Biology subjects

Choi, J.

Publications and source records attributed to Choi, J..

At least 271 records · Page 15Linked to original sources

bioRxiv2019

Chemical precision glyco-mutagenesis by glycosyltransferase engineering in living cells

Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-function modification on cells where the products of individual glycosyltransferases can be selectively characterized or manipulated at will.

synthetic biology↗

bioRxiv2019

Strategies for building computing skills to support microbiome analysis: a five-year perspective from the EDAMAME workshop

Here, we report our educational approach and learner evaluations of the first five years of the Explorations in Data Analysis for Metagenomic Advances in Microbial Ecology (EDAMAME) workshop, held annually at Michigan State Universitys Kellogg Biological Station from 2014-2018. We hope this information will be useful for others who want to organize computing-intensive workshops and encourage quantitative skill development among microbiologists.\n\nImportanceHigh-throughput sequencing and related statistical and bioinformatic analyses have become routine in microbiology in the past decade, but there are few formal training opportunities to develop these skills. A week-long workshop can offer sufficient time for novices to become introduced to best computing practices and common workflows in sequence analysis. We report our experiences in executing such a workshop targeted to professional learners (graduate students, post-doctoral scientists, faculty, and research staff).

scientific communication and education↗

bioRxiv2019

High diagnostic yield and clinical utility of WES for patients with undiagnosed genetic disorder by automating variant interpretation

PurposeEVIDENCE, an automated interpretation system, has been developed to facilitate the entire process of whole exome sequencing (WES) analyses. This study investigated the diagnostic yield of EVIDENCE in patients suspected genetic disorders. MethodsDNA from 330 probands (age range, 0-68 years) with suspected genetic disorders were subjected to WES. Candidate variants were identified by EVIDENCE and confirmed by testing family members and/or clinical reassessments. ResultsThe average number of overlapping organ categories per patient was 4.5 {+/-} 5.0. EVIDENCE reported a total 244 variants in 215 (65.1%) of the 330 probands. After clinical reassessment and/or family member testing, 196 variants were identified in 171 probands (51.8%), including 115 novel variants. These variants were confirmed as being responsible for 146 genetic disorders. One hundred-seven (54.6%) of the 196 variants were categorized as pathogenic or likely pathogenic before, and 146 (74.6%) after, clinical assessment and/or family member testing. Factors associated with a variant being confirmed as causative include rules, such as PVS1, PS1, PM1, PM5, and PP5, and similar symptom scores between that variant and a patients phenotype. ConclusionThis new, automated variant interpretation system facilitated the diagnosis of various genetic diseases with a 51% improvement in diagnostic yield.

genetics↗

bioRxiv2019

Massively parallel reporter assays combined with cell-type specific eQTL informed multiple melanoma loci and identified a pleiotropic function of HIV-1 restriction gene, MX2, in melanoma promotion

Genome-wide association studies (GWAS) have identified [~]20 melanoma susceptibility loci. To identify susceptibility genes and variants simultaneously from multiple GWAS loci, we integrated massively-parallel reporter assays (MPRA) with cell type-specific epigenomic data as well as melanocyte-specific expression quantitative trait loci (eQTL) profiling. Starting from 16 melanoma loci, we selected 832 variants overlapping active regions of chromatin in cells of melanocytic lineage and identified 39 candidate functional variants displaying allelic transcriptional activity by MPRA. For four of these loci, we further identified four colocalizing melanocyte cis-eQTL genes (CTSS, CASP8, MX2, and MAFF) matching the allelic activity of MPRA functional variants. Among these, we further characterized the locus encompassing the HIV-1 restriction gene, MX2, on chromosome band Chr21q22.3 and validated a functional variant, rs398206, among multiple high LD variants. rs398206 mediates allelic transcriptional activity via binding of the transcription factor, YY1. This allelic transcriptional regulation is consistent with a significant cis-eQTL of MX2 in primary human melanocytes, where the melanoma risk-associated A allele of rs398206 is correlated with higher MX2 levels. Melanocyte-specific transgenic expression of human MX2 in a zebrafish model demonstrated accelerated melanoma formation in a BRAFV600E background. Thus, using an efficient scalable approach to streamline GWAS follow-up functional studies, we identified multiple candidate melanoma susceptibility genes and variants, and uncovered a pleiotropic function of MX2 in melanoma susceptibility.

