Biology subjects

Lu, Y.

Publications and source records attributed to Lu, Y..

At least 19 recordsLinked to original sources

bioRxiv2018

Endogenous BioID elucidates TCF7L1 interactome modulation upon GSK-3 inhibition in mouse ESCs

Modulation of Wnt target gene expression via the TCF/LEFs remains poorly understood. We employ proximity-based biotin labeling (BioID) to examine GSK-3 inhibitor effects on the TCF7L1 interactome in mouse ESCs. We generated ESC lines with biotin ligase BirA* fused to TCF7L1 by knocking it into the endogenous TCF7L1 locus or by inserting a doxinducible BirA*-TCF7L1 transgene into the Rosa26 locus. Induction yielded BirA*-TCF7L1 levels 3-fold higher than in the endogenous system, but substantial overlap in biotinylated proteins with high peptide counts were detected by each method. Known TCF7L1 interactors TLE3/4 and {beta}-catenin, and numerous proteins not previously associated with TCF7L1, were identified in both systems. Despite reduced BirA*-TCF7L1 levels, the number of hits identified with both BioID approaches increased after GSK-3 inhibition. We elucidate the network of TCF7L1 proximal proteins regulated by GSK-3 inhibition, validate the utility of endogenous BioID, and provide mechanistic insights into TCF7L1 target gene regulation.\n\nHighlightsO_LIBirA*-TCF7L1 at single-copy physiological levels generates robust BioID data\nC_LIO_LICHIR99021 reduces TCF7L1 levels but increases detectable TCF7L1-proximal proteins\nC_LIO_LIThe TCF7L1 interactome of largely epigenetic/transcription factors fluctuates with GSK-3 inhibition\nC_LIO_LIJMJD1C, SALL4 and BRG1/SMARCA4 are validated as TCF7L-interacting proteins\nC_LI

cell biology↗

bioRxiv2018

Single-cell RNA-seq analysis maps the development of human fetal retina

Vision starts with image formation at the retina, which contains diverse neuronal cell types that extract, process, and relay visual information to higher order processing centers in the brain. Though there has been steady progress in defining retinal cell types, very little is known about retinal development in humans, which starts well before birth. In this study, we performed transcriptomic profiling of developing human fetal retina from gestational weeks 12 to 27 using single-cell RNA-seq (scRNA-seq) and used pseudotime analysis to reconstruct the developmental trajectories of retinogenesis. Our analysis reveals transcriptional programs driving differentiation down four different cell types and suggests that Muller glia (MG) can serve as embryonic progenitors in early retinal development. In addition, we also show that transcriptional differences separate retinal progenitor cells (RPCs) into distinct subtypes and use this information to reconstruct RPC developmental trajectories and cell fate. Our results support a hierarchical program of differentiation governing cell-type diversity in the developing human retina. In summary, our work details comprehensive molecular classification of retinal cells, reconstructs their relationships, and paves the way for future mechanistic studies on the impact of gene regulation upon human retinogenesis.

neuroscience↗

bioRxiv2018

Trans-ethnic genome-wide association study provides insight into effector genes and molecular mechanisms for kidney function and highlights a causal effect on kidney-specific disease aetiologies

Chronic kidney disease (CKD) affects [~]10% of the global population, with considerable ethnic differences in prevalence and aetiology. We assembled genome-wide association studies (GWAS)1-3 of estimated glomerular filtration rate (eGFR), a measure of kidney function that defines CKD, in 312,468 individuals from four ancestry groups. We identified 93 loci (20 novel), which were delineated to 127 distinct association signals. These signals were homogenous across ancestries, and were enriched for protein-coding exons, kidney-specific histone modifications, and transcription factor binding sites for HDAC2 and EZH2. Fine-mapping revealed 40 high-confidence variants driving eGFR associations and highlighted potential causal genes with cell-type specific expression in glomerulus, and proximal and distal nephron. Mendelian randomisation (MR) supported causal effects of eGFR on overall and cause-specific CKD, kidney stone formation, diastolic blood pressure (DBP) and hypertension. These results define novel molecular mechanisms and effector genes for eGFR, offering insight into clinical outcomes and routes to CKD treatment development.

