Biology subjects

Chun, J.

Publications and source records attributed to Chun, J..

30 records · Page 2Linked to original sources

bioRxiv2021

Revealing the transcription factor regulatory context of human specific cortical development using single-cell multi-omics

Human behaviors are at least partially driven by genomic regions that influence human-specific neurodevelopment. This includes genomic regions undergoing human specific sequence acceleration (Human Accelerated Regions or HARs) and regions showing human-specific enhancer activity (Human Gained Enhancers or HGEs) not present in other primates. However, prior studies on HAR/HGE activities involved mixtures of brain cell types and focused only on putative downstream target genes. Here, we directly measured cell type specific HAR/HGE activity in the developing fetal human brain using two independent single-cell chromatin accessibility datasets with matching single-cell gene expression data. Transcription factor (TF) motif analyses identified upstream TFs binding to HARs/HGEs and identified LHX2, a key regulator of forebrain development, as an active HGE regulator in neuronal progenitors. We integrated our TF motif analyses with published chromatin interaction maps to build detailed regulatory networks where TFs are linked to downstream genes via HARs/HGEs. Through these networks, we identified a potential regulatory role for NFIC in human neuronal progenitor networks via modulating the Notch signaling and cell adhesion pathways. Therefore, by using a single cell multi-omics approach, we were able to capture both the upstream and downstream regulatory context of HARs/HGEs, which may provide a more comprehensive picture of the roles HARs/HGEs play amongst diverse fetal cell types of the developing human brain.

genomics↗

bioRxiv2021

Large scale genomic and evolutionary study reveals SARS-CoV-2 virus isolates from Bangladesh strongly correlate with European origin and not with China.

RationaleThe global public health is in serious crisis due to emergence of SARS-CoV-2 virus. Studies are ongoing to reveal the genomic variants of the virus circulating in various parts of the world. However, data generated from low- and middle-income countries are scarce due to resource limitation. This study was focused to perform whole genome sequencing of 151 SARS-CoV-2 isolates from COVID-19 positive Bangladeshi patients. The goal of this study was to identify the genomic variants among the SARS-CoV-2 virus isolates in Bangladesh, to determine the molecular epidemiology and to develop a relationship between host clinical trait with the virus genomic variants. MethodSuspected patients were tested for COVID-19 using one step commercial qPCR kit for SARS-CoV-2 Virus. Viral RNA was extracted from positive patients, converted to cDNA which was amplified using Ion AmpliSeq SARS-CoV-2 Research Panel. Massive parallel sequencing was carried out using Ion AmpliSeq Library Kit Plus. Assembly of raw data is done by aligning the reads to a pre-defined reference genome (NC_045512.2) while retaining the unique variations of the input raw data by creating a consensus genome. A random forest-based association analysis was carried out to correlate the viral genomic variants with the clinical traits present in the host. ResultAmong the 151 viral isolates, we observed the 413 unique variants. Among these 8 variants occurred in more than 80 % of cases which include 241C to T, 1163A to T, 3037C to T,14408C to T, 23403A to G, 28881G to A, 28882 G to A, and finally the 28883G to C. Phylogenetic analysis revealed a predominance of variants belonging to GR clade, which have a strong geographical presence in Europe, indicating possible introduction of the SARS-CoV-2 virus into Bangladesh through a European channel. However, other possibilities like a route of entry from China cannot be ruled out as viral isolate belonging to L clade with a close relationship to Wuhan reference genome was also detected. We observed a total of 37 genomic variants to be strongly associated with clinical symptoms such as fever, sore throat, overall symptomatic status, etc. (Fishers Exact Test p-value<0.05). The most mention-worthy among those were the 3916CtoT (associated with causing sore throat, p-value 0.0005), the 14408C to T (associated with protection from developing cough, p-value= 0.027), and the 28881G to A, 28882G to A, and 28883G to C variant (associated with causing chest pain, p-value 0.025). ConclusionTo our knowledge, this study is the first large scale phylogenomic studies of SARS-CoV-2 virus circulating in Bangladesh. The observed epidemiological and genomic features may inform future research platform for disease management, vaccine development and epidemiological study.

genetics↗

bioRxiv2020

A multimodal cell census and atlas of the mammalian primary motor cortex

We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties.