genomics↗

bioRxiv2019

Benchmarking of alignment-free sequence comparison methods

Alignment-free (AF) sequence comparison is attracting persistent interest driven by data-intensive applications. Hence, many AF procedures have been proposed in recent years, but a lack of a clearly defined benchmarking consensus hampers their performance assessment. Here, we present a community resource (http://afproject.org) to establish standards for comparing alignment-free approaches across different areas of sequence-based research. We characterize 74 AF methods available in 24 software tools for five research applications, namely, protein sequence classification, gene tree inference, regulatory element detection, genome-based phylogenetic inference and reconstruction of species trees under horizontal gene transfer and recombination events. The interactive web service allows researchers to explore the performance of alignment-free tools relevant to their data types and analytical goals. It also allows method developers to assess their own algorithms and compare them with current state-of-the-art tools, accelerating the development of new, more accurate AF solutions.

bioinformatics↗

bioRxiv2019

eIF5B gates the transition from translation initiation to elongation

Translation initiation determines both the quantity and identity of the protein product by establishing the reading frame for protein synthesis. In eukaryotic cells, numerous translation initiation factors (eIFs) prepare ribosomes for polypeptide elongation, yet the underlying dynamics of this process remain enigmatic1-4. A central question is how eukaryotic ribosomes transition from translation initiation to elongation. Here, we applied in vitro single-molecule fluorescence microscopy approaches to monitor directly in real time the pathways of late translation initiation and the transition to elongation using a purified yeast Saccharomyces cerevisiae translation system. This transition was remarkably slower in our eukaryotic system than that reported for Escherichia coli5-7. The slow entry to elongation was defined by a long residence time of eIF5B on the 80S ribosome after joining of individual ribosomal subunits, which is catalyzed by this universally conserved initiation factor. Inhibition of eIF5B GTPase activity following subunit joining prevented eIF5B dissociation from the 80S complex, thereby preventing elongation. Our findings illustrate how eIF5B dissociation serves as a kinetic checkpoint for the transition from initiation to elongation, and its release may be governed by a conformation of the ribosome complex that triggers GTP hydrolysis.

biochemistry↗

bioRxiv2019

Whole-genome and RNA sequencing reveal variation and transcriptomic coordination in the developing human prefrontal cortex

Variation in gene expression underlies neurotypical development, while genomic variants contribute to neuropsychiatric disorders. BrainVar is a unique resource of paired whole-genome sequencing and bulk-tissue RNA-sequencing from the human dorsolateral prefrontal cortex of 176 neurotypical individuals across prenatal and postnatal development, providing the opportunity to assay genomic and transcriptomic variation in tandem. Leveraging this resource, we identified rare premature stop codons with commensurate reduced and allele-specific expression of corresponding genes, and common variants that alter gene expression (expression quantitative trait loci, eQTLs). Categorizing eQTLs by prenatal and postnatal effect, genes affected by temporally-specific eQTLs, compared to constitutive eQTLs, are enriched for haploinsufficiency, protein-protein interactions, and neuropsychiatric disorder risk loci. Expression levels of over 12,000 genes rise or fall in a concerted late-fetal transition, with the transitional genes enriched for cell type specific genes and neuropsychiatric disorder loci, underscoring the importance of cataloguing developmental trajectories in understanding cortical physiology and pathology.\n\nHighlightsO_LIWhole-genome and RNA-sequencing across human prefrontal cortex development in BrainVar\nC_LIO_LIGene-specific developmental trajectories characterize the late-fetal transition\nC_LIO_LIIdentification of constitutive, prenatal-specific, postnatal-specific, and rare eQTLs\nC_LIO_LIIntegrated analysis reveals genetic and developmental influences on CNS traits and disorders\nC_LI