genetics↗

bioRxiv2018

Multiple Toll-Spätzle Pathways in Drosophila melanogaster Immunity

The Drosophila melanogaster Toll-Spatzle pathway plays an important role in development and immunity. Drosophila genome encodes nine Toll receptors and six Spatzle (Spz) proteins, and only the canonical Toll-Spz (Toll-1-Spz-1) pathway has been well investigated. In this study, we compared the nine Drosophila Tolls and showed that similarly to Toll, Toll-7 also strongly activated drosomycin promoter. Importantly, we showed that both Toll and Toll-7 interacted with Spz, Spz-2 and Spz-5, and co-expression of Toll or Toll-7 with Spz, Spz-2 and Spz-5 activated the drosomycin promoter. Furthermore, Toll and Toll-7 both recognized vesicular stomatitis virus (VSV) by binding to the VSV glycoprotein. Septic infection in Toll and Toll-7 mutant flies suggested that Toll and Toll-7 differentially affected defense responses in adult males and females after systemic infection by Enterococcus faecalis, Pseudomonas aeruginosa, Candida albicans or VSV. Our results suggest multiple Toll family members activate the expression of antimicrobial peptides. Our results also provide evidence that Toll and Toll-7 bind multiple Spatzle proteins and differentially affect immune defense against different pathogens in adult male and female flies.

immunology↗

bioRxiv2018

Visualizing ATP-dependent substrate-processing dynamics of the human 26S proteasome at near-atomic resolution

The proteasome is an ATP-dependent 2.5-megadalton machine responsible for ubiquitylated protein degradation in all eukaryotic cells. Here we present cryo-EM structures of the substrate-engaged human 26S proteasome in seven conformational states at 2.8-3.6 [A] resolution, captured during polyubiquitylated protein degradation. These structures visualize a continuum of dynamic substrate-proteasome interactions from ubiquitin recognition to processive substrate translocation, during which ATP hydrolysis sequentially navigate through all six ATPase subunits. Three principle modes of coordinated ATP hydrolysis are observed, featuring hydrolytic events in two oppositely positioned ATPases, in two consecutive ATPases, and in one ATPase at a time. They regulate deubiquitylation, translocation initiation and processive unfolding of substrates, respectively. A collective power stroke in the ATPase motor is generated by synchronized ATP binding and ADP release in the substrate-engaging and disengaging ATPases, respectively. It is amplified largely in the substrate-disengaging ATPase, and propagated unidirectionally by coordinated ATP hydrolysis in the third consecutive ATPase.

biochemistry↗

bioRxiv2018

Glutamate spillover in C. elegans triggers repetitive behavior through presynaptic activation of MGL-2/mGluR5

Glutamate is a major excitatory neurotransmitter, and impaired glutamate clearance following synaptic release promotes spillover, inducing extra-synaptic signaling. The effects of glutamate spillover on animal behavior and its neural correlates are poorly understood. We developed a glutamate spillover model in Caenorhabditis elegans by inactivating the conserved glial glutamate transporter GLT-1. GLT-1 loss drives aberrant repetitive locomotory reversal behavior through uncontrolled oscillatory release of glutamate onto AVA, a major interneuron governing reversals. Repetitive glutamate release and reversal behavior require the glutamate receptor MGL-2/mGluR5, expressed in RIM and other interneurons presynaptic to AVA. mgl-2 loss blocks oscillations and repetitive behavior; while RIM activation is sufficient to induce repetitive reversals in glt-1 mutants. Repetitive AVA firing and reversals require EGL-30/Gq, an mGluR5 effector. Our studies reveal that cyclic autocrine presynaptic activation drives repetitive reversals following glutamate spillover. That mammalian GLT1 and mGluR5 are implicated in pathological motor repetition suggests a common mechanism controlling repetitive behaviors.

