Biology subjects

Xiang, J.

Publications and source records attributed to Xiang, J..

67 records · Page 4Linked to original sources

bioRxiv2020

Dppa2/4 promotes zygotic genome activation by binding to GC-rich region of proximal promoter in signaling pathways

Developmental pluripotency associated 2 (Dppa2) and Dppa4 as positive drivers were helpful for transcriptional regulation of ZGA. Here, we systematically assessed the cooperative interplay between Dppa2 and Dppa4 in regulating cell pluripotency of three cell types and found that simultaneous overexpression of Dppa2/4 can make induced pluripotent stem cells closer to embryonic stem cells. Compared with other pluripotency transcription factors (TFs), Dppa2/4 tends to bind on GC-rich region of proximal promoter (0-500bp). Moreover, there was more potent effect of Dppa2/4 regulation on signaling pathways than other TFs, in which 75% and 85% signaling pathways were significantly activated by Dppa2 and Dppa4, respectively. Notably, Dppa2/4 also can dramatically trigger the decisive signaling pathways for facilitating ZGA, including Hippo, MAPK and TGF-beta signaling pathways and so on. At last, we found that Alkaline phosphatase placental-like 2 (Alppl2) was significantly activated at the 2-cell stage in mouse embryos and 4-8 cell stage in human embryos, further predicted that Alppl2 was directly regulated by Dppa2/4 as a candidate driver of ZGA to regulate pre-embryonic development.

bioinformatics↗

bioRxiv2019

Translationally relevant transcriptomic alterations in mouse ischemic cerebral microvessels

Increasing evidence implicates cerebral microvascular dysfunction in the pathophysiology of numerous central nervous system pathologies, including stroke. Understanding the molecular alterations in cerebral microvessels in these conditions will provide original opportunities for scientific investigation at the pre-clinical and clinical levels. In this study, we conducted a novel genome-wide transcriptomic analysis of microvessels in a mouse model of transient focal cerebral ischemia. Using a publicly available human ischemic stroke dataset, we identified shared alterations in our microvessel dataset with implications for human pathophysiology. From this unbiased analysis, we report predicted alterations in inter- and intra-cellular signaling, emphasizing perturbations in genes involved in blood brain barrier function, endothelial cell activation and metabolism. Furthermore, our study unveiled previously unreported gene expression changes associated with altered sphingolipid metabolism. Altogether, our results have identified microvessel-specific transcriptomic changes in a number of translationally relevant pathways that support the targeting of these pathways in preclinical studies. The data shared here provide a resource for future investigation of translationally relevant pathways in ischemic stroke.

neuroscience↗

bioRxiv2019

AutoPVS1: An automatic classification tool for PVS1 interpretation of null variants

Null variants are prevalent within human genome, and their accurate interpretation is critical for clinical management. In 2018, the ClinGen Sequence Variant Interpretation (SVI) Working Group refined the only criterion (PVS1) for pathogenicity in the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines. The refinement may improve interpretation consistency, but it also brings hurdles to biocurators because of the complicated workflows and multiple bioinformatics sources required. To address these issues, we developed an automatic classification tool called AutoPVS1 to streamline PVS1 interpretation. We assessed the performance of AutoPVS1 using 56 variants manually curated by ClinGens SVI Working Group and achieved an interpretation concordance of 95% (53/56). A further analysis of 28,586 putative loss-of-function variants by AutoPVS1 demonstrated that at least 27.6% of them do not reach a very strong strength level, with 17.4% based on variant-specific issues and 10.2% on disease mechanism considerations. Moreover, 40.7% (1,918/4,717) of splicing variants were assigned a decreased PVS1 strength level, significantly higher than frameshift and nonsense variants. Our results reinforce the necessity of considering variant-specific issues and disease mechanisms in variant interpretation, and demonstrate that AutoPVS1 is an accurate, reproducible, and reliable tool which facilitates PVS1 interpretation and will thus be of great importance to curators.

genetics↗

bioRxiv2019

Human induced pluripotent stem cell-derived neuroectodermal epithelial cells mistaken for blood-brain barrier-forming endothelial cells

