Biology subjects

Yu, J.

Publications and source records attributed to Yu, J..

At least 487 records · Page 27Linked to original sources

bioRxiv2020

Revealing Atomic-scale Molecular Diffusion of a Plant Transcription Factor WRKY domain protein along DNA

Transcription factor (TF) target search on genome is highly essential for gene expression and regulation. High-resolution determination of TF diffusion along DNA remains technically challenging. Here we constructed a TF model system of the plant WRKY domain protein in complex with DNA from crystallography and demonstrated microsecond diffusion dynamics of WRKY on the DNA employing all-atom molecular dynamics (MD) simulations. Notably, we found that WRKY preferentially binds to the Crick strand of DNA with significantly stronger energetic association than to the Watson strand. The preferential binding becomes highly prominent from non-specific to specific DNA binding, but less distinct from static binding to diffusive movements of WRKY on the DNA. Remarkably, without employing acceleration forces or bias, we captured a complete one-base pair (bp) stepping cycle of WRKY tracking along major groove of DNA with homogenous (AT)n sequence, as individual protein-DNA contacts break and reform at the binding interface. Continuous tracking of WRKY forward or backward, with occasional sliding as well as strand crossing to the minor groove of DNA, have also been captured in the simulation. The processive diffusion of WRKY had been confirmed by accompanied single-molecule fluorescence assays and coarse-grained (CG) structural simulations. The study thus provides unprecedented structural dynamics details on the TF diffusion, suggests how TF possibly approaches to gene target, and supports further high-precision experimental follow-up. The stochastic movements revealed in the TF diffusion also provide general clues on how other nucleic acid walkers step and slide along DNA. Significance StatementHow transcription factors search for target genes impact on how quickly and accurately the genes are transcribed and expressed. To locate target sufficiently fast, 1D diffusion of the protein along DNA appears essential. Experimentally, it remains challenging to determine diffusional steps of protein on DNA. Here, we report all-atom equilibrium simulations of a WRKY protein binding and diffusing on DNA, revealing structural dynamics details which have not been identified previously. We unprecedently demonstrate a complete stepping cycle of the protein for one base pair on DNA within microseconds, along with stochastic stepping or sliding, directional switching, and strand crossing. Additionally, we have found preferential DNA strand association of WRKY. These suggest how protein factors approach toward target DNA sequences.

biophysics↗

bioRxiv2020

High-quality de novo genome assembly of Kappaphycus alvarezii based on both PacBio and HiSeq sequencing

The red algae Kappaphycus alvarezii is the most important aquaculture species in Kappaphycus, widely distributed in tropical waters, and it has become the main crop of carrageenan production at present. The mechanisms of adaptation for high temperature, high salinity environments and carbohydrate metabolism may provide an important inspiration for marine algae study. Scientific background knowledge such as genomic data will be also essential to improve disease resistance and production traits of K. alvarezii. 43.28 Gb short paired-end reads and 18.52 Gb single-molecule long reads of K. alvarezii were generated by Illumina HiSeq platform and Pacbio RSII platform respectively. The de novo genome assembly was performed using Falcon_unzip and Canu software, and then improved with Pilon. The final assembled genome (336 Mb) consists of 888 scaffolds with a contig N50 of 849 Kb. Further annotation analyses predicted 21,422 protein-coding genes, with 61.28% functionally annotated. Here we report the draft genome and annotations of K. alvarezii, which are valuable resources for future genomic and genetic studies in Kappaphycus and other algae.

genomics↗

bioRxiv2020

A multilayered post-GWAS assessment on genetic susceptibility to pancreatic cancer

Pancreatic cancer (PC) is a complex disease in which both non-genetic and genetic factors interplay. To-date, 40 GWAS hits have been associated with PC risk in individuals of European descent, explaining 4.1% of the phenotypic variance. Here, we complemented a classical new PC GWAS (1D) with spatial autocorrelation analysis (2D) and Hi-C maps (3D) to gain additional insight into the inherited basis of PC. In-silico functional analysis of public genomic information allowed prioritization of potentially relevant candidate variants. We replicated 17/40 previous PC-GWAS hits and identified novel variants with potential biological functions. The spatial autocorrelation approach prioritized low MAF variants not detected by GWAS. These were further expanded via 3D interactions to 54 target regions with high functional relevance. This multi-step strategy, combined with an in-depth in-silico functional analysis, offers a comprehensive approach to advance the study of PC genetic susceptibility and could be applied to other diseases.