neuroscience↗

bioRxiv2018

Suppression of p16 increases nucleotide synthesis via mTORC1

Reprogrammed metabolism and cell cycle dysregulation are two cancer hallmarks. p16 is a cell cycle inhibitor and tumor suppressor that is upregulated during oncogene-induced senescence (OIS). Loss of p16 allows for uninhibited cell cycle progression, bypass of OIS, and tumorigenesis. Whether p16 loss affects pro-tumorigenic metabolism is unclear. We report that suppression of p16 plays a central role in reprogramming metabolism by increasing nucleotide synthesis. This occurred via activation of mTORC1 signaling, which directly mediated increased translation of the mRNA encoding ribose-5-phosphate isomerase A (RPIA), a pentose phosphate pathway enzyme. p16 loss correlated with activation of the mTORC1-RPIA axis in multiple cancer types. Suppression of RPIA inhibited proliferation only in p16-low cells by inducing senescence both in vitro and in vivo. These data reveal the molecular basis whereby p16 loss modulates pro-tumorigenic metabolism through mTORC1-mediated upregulation of nucleotide synthesis and reveals a metabolic vulnerability of p16-null cancer cells.\n\nHighlightsO_LImTORC1 is activated by p16 knockdown to increase nucleotide synthesis and bypass senescence\nC_LIO_LImTORC1 directly increases translation RPIA to increase ribose-5-phosphate\nC_LIO_LIActivation of mTORC1 pathway downstream of p16 suppression is independent of RB\nC_LIO_LIRPIA suppression induces senescence only in cells and tumors with low p16\nC_LI

cancer biology↗

bioRxiv2018

PROTEIN-CODING VARIANTS IMPLICATE NOVEL GENES RELATED TO LIPID HOMEOSTASIS CONTRIBUTING TO BODY FAT DISTRIBUTION

Body fat distribution is a heritable risk factor for a range of adverse health consequences, including hyperlipidemia and type 2 diabetes. To identify protein-coding variants associated with body fat distribution, assessed by waist-to-hip ratio adjusted for body mass index, we analyzed 228,985 predicted coding and splice site variants available on exome arrays in up to 344,369 individuals from five major ancestries for discovery and 132,177 independent European-ancestry individuals for validation. We identified 15 common (minor allele frequency, MAF[≥]5%) and 9 low frequency or rare (MAF<5%) coding variants that have not been reported previously. Pathway/gene set enrichment analyses of all associated variants highlight lipid particle, adiponectin level, abnormal white adipose tissue physiology, and bone development and morphology as processes affecting fat distribution and body shape. Furthermore, the cross-trait associations and the analyses of variant and gene function highlight a strong connection to lipids, cardiovascular traits, and type 2 diabetes. In functional follow-up analyses, specifically in Drosophila RNAi-knockdown crosses, we observed a significant increase in the total body triglyceride levels for two genes (DNAH10 and PLXND1). By examining variants often poorly tagged or entirely missed by genome-wide association studies, we implicate novel genes in fat distribution, stressing the importance of interrogating low-frequency and protein-coding variants.

genetics↗

bioRxiv2018

BindSpace: decoding transcription factor binding signals by large-scale joint embedding

Decoding transcription factor (TF) binding signals in genomic DNA is a fundamental problem. Here we present a prediction model called BindSpace that learns to embed DNA sequences and TF class/family labels into the same space. By training on binding data for hundreds of TFs and embedding over 1M DNA sequences, BindSpace achieves state-of-the-art multiclass binding prediction performance, in vitro and in vivo, and can distinguish signals of closely related TFs.

bioinformatics↗

bioRxiv2018

Identification of a Nocardia seriolae secreted protein targeting host cell mitochondria and inducing apoptosis in fathead minnow (FHM) cells