Brain microvascular endothelial cells (BMECs) possess unique properties underlying the blood-brain-barrier (BBB), that are crucial for homeostatic brain functions and interactions with the immune system. Modulation of BBB function is essential for treatment of neurological diseases and effective tumor targeting. Studies to-date have been hampered by the lack of physiological models using cultivated human BMECs that sustain BBB properties. Recently, differentiation of induced pluripotent stem cells (iPSCs) into cells with BBB-like properties has been reported, providing a robust in vitro model for drug screening and mechanistic understanding of neurological diseases. However, the precise identity of these iBMECs remains unclear. Employing single-cell RNA sequencing, bioinformatic analysis and immunofluorescence for several pathways, transcription factors (TFs), and surface markers, we examined the molecular and functional properties of iBMECs differentiated either in the absence or presence of retinoic acid. We found that iBMECs lack both endothelial-lineage genes and ETS TFs that are essential for the establishment and maintenance of EC identity. Moreover, iBMECs fail to respond to angiogenic stimuli and form lumenized vessels in vivo. We demonstrate that human iBMECs are not barrier-forming ECs but rather EpCAM+ neuroectodermal epithelial cells (NE-EpiCs) that form tight junctions resembling those present in BBB-forming BMECs. Finally, overexpression of ETS TFs (ETV2, FLI1, and ERG) reprograms NE-EpiCs to become more like the BBB-forming ECs. Thus, although directed differentiation of human iBMECs primarily gives rise to epithelial cells, overexpression of several ETS TFs can divert them toward a vascular BBB in vitro.

cell biology↗

bioRxiv2019

The Norepinephrine Metabolite DOPEGAL Confers Locus Coeruleus Tau Vulnerability and Propagation via Asparagine Endopeptidase

Aberrant Tau inclusions in the locus coeruleus (LC) are the earliest detectable Alzheimers disease (AD)-like neuropathology in the human brain; however, why LC neurons are selectively vulnerable to developing early Tau pathology and degenerating later in disease and whether the LC might seed the stereotypical spread of Tau pathology to the rest of the brain remain unclear. Here we show that 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL), which is produced exclusively in noradrenergic neurons by monoamine oxidase A (MAO-A) metabolism of norepinephrine (NE), activates asparagine endopeptidase (AEP) that cleaves Tau at residue N368 into aggregation- and propagation-prone forms, thereby leading to LC degeneration and the spread of Tau pathology. DOPEGAL triggers AEP-cleaved Tau aggregation in vitro and in intact cells, resulting in LC neurotoxicity and propagation of pathology to the forebrain. Thus, our findings reveal a novel molecular mechanism underlying the selective vulnerability of LC neurons in AD.

neuroscience↗

bioRxiv2019

A Chemical Probe of CARM1 Alters Epigenetic Plasticity against Breast Cancer Cell Invasion

CARM1 is a cancer-relevant protein arginine methyltransferase that regulates many aspects of transcription. Its pharmacological inhibition is a promising anti-cancer strategy. Here SKI-73 is presented as a CARM1 chemical probe with pro-drug properties. SKI-73 can rapidly penetrate cell membranes and then be processed into active inhibitors, which are retained intracellularly with 10-fold enrichment for days. These compounds were characterized for their potency, selectivity, modes of action, and on-target engagement. SKI-73 recapitulates the effect of CARM1 knockout against breast cancer cell invasion. Single-cell RNA-seq analysis revealed that the SKI-73-associated reduction of invasiveness act via altering epigenetic plasticity and suppressing the invasion-prone subpopulation. Interestingly, SKI-73 and CARM1 knockout alter the epigenetic plasticity with remarkable difference, arguing distinct modes of action between the small-molecule and genetic perturbation. We therefore discovered a CARM1-addiction mechanism of cancer metastasis and developed a chemical probe to target this process.

biochemistry↗

bioRxiv2019

A 3-hydroxy-3-methylglutaryl-CoA synthase-based probe for the discovery of the acyltransferase-less type I polyketide synthases

Acyltransferase (AT)-less type I polyketide synthases (PKSs) produce complex natural products due to the presence of many unique tailoring enzymes. The 3-hydroxy-3-methylglutaryl coenzyme A synthases (HCSs) are responsible for {beta}-alkylation of the growing polyketide intermediates in AT-less type I PKSs. In this study, we discovered a large group of HCSs, closely associated with the characterized and orphan AT-less type I PKSs through in silico genome mining, sequence and genome neighborhood network analysis. Using HCS-based probes, the survey of 1207 inhouse strains and 18 soil samples from different geological locations revealed the vast diversity of HCS-containing AT-less type I PKSs. The presence of HCSs in many AT-less type I PKSs suggests their co-evolutionary relationship. Our study should inspire future efforts to discover new polyketide natural products from AT-less type I PKSs.

microbiology↗

bioRxiv2018

Nationwide population genetic screening improves outcomes of newborn screening for hearing loss in China

