Biology subjects

Leung, W.

Publications and source records attributed to Leung, W..

14 recordsLinked to original sources

bioRxiv2024

Cell type specific suppression of hyper-recombination by human RAD18 is linked to PCNA K164 ubiquitination

Homologous recombination (HR) and translesion synthesis (TLS) promote gap-filling DNA synthesis to complete genome replication. One factor involved in both pathways is RAD18, an E3 ubiquitin ligase. Although RAD18s role in promoting TLS through the ubiquitination of PCNA at lysine 164 (K164) is well established, its requirement for HR-based mechanisms is currently less clear. To assess this, we inactivated RAD18 in three human cell lines. Our analyses found that loss of RAD18 in HCT116, but neither hTERT RPE-1 nor DLD1 cell lines, resulted in elevated sister chromatid exchange, gene conversion, and gene targeting, i.e. HCT116 mutants were hyper-recombinogenic (hyper-rec). Loss of RAD18 also impaired TLS activity in HCT116 cells, but unexpectedly, did not reduce clonogenic survival. Interestingly, these phenotypes appear linked to PCNA K164 ubiquitination, as HCT116 PCNAK164R/+ mutants were also hyper-rec and showed reduced TLS activity, consistent with previous studies in rad18-/-or pcnaK164R avian DT40 mutant cells. Importantly, knockdown of UBC9 to prevent PCNA K164 SUMOylation did not affect hyper-recombination, strengthening the link between increased recombination and RAD18-catalyzed PCNA K164 ubiquitination, but not K164 SUMOylation. Taken together, these data suggest that the roles of human RAD18 in directing distinct gap-filling DNA synthesis pathways varies depending on cell type and that these functions are linked to PCNA ubiquitination.

molecular biology↗

bioRxiv2023

Long-read genome assemblies for the study of chromosome expansion: Drosophila kikkawai Drosophila takahashii, Drosophila bipectinata, and Drosophila ananassae

Flow cytometry estimates of genome sizes among species of Drosophila show a 3-fold variation, ranging from [~]127 Mb in Drosophila mercatorum to [~]400 Mb in Drosophila cyrtoloma. However, the assembled portion of the Muller F Element (orthologous to the fourth chromosome in Drosophila melanogaster) shows a nearly 14-fold variation in size, ranging from [~]1.3 Mb to > 18 Mb. Here, we present chromosome-level long read genome assemblies for four Drosophila species with expanded F Elements ranging in size from 2.3 Mb to 20.5 Mb. Each Muller Element is present as a single scaffold in each assembly. These assemblies will enable new insights into the evolutionary causes and consequences of chromosome size expansion.

genomics↗

bioRxiv2020

Automated hiPSC culture and sample preparation for 3D live cell microscopy

Our goal is to identify and understand cellular behaviors using 3D live imaging of cell organization. To do this, we image human inducible pluripotent stem cell (hiPSC) lines expressing fluorescently tagged protein representing specific cellular organelles and structures. To produce large numbers of standardized cell images, we developed an automated hiPSC culture procedure, to maintain, passage and Matrigel coat 6-well plastic plates and 96-well glass plates compatible with high-resolution 3D microscopy. Here we describe this system including optimization procedures and specific values for plate movement, angle of tips, speed of aspiration and dispense, seeding strategies and timing of every step. We validated this approach through a side-by-side comparison of quality control results obtained from manual and automated methods. Additionally, we developed an automated image-based colony segmentation and feature extraction pipeline to predict cell count and select wells with consistent morphology for high resolution 3D microscopy.

cell biology↗

bioRxiv2020

Manual Annotation of Genes within Drosophila Species: the Genomics Education Partnership protocol

Annotating the genomes of multiple organisms allows us to study their genes as well as the evolution of those genes. While many eukaryotic genome assemblies already include computational gene predictions, these predictions can benefit from review and refinement through manual gene annotation. The Genomics Education Partnership (GEP; thegep.org) has developed an annotation protocol for protein-coding genes that enables undergraduate students and other researchers to create high-quality gene annotations that can be utilized in subsequent scientific investigations. For example, this protocol has been utilized by the GEP faculty to engage undergraduate students in the comparative annotation of genes involved in the insulin signaling pathway in 28 Drosophila species, using D. melanogaster as the informant genome. Students construct gene models using multiple lines of computational and experimental evidence including expression data (e.g., RNA-Seq), sequence similarity (e.g., BLAST, multiple sequence alignments), and computational gene predictions. For quality control, each gene is annotated by at least two students working independently, followed by reconciliation of the submitted gene models by a more experienced student. This article provides an overview of the annotation protocol and describes how discrepancies in student submitted gene models are resolved to produce a final, high-quality gene set suitable for subsequent analyses. This annotation protocol can be adapted to other scientific questions (e.g., expansion of the Drosophila Muller F element) and other species (e.g., parasitoid wasps) to provide additional opportunities for undergraduate students to participate in genomics research. These student annotation efforts can substantially improve the quality of gene annotations in publicly available genomic databases.

genomics↗

bioRxiv2020

Robust integrated intracellular organization of the human iPS cell: where, how much, and how variable?