neuroscience↗

bioRxiv2020

Lysophosphatidic acid (LPA)-antibody (504B3)validation by free-solution assay and interferometry identifies off-target binding

Antibody specificity and sensitivity is required in basic and clinical research for ensuring scientific rigor and reproducibility, while off-target cross-reactivity could generate erroneous conclusions. Lysophosphatidic acid (LPA) is a bioactive lipid being targeted clinically by antibody strategies. Here, we reexamined binding properties of a commercially available monoclonal antibody (504B3) reported as specific for LPA using a free-solution assay measured in a compensated interferometric reader. The antibody showed comparable binding affinities to LPA and non-LPA lipids including phosphatidic acid (PA) and lysophosphatidylcholine (LPC). These results may alter conclusions drawn from current and past basic and clinical studies employing anti-LPA antibodies.

molecular biology↗

bioRxiv2020

Sphingosine 1-phosphate activates the MAP3K1-JNK pathway to promote epithelial movement and morphogenesis

MAP 3 kinase 1 (MAP3K1) plays an essential role in embryonic eyelid development. It regulates epithelial morphogenesis through the spatial-temporal activation of Jun N-terminal kinases (JNKs), resulting in forward progression of the embryonic eyelid epithelial cells to enable eyelid closure. The developmental signals that activate the MAP3K1-JNK pathway are still unknown, mainly due to the lack of suitable keratinocyte lines to elucidate the mechanisms of pathway regulation. To address this deficiency, we developed a straightforward method for long-term culture of mouse keratinocytes in feeder-free conditions using Ca2+-free media. Cells grown under these conditions displayed characteristic basal epithelial morphology and keratin 14 expression, but did not form tight- or adherens-junctions. Increased extracellular Ca2+ levels restored the formation of cell-cell junctions. Using keratinocyte lines derived from wild type and Map3k1-deficient mice, we found that sphingosine 1-phosphate (S1P) activated the JNK-c-JUN pathways in a manner dependent on MAP3K1 kinase activity and that this MAP3K1-mediated signaling led to epithelial cell migration. The in vivo roles of this pathway were examined through crossing of genetic mutant mice. Loss-of-function of the S1P receptor (S1pr) 2/3 became haploinsufficient only when combined with Map3k1 and Jnk1 mutations such that the compound mutants displayed eyelid closure defects, suggesting these gene products cooperated in eye morphogenesis. Results of this work establish the S1PR-MAP3K1-JNK pathway as a crucial signaling mechanism for epithelial cell movement and morphogenesis.View Full Text

developmental biology↗

bioRxiv2020

Metagenomic association analysis of gut symbiont Lactobacillus reuteri without host-specific genome isolation

Lactobacillus reuteri is a model symbiont colonizing the guts of vertebrates used for studies on host adaptation of the gut symbiont. Previous studies investigated host-specific phylogenetic and functional properties by isolating its genomic sequence. This dependency on genome isolation is a significant bottleneck. Here we propose a method to study the association between L. reuteri and its hosts directly from metagenomic reads without strain isolation by using pan-genomes. We characterized the host-specificity of L. reuteri in metagenomic samples not only in the previously studied organisms (mice and pigs) but additionally in dogs. For each sample, two types of profiles were generated: (1) genome-based strain type abundance profiles and (2) gene composition profiles. Our profiles showed host-association of L. reuteri in both phylogenetic and functional aspects without depending on the host-specific genome isolation. We could observe not only the presence of host-specific lineages but also the dominant lineages associated with the different hosts. Furthermore, we show that metagenome-assembled genomes provide detailed insights into the host-specificity of L. reuteri. We could infer evolutionary trajectories of host-associative L. reuteri strains in the metagenomic samples by placing the metagenome-assembled genomes into a phylogenetic tree and identify novel host-specific genes which were unannotated in existing pan-genome databases. Our pan-genomic approach drops the need for time-consuming and expensive host-specific genome isolation while producing consistent results with previous host-association findings in mice and pigs. Additionally, we could predict associations that have not yet been studied in dogs.

microbiology↗

bioRxiv2020

Deletion of Sphingosine 1-Phosphate Receptor 1 in cardiomyocytes during development leads to abnormal ventricular conduction and fibrosis