PurposeConcurrent newborn hearing and genetic screening has been reported, but its benefits have not been statistically proven due to limited sample sizes and outcome data. To fill this gap, we analyzed outcomes of a large number of newborns with genetic screening results.\n\nMethodsNewborns in China were screened for 20 hearing-loss-related genetic variants from 2012-2017. Genetic results were categorized as positive, at-risk, inconclusive, or negative. Hearing screening results, risk factors, and up-to-date hearing status were followed-up via phone interviews.\n\nResultsWe completed genetic screening on one million newborns and followed up 12,778. We found that a positive genetic result significantly indicated a higher positive predictive value of the initial hearing screening (60% vs. 5.0%, P<0.001) and a lower rate of loss-to-follow-up (5% vs. 22%, P<0.001) than an inconclusive one. Importantly, 42% of subjects in the positive group with reported or presymptomatic hearing loss were \"missed\" by conventional hearing screening. Furthermore, genetic screening identified 0.23% of subjects predisposed to preventable ototoxicity.\n\nConclusionOur results demonstrate that limited genetic screening identified additional cases, reduced loss-to-follow-up, and informed families of ototoxicity risks, providing convincing evidence to support integrating genetic screening into universal newborn hearing screening programs.

genetics↗

bioRxiv2018

Pre-implantation genetic diagnosis for a family with Usher syndrome through targeted sequencing and haplotype analysis

ObjectiveOur objective was to investigate the applicability of targeted capture massively parallel sequencing in developing personalized pre-implantation genetic diagnosis (PGD) assay.\n\nMethodsOne couple at risk of transmitting Usher Syndrome to their offspring was recruited to this study. The genomics DNA (gDNA) was extracted from the peripheral blood and underwent in vitro fertilization (IVF)-PGD. Prenatal molecular diagnosis was performed in the 20th week of gestation and the chromosomal anomaly was analyzed.\n\nResultsCustomized capture probe targeted at USH2A gene and 350kb flanking region were designed for PGD. Eleven blastocysts were biopsied and amplified by using multiple displacement amplification (MDA) and capture sequencing. A HMM-based haplotype analysis was performed to deduce embryos genotype by using SNPs identified in each sample. Four embryos were diagnosed as free of fathers rare mutation, two were transferred and one achieved a successful pregnancy. The fetal genotype was confirmed by Sanger sequencing of fetal genomic DNA obtained by amniocentesis. The PGD and prenatal diagnosis results were further confirmed by the molecular diagnosis of the babys genomic DNA sample. The auditory test showed that the hearing was normal.\n\nConclusionTargeted capture massively parallel sequencing (MPS) is an effective and convenient strategy to develop customized PGD assay.\n\nKey pointsGenetic counseling session was conducted with a family having Usher patient who was molecularly diagnosed, and a healthy baby was born with the help of successful PGD assay. This is of vast importance in management plans since it is the first report of PGD in Usher syndrome based on targeted capture MPS.

genetics↗

bioRxiv2018

Identifying functional targets from transcription factor binding data using SNP perturbation

Transcription factors (TFs) play a key role in transcriptional regulation by binding to DNA to initiate the transcription of target genes. Techniques such as ChIP-seq and DNase-seq provide a genome-wide map of TF binding sites but do not offer direct evidence that those bindings affect gene expression. Thus, these assays are often followed by TF perturbation experiments to determine functional binding that leads to changes in target gene expression. However, such perturbation experiments are costly and time-consuming, and have a well-known limitation that they cannot distinguish between direct and indirect targets. In this study, we propose to use the naturally occurring perturbation of gene expression by genetic variation captured in population SNP and expression data to determine functional targets from TF binding data. We introduce a computational methodology based on probabilistic graphical models for isolating the perturbation effect of each individual SNP, given a large number of SNPs across genomes perturbing the expression of all genes simultaneously. Our computational approach constructs a gene regulatory network over TFs, their functional targets, and further downstream genes, while at the same time identifying the SNPs perturbing this network. Compared to experimental perturbation, our approach has advantages of identifying direct and indirect targets, and leveraging existing data collected for expression quantitative trait locus mapping, a popular approach for studying the genetic architecture of expression. We apply our approach to determine functional targets from the TF binding data for a lymphoblastoid cell line from the ENCODE Project, using SNP and expression data from the HapMap 3 and 1000 Genomes Project samples. Our results show that from TF binding data, functional target genes can be determined by SNP perturbation of various aspects that impact transcriptional regulation, such as TF concentration and TF-DNA binding affinity.