neuroscience↗

bioRxiv2019

RACK1 on and off the ribosome

Receptor for activated C kinase 1 (RACK1) is a eukaryote-specific ribosomal protein implicated in diverse biological functions. To engineer ribosomes for specific fluorescent labeling, we selected RACK1 as an target given its location on the small ribosomal subunit and other properties. However, prior results suggested that RACK1 has roles both on and off the ribosome, and such an exchange might be related to its various cellular functions and hinder our ability to use RACK1 as a stable fluorescent tag for the ribosome. In addition, the kinetics of spontaneous exchange of RACK1 or any ribosomal protein from a mature ribosome in vitro remain unclear. To address these issues, we engineered fluorescently-labeled human ribosomes via RACK1, and applied bulk and single-molecule biochemical analyses to track RACK1 on and off the human ribosome. Our results demonstrate that, despite its cellular non-essentiality from yeast to humans, RACK1 readily re-associates with the ribosome, displays limited conformational dynamics, and remains stably bound to the ribosome for hours in vitro. This work sheds insight onto the biochemical basis of ribosomal protein exchange on and off a mature ribosome and provides tools for single-molecule analysis of human translation.

biochemistry↗

bioRxiv2019

Short translational ramp determines efficiency of protein synthesis

It is generally assumed that translation efficiency is governed by translation initiation. However, the efficiency of protein synthesis is regulated by multiple factors including tRNA abundance, codon composition, mRNA motifs and amino-acid sequence1-4. These factors influence the rate of protein synthesis beyond the initiation phase of translation, typically by modulating the rate of peptide-bond formation and to a lesser extent that of translocation. The slowdown in translation during the early elongation phase, known as the 5 translational ramp, likely contributes to the efficiency of protein synthesis 5-9. Multiple mechanisms, which could explain the molecular basis for this translational ramp, have been proposed that include tRNA abundance bias6,9, the rate of translation initiation10-15, mRNA and ribosome structure 11,12,14,16-18, or retention of initiation factors during early elongation events 19. Here, we show that the amount of synthesized protein (translation efficiency) depends on a short translational ramp that comprises the first 5 codons in mRNA. Using a library of more than 250,000 reporter sequences combined with in vitro and in vivo protein expression assays, we show that differences in the short ramp can lead to 3 to 4 orders of magnitude changes in protein abundance. The observed difference is not dependent on tRNA abundance, efficiency of translation initiation, or overall mRNA structure. Instead, we show that translation is regulated by amino-acid-sequence composition and local mRNA sequence. Single-molecule measurements of translation kinetics indicate substantial pausing of ribosome and abortion of protein synthesis on the 4th or 5th codon for distinct amino acid or nucleotide compositions. Introduction of preferred sequence motifs, only at the exact positions within the mRNA, improves protein synthesis for recombinant proteins, indicating an evolutionarily conserved mechanism for controlling translational efficiency.

molecular biology↗

bioRxiv2019

EML4-ALK V3 drives cell migration through NEK9 and NEK7 kinases in non-small-cell lung cancer

EML4-ALK is an oncogenic fusion present in [~]5% lung adenocarcinomas. However, distinct EML4-ALK variants differ in the length of the EML4 microtubule-associated protein encoded within the fusion and are associated with a poorly understood variability in disease progression and therapeutic response. Here, we show that EML4-ALK variant 3, which is linked to accelerated metastatic spread and worse patient outcome, causes microtubule stabilization, formation of extended cytoplasmic protrusions, loss of cell polarity and increased cell migration. Strikingly, this is dependent upon the NEK9 kinase that interacts with the N-terminal region of EML4. Overexpression of wild-type EML4, as well as constitutive activation of NEK9, also perturbs cell morphology and accelerates cell migration in a manner that requires the downstream kinase NEK7 but not ALK activity. Moreover, elevated NEK9 is associated in patients with EML4-ALK V3 expression, as well as reduced progression-free and overall survival. Hence, we propose that EML4-ALK V3 promotes microtubule stabilization through recruitment of NEK9 and NEK7 to increase cell migration and that this represents a novel actionable pathway that drives disease progression in lung cancer.