Sphingosine 1-phosphate receptor 1 (S1P1, encoded by S1pr1) is a G protein-coupled receptor that signals in multiple cell types including endothelial cells and cardiomyocytes. Cardiomyocyte-specific deletion of S1pr1 during development leads to ventricular noncompaction, with one-third of mutant mice surviving to adulthood. Adult survivors of embryonic cardiomyocyte S1pr1 deletion showed cardiac hypertrabeculation consistent with ventricular noncompaction. Surprisingly, systolic function in mutant mice was preserved through at least one year of age. Cardiac conduction was abnormal in cardiomyocyte S1pr1 mutant mice, with prolonged QRS intervals in mutants as compared with littermate control mice. Immunostaining of hearts from S1pr1 mutant embryos displayed a zone of intermediate Connexin 40 expression in the trabecular myocardium. However, we observed no significant differences in Connexin 40 and Connexin 43 immunostaining in hearts from adult survivors of embryonic cardiomyocyte S1pr1 deletion, which suggests normalized development of the ventricular conduction system in mutant mice. By contrast, the adult survivors of embryonic cardiomyocyte S1pr1 deletion showed increased cardiac fibrosis as compared with littermate controls. These results demonstrate that ventricular hypertrabeculation caused by embryonic deletion of cardiomyocyte S1pr1 leads to cardiac fibrosis, which contributes to abnormal ventricular conduction. These results also reveal conduction abnormalities in the setting of hypertrabeculation with normal systolic function, which may be of clinical relevance in humans with ventricular hypertrabeculation.

physiology↗

bioRxiv2020

Conditional Lpar1 gene targeting identifies cell types mediating neuropathic pain

LPA1 is one of the six known receptors (LPA1-6) for lysophosphatidic acid (LPA). Constitutive Lpar1 null mutant mice have been instrumental in identifying roles for LPA-LPA1 signaling in neurobiological processes, brain development, and behavior as well as modelling human neurological diseases like neuropathic pain. Constitutive Lpar1 null mutant mice are protected from partial sciatic nerve ligation (PSNL)-induced neuropathic pain, however Lpar1 expressing cell types that are functionally responsible for mediating this protective effect are unknown. Here we report generation of a Lpar1flox/flox conditional null mutant mouse that allows cre-mediated conditional deletion combined with its use in a PSNL pain model. Lpar1flox/flox mice were crossed with cre transgenic lines driven by neural gene promoters for nestin (all neural cells), synapsin (neurons), or P0 (Schwann cells). CD11b-cre transgenic mice were also used to delete Lpar1 in microglia. PSNL-initiated pain responses were reduced following cre-mediated Lpar1 deletion with all 3 neural promoters but not the microglial promoter, supporting involvement of Schwann cells and central and/or peripheral neurons in mediating pain. Interestingly, rescue responses that were due to conditional deletion were non-identical, implicating distinct roles for Lpar1-expressing cell types. Our results with a new Lpar1 conditional mouse mutant expand an understanding of LPA1 signaling in the PSNL model of neuropathic pain.

pharmacology and toxicology↗

bioRxiv2019

Activation of macrophages by lysophosphatidic acid through the lysophosphatidic acid receptor 1 as a novel mechanism in multiple sclerosis pathogenesis.

Multiple sclerosis (MS) is a neuro-inflammatory disease for which the pathogenesis remains largely unclear. Lysophosphatidic acid (LPA) is an endogenous phospholipid that is involved in multiple immune cell functions and is dysregulated in MS. Its receptor LPA1 is expressed in macrophages and regulates their activation, which is of interest due to the role of macrophage activation in MS in both destruction and repair. In this study, we studied the viable Malaga variant of LPA1-null mutation as well as pharmaceutical inhibition of LPA1 in mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. LPA1 expression was also analyzed in both wild-type EAE mice and MS patient immune cells. The effect of LPA and LPA1 on macrophage activation was studied in human monocyte-derived macrophages. We show that lack of LPA1 activity induces a milder clinical course in EAE, and that Lpar1 expression in peripheral blood mononuclear cells (PBMCs) correlates with onset of relapses and severity in wild-type EAE mice. We see the same over-expression in PBMCs from MS patients during relapse compared to progressive forms of the disease, and in monocyte-derived macrophages after exposure to pro-inflammatory stimuli. In addition, LPA induced a pro-inflammatory-like response in macrophages through LPA1, providing a plausible way in which LPA and LPA1 dysregulation can lead to the inflammation seen in MS. These data show a new mechanism of LPA signaling in the pathogenesis of MS, prompting further research into its use as a therapeutic target biomarker.