bioinformatics↗

bioRxiv2018

The cargo receptor SURF4 promotes the efficient cellular secretion of PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that plays an important role in regulating plasma cholesterol and cardiovascular disease risk. PCSK9 secretion uniquely depends on the cytoplasmic COPII protein SEC24A, suggesting the presence of a transmembrane ER cargo receptor mediating this interaction. Here, we report a novel approach that combines proximity-dependent biotinylation and proteomics together with genome-scale CRISPR screening to identify proteins that facilitate the efficient secretion of PCSK9 heterologously expressed in HEK293T cells. We first identified 35 candidate proteins that were labeled by BirA* fusions to PCSK9 and either COPII component SAR1A or SAR1B. We then performed genome-scale pooled CRISPR mutagenesis to identify genes whose perturbation resulted in intracellular accumulation of PCSK9-eGFP but not the control A1AT-mCherry. The 4 most enriched sgRNAs in this screen all targeted SURF4, a homologue of the yeast endoplasmic reticulum (ER) cargo receptor Erv29p and the only candidate also identified by proximity-dependent biotinylation. The functional contribution of SURF4 to PCSK9 secretion was confirmed with multiple independent SURF4-targeting sgRNAs, clonal SURF4-deficient cell lines, and functional rescue with SURF4 cDNA. Compatible with a function of SURF4 as a cargo receptor for PCSK9, fluorescence microscopy localized SURF4 to the early secretory pathway, coimmunoprecipitation revealed a physical interaction between SURF4 and PCSK9, and SURF4 deletion resulted in decreased extracellular secretion of PCSK9 and PCSK9 accumulation in the ER. Taken together, these findings support a model in which SURF4 functions as an ER cargo receptor for the efficient cellular secretion of PCSK9.

cell biology↗

bioRxiv2018

Detection of Pro-apoptotic BaxΔ2 Proteins in the Human Cerebellum

Bax{Delta}2 is a pro-apoptotic protein originally discovered in colon cancer patients with high microsatellite instability. Unlike most pro-apoptotic Bax family members, Bax{Delta}2 mediates cell death through a non-mitochondrial caspase 8-dependent pathway. In the scope of analyzing the distribution of Bax{Delta}2 expression in human tissues, we examined a panel of human brain samples. Here, we report 4 cerebellar cases in which the subjects had no neurological disorder or disease documented. We found Bax{Delta}2 positive cells scattered in all areas of the cerebellum, but most strikingly concentrated in Purkinje cell bodies and dendrites. Two out the four subjects tested had strong Bax{Delta}2- positive staining in nearly all Purkinje cells; one was mainly negative; and one had various levels of positive staining within the same sample. Further genetic analysis of the Purkinje cell layer, collected by microdissection from two subjects, showed that the samples contained G7 and G9 Bax microsatellite mutations. Both subjects were young and had no diseases reported at the time of death. As the distribution of Bax{Delta}2 is consistent with that known for Baxa, but in a less ubiquitous manner, these results may imply a potential function of Bax{Delta}2 in Purkinje cells.

cell biology↗

bioRxiv2017

Discovery and reporting of clinically-relevant germline variants in advanced cancer patients assessed using whole-exome sequencing

PurposeIn precision cancer care, WES-based analysis of tumor-normal samples helps reveal somatic alterations but can also identify cancer-associated germline variants important for disease surveillance, treatment choice and cancer prevention. WES can also identify germline secondary findings impacting risk of cardiac, neurodegenerative or metabolic diseases. In patients with advanced cancer, the frequency of reportable secondary findings encountered with WES is not well defined.\n\nMethodsTo address this question, we analyzed a cohort of 343 patients with advanced, metastatic cancer for whom we have performed tumor and germline WES interrogating more than 21,000 genes using a CLIA/CLEP approved assay.\n\nResults17% of patients in our cohort have one or more reportable germline variants, including patients with pathogenic variants in the BRCA1 and BRCA2 genes. The frequency of non-cancer clinically relevant germline variants (8.8%) was within the range of two control non-cancer cohorts (11.0% and 6.5%). The frequency of variants in cancer-associated genes was significantly higher (p<0.0005) in our advanced cancer cohort (8.2%) compared to control cohorts (2.7% and 3.8%). More than 50% of patients with reportable germline cancer variants had a family history of cancer.\n\nConclusionthese results stress the importance of returning germline results found during somatic genomic tumor testing.

genomics↗