Nocardia seriolae, is a Gram-positive, partially acid-fast, aerobic, and filamentous bacterium. This bacterium is the main pathogen of fish nocardiosis. A bioinformatic analysis based on the genomic sequence of the N. seriolae strain ZJ0503 showed that ORF3141 encoded a secreted protein with a signal peptide at the N-terminate which may target the mitochondria in the host cell. However, the functions of this protein and its homologs remain unknown. In this study, we experimentally tested the bioinformatic prediction on this protein. Mass spectrometry analysis of the extracellular products from N. seriolae showed that ORF3141 was a secreted protein. Subcellular localization of the ORF3141-GFP fusion protein revealed that the green fluorescence protein co-localized with the mitochondria, while ORF3141{Delta}sig-GFP (with the signal peptide deleted) fusion protein was evenly distributed in the whole cell of fathead minnow (FHM) cells. Thus, the N-terminate signal peptide had a significant impact on mitochondrial targeting. Notably, the expression of ORF3141 protein changed the distribution of mitochondria from perinuclear halo into lumps in the transfected FHM cells. In addition, apoptotic features were found in the transfected FHM cells by overexpression of ORF3141 and ORF3141{Delta}sig proteins, respectively. Quantitative assays of mitochondrial membrane potential value, caspase-3 activity and apoptosis-related gene mRNA expression suggested that cell apoptosis was induced in the transfected FHM cells. In conclusion, the ORF3141 was a secreted protein of N. seriolae that targeted host cell mitochondria and induced apoptosis in FHM cells. This protein may participate in the cell apoptosis regulation and plays an important role in the pathogenesis of N. seriolae.\n\nAuthor summaryNocardia seriolae is the causative pathogen responsible for fish nocardiosis. This facultative intercellular bacterium, adapts to survive and colonize by evading intracellular killing after being engulfed with macrophages in the host. Despite considerable economic losses caused by N. seriolae in fish infection, the pathogenic mechanism and specific virulence factor of this bacterium remain ambiguous. In this study, the characteristic of ORF3141 protein function was investigated by subcellular localization and its possible contributions on the ability of N. seriolae to induce apoptosis in transfected fathead minnow (FHM) cells was investigated. Here, we confirmed that ORF3141 was a secreted protein that targeted host cell mitochondria and induced cell apoptosis in FHM cells. Interestingly, after deleting the signal peptide, ORF3141{Delta}sig protein was evenly distributed in the whole host cell and did not co-localize with the mitochondria which could also induce cell apoptosis. Thus, the N-terminate signal peptide played an important role in mitochondrial targeting, and the domain part without the signal peptide had a critical relationship with cell apoptosis. These results demonstrated that ORF3141 mays act as a potential virulence factor that induces apoptosis in fish cells. This protein is significant to elucidate the pathogenic mechanism of N. seriolae and this study mays provide beneficial insight to prevent and treat fish nocardiosis.

pathology↗

bioRxiv2018

Collagen assembly and turnover imaged with a CRISPR-Cas9 engineered Dendra2 tag

Electron microscopy has been the \"gold standard\" for studying collagen networks but dynamic information on how cells synthesise the networks has been lacking. Live imaging methods have been unable to distinguish newly-synthesised fibrils from pre-existing fibrils and intracellular collagen. Here, we tagged endogenous collagen-I using CRISPR-Cas9 with photoswitchable Dendra2 and demonstrate live cells synthesising, migrating on, and interacting with, collagen fibrils. This strategy is applicable for other long half-life proteins.

cell biology↗

bioRxiv2018

Circadian Clock Regulation of the Secretory Pathway

Collagen is the most abundant secreted protein in vertebrates that persists throughout life without renewal. The unchanging nature of collagen contrasts with observed continued collagen synthesis throughout adulthood and with conventional transcriptional and translational homeostatic mechanisms that replace damaged proteins with new copies. Here we show circadian clock regulation of procollagen transport from ER-to-Golgi and Golgi-to-plasma membrane by sequential rhythmic expression of SEC61, TANGO1, PDE4D and VPS33B. The result is nocturnal procollagen synthesis and daytime collagen fibril assembly in mice. Rhythmic collagen degradation by CTSK maintains collagen homeostasis. This circadian cycle of collagen synthesis, assembly and degradation affects only a pool of newly-synthesized collagen whilst maintaining the persistent collagen network. Disabling the circadian clock causes collagen accumulation and abnormal fibrils in vivo. In conclusion, our study has identified a circadian clock mechanism of protein homeostasis in which a sacrificial pool of collagen is synthesized and removed to maintain tissue function.