genetics↗

bioRxiv2020

Identifying Nootropic Drug Targets via Large-Scale Cognitive GWAS and Transcriptomics

BackgroundCognitive traits demonstrate significant genetic correlations with many psychiatric disorders and other health-related traits. Many neuropsychiatric and neurodegenerative disorders are marked by cognitive deficits. Therefore, genome-wide association studies (GWAS) of general cognitive ability might suggest potential targets for nootropic drug repurposing. Our previous effort to identify "druggable genes" (i.e., GWAS-identified genes that produce proteins targeted by known small molecules) was modestly powered due to the small cognitive GWAS sample available at the time. Since then, two large cognitive GWAS meta-analyses have reported 148 and 205 genome-wide significant loci, respectively. Additionally, large-scale gene expression databases, derived from post-mortem human brain, have recently been made available for GWAS annotation. Here, we 1) reconcile results from these two cognitive GWAS meta-analyses to further enhance power for locus discovery; 2) employ several complementary transcriptomic methods to identify genes in these loci with variants that are credibly associated with cognition; and 3) further annotate the resulting genes to identify "druggable" targets. MethodsGWAS summary statistics were harmonized and jointly analysed using Multi-Trait Analysis of GWAS [MTAG], which is optimized for handling sample overlaps. Downstream gene identification was carried out using MAGMA, S-PrediXcan/S-TissueXcan Transcriptomic Wide Analysis, and eQTL mapping, as well as more recently developed methods that integrate GWAS and eQTL data via Summary-statistics Mendelian Randomization [SMR] and linkage methods [HEIDI], Available brain-specific eQTL databases included GTEXv7, BrainEAC, CommonMind, ROSMAP, and PsychENCODE. Intersecting credible genes were then annotated against multiple chemoinformatic databases [DGIdb, KI, and a published review on "druggability"]. ResultsUsing our meta-analytic data set (N = 373,617) we identified 241 independent cognition-associated loci (29 novel), and 76 genes were identified by 2 or more methods of gene identification. 26 genes were associated with general cognitive ability via SMR, 16 genes via STissueXcan/S-PrediXcan, 47 genes via eQTL mapping, and 68 genes via MAGMA pathway analysis. The use of the HEIDI test permitted the exclusion of candidate genes that may have been artifactually associated to cognition due to linkage, rather than direct causal or indirect pleiotropic effects. Actin and chromatin binding gene sets were identified as novel pathways that could be targeted via drug repurposing. Leveraging on our various transcriptome and pathway analyses, as well as available chemoinformatic databases, we identified 16 putative genes that may suggest drug targets with nootropic properties. DiscussionResults converged on several categories of significant drug targets, including serotonergic and glutamatergic genes, voltage-gated ion channel genes, carbonic anhydrase genes, and phosphodiesterase genes. The current results represent the first efforts to apply a multi-method approach to integrate gene expression and SNP level data to identify credible actionable genes for general cognitive ability.

genomics↗

bioRxiv2020

RBMS1 suppresses colon cancer metastasis through targeted stabilization of its mRNA regulon

Broad dysregulation of gene expression control is a hallmark of cancer progression. Identifying the underlying master regulators that drive pathological gene expression is a key challenge in precision oncology. Here, we have developed a network analytical framework, named PRADA, that identifies oncogenic RNA-binding proteins through the systematic detection of coordinated changes in their target regulons. Application of this approach to data collected from clinical samples, patient-derived xenografts, and cell line models of colon cancer metastasis revealed the RNA-binding protein RBMS1 as a suppressor of colon cancer progression. We observed that silencing RBMS1 results in increased metastatic capacity in xenograft mouse models, and that restoring its expression blunts metastatic liver colonization. We have found that RBMS1 functions as a post-transcriptional regulator of RNA stability by directly binding and stabilizing ~80 target mRNAs. Measurements in more than 180 clinical samples as well as survival analyses in publicly available datasets, have shown that RBMS1 silencing and the subsequent downregulation of its targets are strongly associated with disease progression and poor survival in colon cancer patients. Together, our findings establish a role for RBMS1 as a previously unknown regulator of RNA stability and as a suppressor of colon cancer metastasis with clinical utility for risk stratification of patients. SignificanceBy applying a new analytical approach to transcriptomic data from clinical samples and models of colon cancer progression, we have uncovered RBMS1 as a suppressor of metastasis and as a post-transcriptional regulator of RNA stability. Notably, RBMS1 silencing and downregulation of its targets are negatively associated with patient survival.