neuroscience↗

bioRxiv2019

Reply: Evidence that APP gene copy number changes reflect recombinant vector contamination

In the accompanying comment1, Kim et al. concluded that somatic gene recombination (SGR) and amyloid precursor protein (APP) genomic complementary DNAs (gencDNAs) in brain are contamination artifacts and do not naturally exist. We disagree. Here we address the three types of analyses used by Kim et al. to reach their conclusions: informatic contaminant identification, plasmid PCR, and single-cell sequencing. Additionally, Kim et al. requested "reads supporting novel APP insertion breakpoints," and we now provide 10 different examples that support APP gencDNA insertion within eight chromosomes beyond wildtype APP on chromosome 21 from Alzheimers disease (AD) samples. If SGR exists as experimentally supported here and previously2,3, contamination scenarios become moot. Our informatic analyses of data generated by an independent laboratory (Park et al.)4, complement and are entirely consistent with what Lee et al.2 presented via nine distinct lines of evidence, in addition to three from a prior publication3. Plasmid contamination was identified in a single pull-down dataset after publication of Lee et al.2; however subsequent analyses did not alter any of our conclusions including those of our prior publications3,5 and plasmid contamination-free replication of this approach by ourselves and others supported the original conclusions. Novel retro-insertion sites, alterations of APP gencDNA number and form with cell type from the same brain and pathogenic SNVs occurring only in AD, all support the existence of APP gencDNAs produced by SGR.

genomics↗

bioRxiv2019

Proteolytic program-dependent functions are impaired in INF2-mediated focal segmental glomerulosclerosis.

Regulation of the actin cytoskeleton is critical for normal glomerular podocyte structure and function. Altered regulation of the podocyte cytoskeleton can lead to proteinuria, reduced kidney filtration function and focal segmental glomerulosclerosis (FSGS). Mutations in inverted formin 2 (INF2), a member of the formin family of actin regulatory proteins, are the most common cause of autosomal dominant FSGS. INF2 is a multi-domain protein regulated by interaction between its N-terminal Diaphanous Inhibitory Domain (DID) and its C-terminal Diaphanous Auto-regulatory Domain (DAD). Although many aspects of the INF2 DID-DAD interaction are understood, it remains unclear why disease-causing mutations are restricted to the DID and how these mutations cause human disease. Here we report a proteolytic cleavage in INF2 that liberates the INF2 N-terminal DID to function independently of the INF2 C-terminal fragment containing the DAD domain. N-terminal DID region epitopes are differentially localized to podocyte foot process structures in normal glomeruli. This N-terminal fragment localization is lost in INF2-mediated FSGS, whereas INF2 C-terminal fragment epitopes localize to the podocyte cell body in both normal and disease conditions. INF2 cleavage is mediated by cathepsin proteases. In cultured podocytes, the wild-type INF2 N-terminal fragment localizes to membrane regions and promotes cell spreading, while these functions are impaired in a disease-associated INF2 mutant R218Q in the DID. These features are dependent on INF2-cleavage, with accompanying interaction of INF2 N-fragment with mDIA1. Our data suggest a unique cellular function of the DID dependent on INF2 cleavage and help explain the altered localization of FSGS-associated INF2 mutant polypeptides.

pathology↗

bioRxiv2017

Integrative Single-Cell Analysis By Transcriptional And Epigenetic States In Human Adult Brain

Detailed characterization of the cell types comprising the highly complex human brain is essential to understanding its function. Such tasks require highly scalable experimental approaches to examine different aspects of the molecular state of individual cells, as well as the computational integration to produce unified cell state annotations. Here we report the development of two highly scalable methods (snDrop-Seq and scTHS-Seq), that we have used to acquire nuclear transcriptome and DNA accessibility maps for thousands of single cells from the human adult visual and frontal cortex. This has led to the best-resolved human neuronal subtypes to date, identification of a majority of the non-neuronal cell types, as well as the cell-type specific nuclear transcriptome and DNA accessibility maps. Integrative analysis allowed us to identify transcription factors and regulatory elements shaping the state of different brain cell types, and to map genetic risk factors of human brain common diseases to specific pathogenic cell types and subtypes.

genomics↗