv2022

Structure of Importin-4 bound to the H3-H4·ASF1 histone·histone chaperone complex

Importin-4 is the primary nuclear import receptor of core histones H3 and H4. Importin-4 binds the H3-H4 dimer and histone-chaperone ASF1 prior to nuclear import, but available structures of Importin-4{middle dot}histone tail complexes do not explain how Importin-4 recognizes the biologically relevant heterotrimeric H3-H4{middle dot}ASF1 cargo. Our 3.5 [A] Importin-4{middle dot}H3-H4{middle dot}ASF1 cryo-electron microscopy structure revealed interactions with H3-H4{middle dot}ASF1 different those suggested by previous Importin-H3 tail peptide structures. The N-terminal half of Importin-4 clamps the globular histone domain and the H3 N helix while its C-terminal half binds the H3 N-terminal tail weakly, with negligible tail contribution to binding energy; ASF1 binds H3-H4 without contacting Importin-4. Together, ASF1 and Importin-4 shield nucleosomal interfaces of H3-H4 to chaperone and import it into the nucleus, where Importin-4 undergoes large conformational changes as RanGTP binds to release H3-H4{middle dot}ASF1. This work explains the mechanisms of nuclear import of full-length H3-H4.

molecular biology↗

bioRxiv2022

Salicin modifies osteoarthritis progression by binding on IRE1α and inhibiting IRE1α mediated endoplasmic reticulum stress

ObjectivesTo investigate the effect and mechanisms of salicin (SA) on osteoarthritis (OA) progression. MethodsPrimary rat chondrocytes were stimulated with TNF- and treated with or without SA. CCK-8 was utilized to determine the cytotoxicity of SA. RT-qPCR, Western Blotting and immunofluorescence staining were used to detect inflammatory factors, cartilage matrix degeneration markers, cell proliferation and apoptosis markers expression at mRNA and protein levels respectively. EdU assay and flow cytometer analysis were utilized for evaluating cell proliferation and apoptosis. RNA-sequencing, molecular docking, drug affinity responsive target stability and WB were applied to clarify mechanisms. Rat OA model was used to evaluate the effect of intra-articular injection of SA on OA progression. ResultsNo obvious cytotoxicity was found with the treatment of 10 M SA. SA rescued TNF- induced degeneration of cartilage matrix, inhibition of chondrocytes proliferation, and promotion of chondrocytes apoptosis. In mechanism, we clarified SA could directly bind on IRE1 and occupy IRE1 phosphorylation site, followed with inhibiting IRE1 phosphorylation and regulating IRE1 mediated endoplasmic reticulum (ER) stress by IRE1-I{kappa}B-p65 signaling. Finally, intra-articular injection of SA loaded PLGA could ameliorate OA progression by inhibiting IRE1 mediated ER stress in OA model. ConclusionsSA alleviates OA by directly binding on ER stress regulator IRE1 and inhibits IRE1 mediated ER stress by IRE1-I{kappa}B-p65 signaling. Topical use of small molecular drug SA holds the potential of modifying OA progression.

cell biology↗