systems biology↗

bioRxiv2019

Delineating the Organization of Projection Neuron Subsets with Multi-fluorescent Rabies Virus Tracing Tool

The elegant functions of the brain are facilitated by sophisticated connections between neurons, the architecture of which is frequently characterized by one nucleus connecting to multiple targets via projection neurons. Delineating the sub-nucleus fine architecture of projection neurons in a certain nucleus could greatly facilitate its circuit, computational, and functional resolution. Here, we developed multi-fluorescent rabies virus to delineate the fine organization of corticothalamic projection neuron subsets in the primary visual cortex (V1). By simultaneously labeling multiple distinct subsets of corticothalamic projection neurons in V1 from their target nuclei in thalamus (dLGN, LP, LD), we observed that V1-dLGN corticothalamic neurons were densely concentrated in layer VI, except for several sparsely scattered neurons in layer V, while V1-LP and V1-LD corticothalamic neurons were localized to both layers V and VI. Meanwhile, we observed a fraction of V1 corticothalamic neurons targeting multiple thalamic nuclei, which was further confirmed by fMOST whole-brain imaging. We further conceptually proposed an upgraded sub-nucleus tracing system with higher throughput (21 subsets) for more complex architectural tracing. The multi-fluorescent RV tracing tool can be extensively applied to resolve architecture of projection neuron subsets, with a strong potential to delineate the computational and functional organization of these nuclei.

neuroscience↗

bioRxiv2019

A Holistic Analysis of the Intestinal Stem Cell Niche Network

Although many studies into the intestinal stem cell (ISC) niche have been carried out, they have focused on the role of a single cell type or molecular signal. However, no holistic comparisons of the predominant cell types and signals present within the intestinal mucosa have been conducted to date. We utilize bulk RNA sequencing to profile 20 different mucosal cell types covering four major cell categories: epithelial, stromal, endothelial and immune. We further examined the stromal signaling environment using scRNAseq to provide a more comprehensive view of the signaling microenvironment within the intestinal mucosa. We identified the primary signals for the major ISC regulatory pathways and their respective cellular sources. Our analysis suggests that a niche network exists, with no single cell type being responsible for ISC self-renewal, proliferation, or differentiation; rather, each cell type within the network carries out specific functions in a highly cooperative and coordinated manner.

cell biology↗

bioRxiv2019

CoproID predicts the source of coprolites and paleofeces using microbiome composition and host DNA content

Shotgun metagenomics applied to archaeological feces (paleofeces) can bring new insights into the composition and functions of human and animal gut microbiota from the past. However, paleofeces often undergo physical distortions in archaeological sediments, making their source species difficult to identify on the basis of fecal morphology or microscopic features alone. Here we present a reproducible and scalable pipeline using both host and microbial DNA to infer the host source of fecal material. We apply this pipeline to newly sequenced archaeological specimens and show that we are able to distinguish morphologically similar human and canine paleofeces, as well as non-fecal sediments, from a range of archaeological contexts.

microbiology↗

bioRxiv2019

Na2CO3-Responsive Photosynthetic and ROS Scavenging Mechanisms in Chloroplasts of Alkaligrass Revealed by Phosphoproteomics