cancer biology↗

bioRxiv2019

DNA methylation and histone H1 cooperatively repress transposable elements and aberrant intragenic transcripts

DNA methylation and histone H1 mediate transcriptional silencing of genes and transposable elements, but how they interact is unclear. In plants and animals with mosaic genomic methylation, functionally mysterious methylation is also common within constitutively active housekeeping genes. Here we show that H1 is enriched in methylated sequences, including genes, of Arabidopsis thaliana, yet this enrichment is independent of DNA methylation. Loss of H1 disperses heterochromatin, globally alters nucleosome organization, and activates H1-bound genes, but only weakly de-represses transposable elements. However, H1 loss strongly activates transposable elements hypomethylated through mutation of DNA methyltransferase MET1. Loss of H1 also activates antisense transcripts within demethylated genes. Our results demonstrate that H1 and DNA methylation cooperatively maintain transcriptional homeostasis by silencing transposable elements and aberrant intragenic transcripts. Such functionality plausibly explains why DNA methylation, a well-known mutagen, has been maintained within coding sequences of crucial plant and animal genes. HighlightsO_LIHistone H1 is enriched in methylated DNA independently of methylation C_LIO_LILoss of H1 activates genes, alters nucleosome organization and disperses heterochromatin C_LIO_LIDNA methylation and H1 jointly silence transposons C_LIO_LIDNA methylation and H1 cooperatively suppress intragenic antisense transcripts C_LI

genetics↗

bioRxiv2019

Mitochondrial volume fraction controls translation of nuclear-encoded mitochondrial proteins

Mitochondria are dynamic in their size and morphology yet must also precisely control their protein composition according to cellular energy demand. This control is particularly complicated for mitochondria, as they must coordinate gene expression from both the nuclear and mitochondrial genome. We have found that cells are able to use this dynamic morphology to post-transcriptionally coordinate protein expression with the metabolic demands of the cell through enhanced mRNA localization to the mitochondria. As yeast switch to respiratory metabolism, they increase their mitochondrial volume fraction that is, the ratio of mitochondrial volume to intracellular volume which drives the localization of nuclear-encoded mitochondrial mRNAs to the surface of the mitochondria. Through artificial tethering experiments, we show that this mitochondrial localization is sufficient to increase protein production, whereas sequestering mRNAs away from the mitochondrial surface decreases protein production, and those cells are deficient in growth in respiratory conditions. Furthermore, we find that this mRNA sensitivity to mitochondrial volume fraction is driven by the speed of translation downstream of the mitochondrial targeting sequence (MTS), as local ribosome stalling through a stretch of polyprolines in the nascent peptide can drive constitutive localization of mRNAs to the mitochondria. This points to a mechanism by which organelle volume fraction provides feedback to regulate organelle-specific gene expression through mRNA localization while potentially circumventing the need to directly coordinate with the nuclear genome.

cell biology↗

bioRxiv2018

Acidity promotes tumor progression by altering macrophage phenotype in prostate cancer

Tumors rapidly ferment glucose to lactic acid even in the presence of oxygen, and coupling high glycolysis with poor perfusion leads to extracellular acidification. Here we demonstrate that acidity, independent from lactate, augments the pro-tumor phenotype of macrophages. We used zwitterionic buffers to show that activating macrophages at pH 6.8 in vitro enhanced an IL-4-driven phenotype as measured by gene expression, cytokine profiling, and functional assays. These results were recapitulated in vivo wherein neutralizing intratumoral acidity reduced the pro-tumor phenotype of macrophages, while also decreasing tumor incidence and invasion in the TRAMP model of prostate cancer. These results were recapitulated using an in silico mathematical model that simulate macrophage responses to environmental signals. By turning off acid-induced cellular responses, our in silico mathematical modeling shows that acid-resistant macrophages can limit tumor progression. In summary, this study suggests that tumor acidity contributes to prostate carcinogenesis by altering the state of macrophage activation.