Despite the intimate link between cell organization and function, the principles underlying intracellular organization and the relation between organization, gene expression and phenotype are not well understood. We address this by creating a benchmark for mean cell organization and the natural range of cell-to-cell variation. This benchmark can be used for comparison to other normal or abnormal cell states. To do this, we developed a reproducible microscope imaging pipeline to generate a high-quality dataset of 3D, high-resolution images of over 200,000 live cells from 25 isogenic human induced pluripotent stem cell (hiPSC) lines from the Allen Cell Collection. Each line contains one fluorescently tagged protein, created via endogenous CRISPR/Cas9 gene editing, representing a key cellular structure or organelle. We used these images to develop a new multi-part and generalizable analysis approach of the locations, amounts, and variation of these 25 cellular structures. Taking an integrated approach, we found that both the extent to which a structures individual location varied ("stereotypy") and the extent to which the structure localized relative to all the other cellular structures ("concordance") were robust to a wide range of cell shape variation, from flatter to taller, smaller to larger, or less to more polarized cells. We also found that these cellular structures varied greatly in how their volumes scaled with cell and nuclear size. These analyses create a data-driven set of quantitative rules for the locations, amounts, and variation of 25 cellular structures within the hiPSC as a normal baseline for cell organization.

cell biology↗

bioRxiv2020

Consensus-based somatic variant-calling method correlates FBXW7 mutations with poor prognosis in canine B-cell lymphoma

INTRODUCTIONCanine Lymphoma (CL) is the most commonly diagnosed malignancy in the domestic dog, with estimates reaching 80,000 new cases per year in the United States. Understanding of genetic factors involved in development and progression of canine B-Cell Lymphoma (cBCL), the most common of the two major subtypes of CL, can help guide efforts to prevent, diagnose, and treat disease in dogs. Such findings also have implications for human Non-Hodgkin Lymphoma (NHL), as pet dogs have recently emerged as an important translational model due to the many shared histopathological, biological, and clinical characteristics between cBCL and NHL. OBJECTIVESWe aimed to identify potential driver mutations in cBCL and detect associations between affected genes and differential clinical outcomes. METHODSUsing exome sequencing of paired normal and tumor tissues from 71 dogs of various breeds with cBCL, we identified somatic variants with a consensus approach: keeping variants called by both MuTect2 and with high-confidence by VarScan 2. We predicted effects of these variants using SnpEff then measured associations between mutated genes and survival times from clinical data available for 62 cohort dogs using a multivariate Cox Proportional Hazards Model. RESULTSMutations in FBXW7, a gene commonly mutated in both human and canine cancers including lymphoma, were associated with shorter overall survival (OS; p=0.01, HR 3.3 [1.4-7.6]). The two most frequently mutated codons of FBXW7 in our cohort correspond to the most frequently mutated codons in human cancers. CONCLUSIONSOur findings show that exome sequencing results can be combined with clinical data to identify key mutations associated with prognosis in cBCL. These results may have implications for precision medicine in dogs and also allow subsets of dogs to serve as models for specific subtypes of human lymphoma. Translational RelevanceIdentifying tumor biomarkers associated with clinical outcomes has been a major driver in improved success in treating many types of human cancers, including Non-Hodgkin lymphoma (NHL). Since canine B-cell Lymphoma (cBCL) shares many clinically identifiable characteristics with NHL, our detection of recurring mutations in certain genes in cBCL and their association with clinical outcomes stands to benefit both humans and dogs. If common canine lymphoma subtypes show mutational similarity to certain human subtypes, then therapies found to be effective for a subtype in one species may be more likely to improve treatment response in the analogous subtype in the other.