cell biology↗

bioRxiv2018

Genome-wide analysis of adolescent psychotic-like experiences shows genetic overlap with psychiatric disorders

This study aimed to test for overlap in genetic influences between psychotic experience traits shown by adolescents in the community, and clinically-recognized psychiatric disorders in adulthood, specifically schizophrenia, bipolar disorder, and major depression. The full spectra of psychotic experience domains, both in terms of their severity and type (positive, cognitive, and negative), were assessed using self- and parent-ratings in three European community samples aged 15-19 years (Final N incl. siblings = 6,297-10,098). A mega-genome-wide association study (mega-GWAS) for each psychotic experience domain was performed. SNP-heritability of each psychotic experience domain was estimated using genomic-relatedness-based restricted maximum-likelihood (GREML) and linkage disequilibrium-(LD-) score regression. Genetic overlap between specific psychotic experience domains and schizophrenia, bipolar disorder, and major depression was assessed using polygenic risk scoring (PRS) and LD-score regression. GREML returned SNP-heritability estimates of 3-9% for psychotic experience trait domains, with higher estimates for less skewed traits (Anhedonia, Cognitive Disorganization) than for more skewed traits (Paranoia and Hallucinations, Parent-rated Negative Symptoms). Mega-GWAS analysis identified one genome-wide significant association for Anhedonia within IDO2 but which did not replicate in an independent sample. PRS analysis revealed that the schizophrenia PRS significantly predicted all adolescent psychotic experience trait domains (Paranoia and Hallucinations only in non-zero scorers). The major depression PRS significantly predicted Anhedonia and Parent-rated Negative Symptoms in adolescence. Psychotic experiences during adolescence in the community show additive genetic effects and partly share genetic influences with clinically-recognized psychiatric disorders, specifically schizophrenia and major depression.

genetics↗

bioRxiv2018

The critical role of ASD-related gene CNTNAP3 in regulating synaptic development and social behavior in mice

Accumulated genetic evidences indicate that the contactin associated protein-like (CNTNAP) family is implicated in autism spectrum disorders (ASD). In this study, we identified genetic mutations in the CNTNAP3 gene from Chinese Han ASD cohorts and Simons Simplex Collections. We found that CNTNAP3 interacted with synaptic adhesion proteins Neuroligin1 and Neuroligin2, as well as scaffolding proteins PSD95 and Gephyrin. Significantly, we found that CNTNAP3 played an opposite role in controlling the development of excitatory and inhibitory synapses in vitro and in vivo, in which ASD mutants exhibited loss-of-function effects. In this study, we showed that Cntnap3-null mice exhibited deficits in social interaction, spatial learning and prominent repetitive behaviors. These evidences elucidate the pivotal role of CNTNAP3 in synapse development and social behaviors, providing the mechanistic insights for ASD.

neuroscience↗

bioRxiv2018

Inhibition of Hsp70 suppresses neuronal hyperexcitability and attenuates seizures by enhancing A-type potassium currents

The heat shock protein 70 (Hsp70) is upregulated in response to stress and has been implicated as a stress marker in temporal lobe epilepsy (TLE). However, whether Hsp70 plays a pathologic or protective role in TLE remains unclear. Here we report that Hsp70 exerts an unexpected deleterious role in kainic acid (KA)-induced seizures, and inhibition of Hsp70 suppresses neuronal hyperexcitability and attenuates both acute and chronic seizures via enhancing A-type potassium currents primarily formed by Kv4 -subunits and auxiliary KChIPs. Proteosomal degradation of Kv4-KChIP4a channel complexes is enhanced by Hsp70, which can be reversed by the Hsp70 inhibitors, 2-phenylethynesulfonamide (PES) and VER-155008 (VER). In cultured hippocampal neurons, either PES or VER can increase A-type Kv4 current to suppress neuronal hyperexcitability. Mechanistically, Hsp70-CHIP complexes directly bind to the N-terminus of auxiliary KChIP4a and target Kv4-KChIP4a complexes to the proteasome. Our findings reveal a previously unrecognized role of Hsp70 in mediating degradation of Kv4-KChIP4a complexes and regulating neuronal excitability, thus highlighting a therapeutic potential for hyperexcitability-related neurological disorders through Hsp70 inhibition.