immunology↗

bioRxiv2018

Comparisons among Vestibular Examinations and Symptoms of Vertigo in Sudden Sensorineural Hearing Loss Patients

ObjectiveVertigo in sudden sensorineural hearing loss (SSNHL) is hypothesized as an extension of the disease caused by the anatomical proximity of the cochlea and vestibule. The present study aimed to demonstrate the association of vestibular function test (VFT) results with SSNHL disease severity and prognosis.\n\nSubjects and methodsThis study assessed clinical records of 263 SSNHL patients admitted to our hospital, between January 2010 and October 2017. Steroid treatment comprised high-dose intravenous dexamethasone (16 mg/d) or oral methylprednisolone (64 mg/d) for 4 days and tapered oral methylprednisolone for 8 days after discharge. Caloric tests were performed in all patients, and cervical vestibular-evoked myogenic potential (c-VEMP) and ocular VEMP (o-VEMP) tests were performed in 209 and 144 patients, respectively.\n\nResultsNinety six patients had vertigo, and caloric abnormalities were observed in 119 patients. Initial PTA in patients with vertigo were worse than in those without vertigo (63.0 dB vs 72.7 dB, P=.002). Initial PTA in patients with abnormal o-VEMP was worse than in those with normal o-VEMP (61.4 dB vs 73.0 dB, P=.004). PTA improvement after steroid treatment in patients with vertigo was lower than in those without vertigo (25.0 dB vs 20.9 dB, P=.028). PTA improvement after treatment in patients with abnormal caloric results was lower than in those with normal caloric results (26.0 dB vs 18.4 dB, P=.013).\n\nConclusionsThe functions of vestibular organs, particularly the utricle and lateral semicircular canal, are associated with disease severity and hearing outcome in SSNHL patients.

bioinformatics↗

bioRxiv2018

RefSoil+: A reference for antimicrobial resistance genes on soil plasmids

Plasmids harbor transferable genes that contribute to the functional repertoire of microbial communities, yet their contributions to metagenomes are often overlooked. Environmental plasmids have the potential to spread antibiotic resistance to clinical microbial strains. In soils, high microbiome diversity and high variability in plasmid characteristics present a challenge for studying plasmids. To improve understanding of soil plasmids, we present RefSoil+, a database containing plasmid sequences from 922 soil microorganisms. Soil plasmids were relatively larger than other described plasmids, which is a trait associated with plasmid mobility. There was no relationship between chromosome size and plasmid size or number, suggesting that these genomic traits are independent in soil. Soil-associated plasmids, but not chromosomes, had fewer antibiotic resistance genes than other microorganisms. These data suggest that soils may offer limited opportunity for plasmid-mediated transfer of described antibiotic resistance genes. RefSoil+ can serve as a baseline for the diversity, composition, and host-associations of plasmid-borne functional genes in soil, a utility that will be enhanced as the database expands. Our study improves understanding of soil plasmids and provides a resource for assessing the dynamics of the genes that they carry, especially genes conferring antibiotic resistances.\n\nImportanceSoil-associated plasmids have the potential to transfer antibiotic resistance genes from environmental to clinical microbial strains, which is a public health concern. A specific resource is needed to aggregate knowledge of soil plasmid characteristics so that the content, host-associations, and dynamics of antibiotic resistance genes can be assessed and then tracked between the environment and the clinic. Here, we present RefSoil+, a database of soil-associated plasmids. RefSoil+ presents a contemporary snapshot of antibiotic resistance genes in soil that can serve as a reference as novel plasmids and transferred antibiotic resistances are discovered. Our study broadens our understanding of plasmids in soil and provides a community resource for investigating clinic-environment dynamics of important plasmid-associated genes, including antibiotic resistance genes.