genomics↗

bioRxiv2020

PCNA-K164 ubiquitination facilitates origin licensing and mitotic DNA synthesis

Ubiquitination of the replication clamp proliferating cell nuclear antigen (PCNA) at the conserved residue lysine 164 (K164) occurs during normal S phase progression and increases after DNA damage induced replication stress. PCNA-K164 ubiquitination is critical for Okazaki fragment maturation and the activation of DNA damage tolerance pathways. Moreover, ubiquitinated PCNA operates in a fork protection pathway parallel to BRCA-RAD51. Whether PCNA ubiquitination regulates other genome maintenance mechanisms is unclear. Utilizing PCNAK164R cells generated by CRISPR-Cas9, we demonstrate that this mutation causes DNA replication defects that impact origin activation. PCNAK164R cells accumulate single-stranded DNA gaps during replication that persist throughout mitosis due to compromised mitotic DNA synthesis (MiDAS). We uncover a novel role for PCNA-K164 ubiquitination in regulating FANCD2 to initiate MiDAS. Persistent gaps hence interfere with MCM2-7 double hexamer loading in the subsequent G1 phase. Our findings demonstrate that the impact of PCNAK164-Ub is not limited to S/G2 phases but extends to mitosis and G1 phase. SUMMARYPCNA-K164 ubiquitination promotes DNA gap filling during S/G2 phases of the cell cycle. This study identifies a novel role for K164 ubiquitination in replication dynamics and mitotic DNA synthesis and thus provides new insight into the players involved in counteracting under-replication.

molecular biology↗

bioRxiv2019

Chronic Mcm10 deficiency causes defects in telomere maintenance in human cells

Minichromosome maintenance protein 10 (Mcm10) is essential for eukaryotic DNA replication. Here, we describe compound heterozygous MCM10 mutations in patients with distinctive but overlapping clinical phenotypes - natural killer (NK) cell deficiency (NKD) and restrictive cardiomyopathy (RCM) with hypoplasia of the spleen and thymus. To understand the mechanism of Mcm10-associated disease, we modeled these mutations in human cell lines. Mcm10 deficiency causes chronic replication stress that reduces cell viability due to increased genomic instability and telomere erosion. Our data suggest that loss of Mcm10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with single-stranded DNA. Terminally-arrested replication forks in Mcm10-deficient cells require endonucleolytic processing by Mus81, as MCM10:MUS81 double mutants display decreased viability and accelerated telomere shortening. We propose that these bi-allelic mutations in MCM10 predispose specific cardiac and immune cell lineages to prematurely arrest during differentiation, causing the clinical phenotypes in both NKD and CM patients.

molecular biology↗

bioRxiv2019

G-OnRamp: Generating genome browsers to facilitate undergraduate-driven collaborative genome annotation

Scientists are sequencing new genomes at an increasing rate with the goal of associating genome contents with phenotypic traits. After a new genome is sequenced and assembled, structural gene annotation is often the first step in analysis. Despite advances in computational gene prediction algorithms, most eukaryotic genomes still benefit from manual gene annotation. Undergraduates can become skilled annotators, and in the process learn both about genes/genomes and about how to utilize large datasets. Data visualizations provided by a genome browser are essential for manual gene annotation, enabling annotators to quickly evaluate multiple lines of evidence (e.g., sequence similarity, RNA-Seq, gene predictions, repeats). However, creating genome browsers requires extensive computational skills; lack of the expertise required remains a major barrier for many biomedical researchers and educators.\n\nTo address these challenges, the Genomics Education Partnership (GEP; https://gep.wustl.edu/) has partnered with the Galaxy Project (https://galaxyproject.org) to develop G-OnRamp (http://g-onramp.org), a web-based platform for creating UCSC Assembly Hubs and JBrowse genome browsers. G-OnRamp can also convert a JBrowse instance into an Apollo instance for collaborative genome annotations in research and educational settings. G-OnRamp enables researchers to easily visualize their experimental results, educators to create Course-based Undergraduate Research Experiences (CUREs) centered on genome annotation, and students to participate in genomics research.\n\nDevelopment of G-OnRamp was guided by extensive user feedback from in-person workshops. Sixty-five researchers and educators from over 40 institutions participated in these workshops, which produced over 20 genome browsers now available for research and education. For example, genome browsers for four parasitoid wasp species were used in a CURE engaging 142 students taught by 13 faculty members -- producing a total of 192 gene models. G-OnRamp can be deployed on a personal computer or on cloud computing platforms, and the genome browsers produced can be transferred to the CyVerse Data Store for long-term access.

scientific communication and education↗

bioRxiv2019

PCNA ubiquitination protects stalled replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly

Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing ubiquitination-deficient 293T and RPE1 cells generated through CRISPR/Cas9 genome editing, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-mediated but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork, and incomplete Okazaki fragment synthesis and maturation, thus interfering with efficient PCNA unloading by ATAD5 and subsequent nucleosomal deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and BRCA2 results in a synergistic increase in nascent DNA degradation and sensitivity to PARP-inhibitors. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors.