Alkali-salinity exerts severe osmotic, ionic and high-pH stresses to plants. To understand the alkali-salinity responsive mechanisms underlying photosynthetic modulation and reactive oxygen species (ROS) homeostasis, physiological and diverse quantitative proteomics analyses of alkaligrass (Puccinellia tenuiflora) under Na2CO3 stress were conducted. In addition, Western blot, real-time PCR, and transgenic techniques were applied to validate the proteomic results and test the functions of the Na2CO3-responsive proteins. A total of 104 and 102 Na2CO3-responsive proteins were identified in leaves and chloroplasts, respectively. In addition, 84 Na2CO3-responsive phosphoproteins were identified, including 56 new phosphorylation sites in 56 phosphoproteins from chloroplasts, which are crucial for the regulation of photosynthesis, ion transport, signal transduction and energy homeostasis. A full-length PtFBA encoding an alkaligrass chloroplastic fructose-bisphosphate aldolase (FBA) was overexpressed in wild-type cells of cyanobacterium Synechocystis sp. Strain PCC 6803, leading to enhanced Na2CO3 tolerance. All these results indicate that thermal dissipation, state transition, cyclic electron transport, photorespiration, repair of photosystem (PS) II, PSI activity, and ROS homeostasis were altered in response to Na2CO3 stress, and they have improved our understanding of the Na2CO3-responsive mechanisms in halophytes.

plant biology↗

bioRxiv2019

Large-scale transgenic Drosophila resource collections for loss- and gain-of-function studies

The Transgenic RNAi Project (TRiP), a Drosophila functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNAi fly stocks. To date, the TRiP has generated >15,000 RNAi fly stocks. As this covers most Drosophila genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express sgRNAs targeting upstream of a gene transcription start site. Gene activation is triggered by co-expression of catalytically dead Cas9 (dCas9) fused to an activator domain, either VP64-p65-Rta (VPR) or Synergistic Activation Mediator (SAM). TRiP-KO stocks express one or two sgRNAs targeting the coding sequence of a gene or genes, allowing for generation of indels in both germline and somatic tissue. To date, we have generated more than 5,000 CRISPR-OE or -KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.

genetics↗

bioRxiv2019

Up-regulation of autophagy by low concentration of salicylic acid delays methyl jasmonate-induced leaf senescence

Crosstalk between salicylic acid (SA) and jasmonic acid (JA) signaling plays an important role in molecular regulation of plant senescence. Our previous works found that SA could delay methyl jasmonate (MeJA)-induced leaf senescence in a concentration-dependent manner. Here, the effect of low concentration of SA (LCSA) application on MeJA-induced leaf senescence was further assessed. High-throughput sequencing (RNA-Seq) results showed that LCSA did not have dominant effects on the genetic regulatory pathways of basal metabolism like nitrogen metabolism, photosynthesis and glycolysis. The ClusterONE was applied to identify discrete gene modules based on protein-protein interaction (PPI) network. Interestingly, an autophagy-related (ATG) module was identified in the differentially expressed genes (DEGs) that exclusively induced by MeJA together with LCSA. RT-qPCR confirmed that the expression of most of the determined ATG genes were upregulated by LCSA. Remarkably, in contrast to wild type (Col-0), LCSA cannot alleviate the leaf yellowing phenotype in autophagy defective mutants (atg5-1 and atg7-2) upon MeJA treatment. Confocal and western blot results showed that LCSA increased the number of autophagic bodies and autophagic flux during MeJA-induced leaf senescence. Collectively, our work revealed up-regulation of autophagy by LCSA as a key regulator to alleviate MeJA-induced leaf senescence.

plant biology↗

bioRxiv2019

The usage of six human IGHJ genes follows a particular nonrandom selection: The recombination signal sequence affects the usage frequency of six human IGHJ genes

The formation of the B cell receptor (BCR) heavy chain variable region is derived from the germline V(D)J gene rearrangement according to the \"12/23\" rule and the \"beyond 12/23\" rule. The usage frequency of each V(D)J gene in the peripheral BCR repertoires is related to the initial recombination, self-tolerance selection, and the clonal proliferative response. However, their specific differences and possible mechanisms are still unknown. We analyzed in-frame and out-of-frame BCR-H repertoires from human samples with physiological and various pathological conditions by high-throughput sequencing. Our results showed that IGHJ gene frequency follows a similar pattern where IGHJ4 is used at high frequency (>40%), IGHJ6/IGHJ3/IGHJ5 is used at medium frequencies (10%[~]20%), and IGH2/IGHJ1 is used at low frequency (<4%) under whether physiological or various pathological conditions. Furthermore, analysis of the recombination signal sequences suggested that the conserved nonamer and heptamer and certain 23 bp spacer length may affect the initial IGHD-IGHJ recombination, which results in different frequencies of IGHJ genes among the initial BCR-H repertoire. Based on this \"background repertoire\", we recommend that re-evaluation and further investigation are needed when analyzing the significance and mechanism of IGHJ gene frequency in self-tolerance selection and the clonal proliferative response.