microbiology↗

bioRxiv2018

Critical role of miR-10b in BRafV600E dependent anchorage independent growth and invasion of melanoma cells

Recent high-throughput-sequencing of cancer genomes has identified oncogenic mutations in the BRaf genetic locus as one of the critical events in melanomagenesis. BRaf encodes a serine/threonine kinase that regulates the MAPK/ERK kinase (MEK) and extracellular signal-regulated kinase (ERK) protein kinase cascade. In normal cells, the activity of BRaf is tightly regulated and is required for cell growth and survival. BRaf gain-of-function mutations in melanoma frequently lead to unrestrained growth, enhanced cell invasion and increased viability of cancer cells. Although it is clear that the invasive phenotypes of BRaf mutated melanoma cells are stringently dependent on BRaf-MEK-ERK activation, the downstream effector targets that are required for oncogenic BRaf-mediated melanomagenesis are not well defined. miRNAs have regulatory functions towards the expression of genes that are important in carcinogenesis. We observed that miR-10b expression correlates with the presence of the oncogenic BRaf (BRafV600E) mutation in melanoma cells. While expression of miR-10b enhances anchorage-independent growth of BRaf wild-type melanoma cells, miR-10b silencing decreases BRafV600E cancer cell invasion in vitro. Importantly, the expression of miR-10b is required for BRafV600E-mediated anchorage-independent growth and invasion of melanoma cells in vitro. Taken together our results suggest that miR-10b is an important mediator of oncogenic BRafV600E activity in melanoma.

cancer biology↗

bioRxiv2018

Identification of deleterious and regulatory genomic variations in known asthma loci

BackgroundCandidate gene and genome-wide association studies have identified hundreds of asthma risk loci. The majority of associated variants, however, are not known to have any biological function and are believed to represent markers rather than true causative mutations. We hypothesized that many of these associated markers are in linkage disequilibrium (LD) with the elusive causative variants.\n\nMethodsWe compiled a comprehensive list of 447 asthma-associated variants previously reported in candidate gene and genome-wide association studies. Next, we identified all sequence variants located within the 304 unique genes using whole-genome sequencing data from the 1000 Genomes Project. Then, we calculated the LD between known asthma variants and the sequence variants within each gene. LD variants identified were then annotated to determine those that are potentially deleterious and/or functional (i.e. coding or regulatory effects on the encoded transcript or protein).\n\nResultsWe identified 10,048 variants in LD (r2 > 0.6) with known asthma variants. Annotations of these LD variants revealed that several have potentially deleterious effects including frameshift, alternate splice site, stop-lost, and missense. Moreover, 24 of the LD variants have been reported to regulate gene expression as expression quantitative trait loci (eQTLs).\n\nConclusionsThis study is proof of concept that many of the genetic loci previously associated with complex diseases such as asthma are not causative but represent markers of disease, which are in LD with the elusive causative variants. We hereby report a number of potentially deleterious and regulatory variants that are in LD with the reported asthma loci. These reported LD variants could account for the original association signals with asthma and represent the true causative mutations at these loci.

bioinformatics↗

bioRxiv2018

Whole genome sequencing identifies high-impact variants in well-known pharmacogenomic genes

More than 1,100 genetic loci have been correlated with drug response outcomes but disproportionately few have been translated into clinical practice. One explanation for the low rate of clinical implementation is that the majority of associated variants may be in linkage disequilibrium (LD) with the causal variants, which are often elusive. This study aims to identify and characterize likely causal variants within well-established pharmacogenomic genes using next-generation sequencing data from the 1000 Genomes Project. We identified 69,319 genetic variations within 160 pharmacogenomic genes, of which 8,207 variants are in strong LD (r2 > 0.8) with known pharmacogenomic variants. Of the latter, 8 are coding or structural variants predicted to have high-impact, with 19 additional missense variants that are predicted to have moderate-impact. In conclusion, we identified putatively functional variants within known pharmacogenomics loci that could account for the association signals and represent the missing causative variants underlying drug response phenotypes.

bioinformatics↗