neuroscience↗

bioRxiv2018

miRge 2.0: An updated tool to comprehensively analyze microRNA sequencing data

miRNAs play important roles in the regulation of gene expression. The rapidly developing field of microRNA sequencing (miRNA-seq; small RNA-seq) needs comprehensive bioinformatics tools to analyze these large datasets. We present the second iteration of miRge, miRge 2.0, with multiple enhancements. miRge 2.0 adds new functionality including novel miRNA detection, A-to-I editing analysis, better output files, and improved alignment to miRNAs. Our novel miRNA detection method is the first to use both miRNA hairpin sequence structure and composition of isomiRs resulting in a more specific capture of potential miRNAs. Using known miRNA data, our support vector machine (SVM) model predicted miRNAs with an average Matthews correlation coefficient (MCC) of 0.939 over 32 human cell datasets and outperformed miRDeep2 and miRAnalyzer regarding phylogenetic conservation. The A-to-I editing analysis implementation strongly correlated with a reference datasets prior analysis with adjusted R2 = 0.96. miRge 2.0 comes with alignment libraries to both miRBase v21 and MirGeneDB for 6 species: human, mouse, rat, fruit fly, nematode and zebrafish; and has a tool to create custom libraries. With the redevelopment of the tool in Python, it is now incorporated into bcbio-nextgen and implementable through Bioconda. miRge 2.0 is freely available at: https://github.com/mhalushka/miRge.

bioinformatics↗

bioRxiv2018

Genetic Diversity and Distributional Pattern of Ammonia Oxidizing Archaea Lineages in the Global Oceans

In the study, we used miTAG approach to analyse the distributional pattern of the ammonium oxidizing archaea (AOA) lineages in the global oceans using the metagenomics datasets of the Tara Oceans global expedition (2009-2013). Using ammonium monooxygenase alpha subunit gene as biomarker, the AOA communities were obviously segregated with water depth, except the upwelling regions. Besides, the AOA communities in the euphotic zones are more heterogeneous than in the mesopelagic zones (MPZs). Overall, water column A clade (WCA) distributes more evenly and widely in the euphotic zone and MPZs, while water column B clade (WCB) and SCM-like clade mainly distribute in MPZ and high latitude waters, respectively. At fine-scale genetic diversity, SCM1-like and 2 WCA subclades showed distinctive niche separation of distributional pattern. The AOA subclades were further divided into ecological significant taxonomic units (ESTUs), which were delineated from the distribution pattern of their corresponding subclades. For examples, ESTUs of WCA have different correlation with depth, nitrate to silicate ratio and salinity; SCM1-like-A was negatively correlated with irradiation; the other SCM-like ESTUs preferred low temperature and high nutrient conditions, etc. Our study provides new insight to the genetic diversity of AOA in global scale and its connections with environmental factors.

ecology↗

bioRxiv2018

Spatial cell firing during virtual navigation of open arenas by head-restrained mice

We present a mouse virtual reality (VR) system which restrains head-movements to horizontal rotations, potentially compatible with multi-photon imaging. We show that this system allows expression of the spatial navigational behaviour and neuronal firing patterns characteristic of real open arenas (R). Place and grid, but not head-direction, cell firing had broader spatial tuning in VR than R. Theta frequency increased less with running speed in VR than in R, while firing rates increased similarly in both. Place, but not grid, cell firing was more directional in VR than R. These results suggest that the scale of grid and place cell firing patterns, and the frequency of theta, reflect translational motion inferred from both virtual (visual and proprioceptive) cues and uncontrolled static (vestibular translation and extra-maze) cues, while firing rates predominantly reflect visual and proprioceptive motion. They also suggest that omni-directional place cell firing in R reflects local-cues unavailable in VR.

neuroscience↗