genomics↗

bioRxiv2019

Defining relative mutational difficulty to understand cancer formation and prevention

Most mutations in human cancer are low-frequency missense mutations, whose functional status remains hard to predict. Here we show that depending on the type of nucleotide change and the surrounding sequences, the tendency to generate each type of nucleotide mutations varies greatly, even by several hundred folds. Therefore, a cancer-promoting mutation may appear only in a small number of cancer cases, if the underlying nucleotide change is too difficult to generate. We propose a method that integrates both the original mutation counts and their relative mutational difficulty. Using this method, we can accurately predict the functionality of hundreds of low-frequency missense mutations in p53, PTEN and INK4A. Many loss-of-function p53 mutations with dominant negative effects were identified, and the functional importance of several regions in p53 structure were highlighted by this analysis. Furthermore, mutational difficulty analysis also points to potential means of cancer prevention. Our study not only established relative mutational difficulties for different types of mutations in human cancer, but also showed that by incorporating such parameter, we can bring new angles to understanding cancer formation and prevention.

cancer biology↗

bioRxiv2019

Mechanism of gating and partial agonist action in the glycine receptor

The glycine receptor is a pentameric, neurotransmitter-activated ion channel that transitions between closed/resting, open and desensitized states. Glycine, a full agonist, produces an open channel probability (Po) of [~]1.0 while partial agonists, such as taurine and {gamma}-amino butyric acid (GABA) yield submaximal Po values. Despite extensive studies of pentameric Cys-loop receptors, there is little knowledge of the molecular mechanisms underpinning partial agonist action and how the receptor transitions from the closed to open and to desensitized conformations. Here we use electrophysiology and molecular dynamics (MD) simulations, together with a large ensemble of single particle cryo-EM reconstructions, to show how agonists populate agonist-bound yet closed channel states, thus explaining their lesser efficacy, yet also populate agonist-bound open and desensitized states. Measurements within the neurotransmitter binding pocket, as a function of bound agonist, provide a metric to correlate the extent of agonist-induced conformational changes to open channel probability across the Cys-loop receptor family.

neuroscience↗

bioRxiv2019

ASK1/p38-mediated NLRP3 inflammasome signaling pathway contributed to aberrant retinal angiogenesis in diabetic retinopathy

AbstractsDiabetic retinopathy is the leading cause of blindness in the working-age population in many countries. Despite the available treatments, some patients present late in the course of the disease when treatment is more difficult. Hence, it is crucial that the new targets are found and utilized in clinical therapy of diabetic retinopathy. In this study, we constructed the DR animal model and the high model in HRMEC cell to investigate the relationship between ASK1/p38 and NLRP3 in DR. The results showed that DR could cause the inflammatory response and microvascular proliferation. NLRP3 contributed to DR-mediated inflammatory development and progression, which promoted the inflammatory related cytokine expression. Meanwhile, it could promote the tube formation of retinal microvascular endothelial and angiogenesis. Moreover, further research showed that NLRP3 mediated aberrant retinal angiogenesis in diabetic retinopathy was regulated by ASK1 and p38. It suggested that ASK1/p38 could become a new target in DR treatment.

cell biology↗

bioRxiv2019

Rapid phylogenetic analysis of African swine fever virus from metagenomic sequences

African swine fever virus (ASFV) has devastating impacts on swine health and the world economy. Rapid and accurate phylogenetic analysis of ASFV causing outbreaks is important to reveal diversity and evolutionary of ASFV. Because it is time-consuming and needs biosafety laboratories to isolate ASFV, here we present a new way to perform rapid genome-wide phylogenetic analysis of ASFV using an allele calling based on gene by gene approach directly from genome drafts assembled from metagenomic sequences. Using open-accessed chewBBACA software, 41 publicly available ASFV genomes were analyzed to optimize the parameters and find the alleles. Alleles as many as 94 were found for building the phylogenetic trees, which covered more than 56% of the whole genome. Based on the alleles, current ASFV isolates could be divided into two major clades and a few subclades. Then the method is used to analyze two ASFV genome drafts assembled from two metagenomic sequences of a swine whole blood and a swine spleen tissue collected in Wuhan, China. It shows that the two ASFV genomes showed highest similarity to that of Pig/HLJ/2018 strain and DB/LN/2018 strain, which isolated recently in China. This proved that the ASFV in Wuhan originate from the same source causing the earlier outbreaks in Helongjiang and Liaoning province of China. This method makes it possible to analyze phylogenetic analysis of ASFV draft genomes flexibly without the need of ASFV isolation. Furthermore, because it is based on Allele calling, the ASFV-specific genetic markers found could be translated into clinical diagnostics or can be used broadly to identify conserved putative therapeutic candidates.

bioinformatics↗

bioRxiv2019

High-throughput living-cell protein crosslinking analysis uncovers the physiological relevance of forming the \"inserted\" state of the ATP synthase ϵ-subunit

ATP synthase, a highly conserved multi-subunit enzyme complex having a common stoichiometry of 3{beta}3{gamma}{delta}{varepsilon}ab2c8-15, functions to supply ATP as the universal energy currency for cells. It comprises of the peripheral F1 sector (3{beta}3{gamma}{delta}{varepsilon}) and the membrane-integrated Fo sector (ab2c8-15). In vitro structural analyses revealed that the C-terminal domain of the {varepsilon}-subunit could adopt either an \"inserted\" or \"non-inserted\" state (with or without interacting with the /{beta}-subunits), with the former being viewed as inhibitory for the ATP hydrolysis activity of ATP synthase. Nevertheless, as common in current protein researches, the physiological relevance of such an \"inserted\" state for ATP synthase functioning is hardly known. To decipher this, designed an unnatural amino acid-mediated living-cell protein photocrosslinking analysis pipeline by developing the scarless genome-targeted site-directed mutagenesis and the high-throughput gel polyacrylamide gel electrophoresis (HT-PAGE) techniques. Employing this powerful approach, we systematically examined the interactions involving the C-terminal helix of the {varepsilon}-subunit in cells living under a variety of experimental conditions. These studies enabled us to uncover that the \"inserted\" and \"non-inserted\" states of the {varepsilon}-subunit exist as an equilibrium in cells cultured under common experimental conditions, shifting to the former upon the appearance of unfavorable conditions, acting as a low-gear state to strengthen the ATP synthesis function. Such a fine-tuning mechanism allows the ATP synthase to reversibly and instantly switch between two functional states. Further, the two powerful techniques that we developed here might be applied to many aspects of protein researches.

biochemistry↗

bioRxiv2019

Distinct features of nucleolus-associated domains in mouse embryonic stem cells

BackgroundHeterochromatin in eukaryotic interphase cells frequently localizes to the nucleolar periphery (nucleolus-associated domains, NADs) and the nuclear lamina (lamina-associated domains, LADs). Gene expression in somatic cell NADs is generally low, but NADs have not been characterized in mammalian stem cells.\n\nResultsHere, we generated the first genome-wide map of NADs in mouse embryonic stem cells (mESCs) via deep sequencing of chromatin associated with biochemically-purified nucleoli. As we had observed in mouse embryonic fibroblasts (MEFs), the large Type I subset of NADs overlaps with constitutive LADs and is enriched for features of constitutive heterochromatin, including late replication timing and low gene density and expression levels. Conversely, the Type II NAD subset overlaps with loci that are not lamina-associated, but in mESCs, Type II NADs are much less abundant than in MEFs. mESC NADs are also much less enriched in H3K27me3 modified regions than are NADs in MEFs. Additionally, comparision of MEF and mESC NADs revealed enrichment of developmentally regulated genes in cell type-specific NADs. Together, these data indicate that NADs are a developmentally dynamic component of heterochromatin.\n\nConclusionsThese studies implicate association with the nucleolar periphery as a mechanism for developmentally-regulated gene silencing, and will facilitate future studies of NADs during mESC differentiation.

genomics↗