molecular biology↗

bioRxiv2018

G-OnRamp: A Galaxy-based platform for creating genome browsers for collaborative genome annotation

SummaryG-OnRamp provides a user-friendly, web-based platform for collaborative, end-to-end annotation of eukaryotic genomes using UCSC Assembly Hubs and JBrowse/Apollo genome browsers with evidence tracks derived from sequence alignments, ab initio gene predictors, RNA-Seq data, and repeat finders. Researchers can use the G-OnRamp output to visualize large genomics datasets, and can utilize the output to drive collaborative genome annotation projects in both research and educational settings.\n\nAvailability and ImplementationThe G-OnRamp virtual machine images and tutorials are freely available on the G-OnRamp web site (http://g-onramp.org/deployments). The G-OnRamp source code is freely available under an Academic Free License version 3.0 through the goeckslab GitHub repository (https://github.com/goeckslab).\n\nContactgoecksj@ohsu.edu\n\nSupplementary information:

genomics↗

bioRxiv2018

Mass media promotion of a smartphone smoking cessation app: Modeled health and cost impacts

BackgroundSmartphones are increasingly available and some high quality apps are available for smoking cessation. However, the cost-effectiveness of promoting such apps has never been studied.\n\nObjectiveTo estimate the health gain, inequality impacts and cost-utility from a five-year promotion campaign of a smoking cessation smartphone app compared to business-as-usual (no app use for quitting).\n\nMethodsA well-established Markov macro-simulation model utilising a multi-state life-table was adapted to the intervention (lifetime horizon, 3% discount rate). The setting was the New Zealand (NZ) population (N = 4.4 million). The intervention effect size was from a multi-country randomised trial: relative risk for quitting at six months = 2.23 (95%CI: 1.08 to 4.77). Intervention costs were based on NZ mass media promotion data and the NZ cost of attracting a smoker to smoking cessation services ($NZ64 per person).\n\nResultsThe five-year intervention was estimated to generate 6760 QALYs (95%UI: 5420 to 8420) over the remaining lifetime of the population. For M[a]ori (Indigenous population) there was 2.8 times the per capita age-standardized QALYs relative to non-M[a]ori. The intervention was also estimated to be cost-saving to the health system (saving NZ$115, 95%UI: 72.5 million [m] to 171m; US$81.8m). The cost-saving aspect of the intervention was maintained in scenario and sensitivity analyses where the discount rate was doubled to 6%, the effect size halved, and the intervention run for just one year.\n\nConclusionsThis study provides modelling-level evidence that mass-media promotion of a smartphone app for smoking cessation is likely to generate health gain, reduce ethnic inequalities in health and save health system costs. Nevertheless, there are other tobacco control which generate considerably larger health gains and cost-savings such as raising tobacco taxes.

epidemiology↗

bioRxiv2018

Engineered developmental niche enables predictive phenotypic screening in human dystrophic cardiomyopathy

Directed differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes typically produces cells with structural, functional, and biochemical properties that most closely resemble those present in the fetal heart. Here we establish an in vitro engineered developmental cardiac niche to produce matured hPSC-derived cardiomyocytes (hPSC-CMs) with enhanced sarcomere development, electrophysiology, contractile function, mitochondrial capacity, and a more mature transcriptome. When this developmental cardiac niche was applied to dystrophin mutant hPSC-CMs, a robust disease phenotype emerged, which was not observed in non-matured diseased hPSC-CMs. Matured dystrophin mutant hPSC-CMs exhibited a greater propensity for arrhythmia as measured via beat rate variability, most likely due to higher resting cytosolic calcium content. Using a custom nanopatterned microelectrode array platform to screen functional output in hPSC-CMs exposed to our engineered developmental cardiac niche, we identified calcium channel blocker, nitrendipine, mitigated hPSC-CM arrhythmogenic behavior and correctly identified sildenafil as a false positive. Taken together, we demonstrate our developmental cardiac niche platform enables robust hPSC-CM maturation allowing for more accurate disease modeling and predictive drug screening.

bioengineering↗

bioRxiv2016

Cell cycle dynamics of chromosomal organisation at single-cell resolution

Chromosomes in proliferating metazoan cells undergo dramatic structural metamorphoses every cell cycle, alternating between a highly condensed mitotic structure facilitating chromosome segregation, and a decondensed interphase structure accommodating transcription, gene silencing and DNA replication. These cyclical structural transformations have been evident under the microscope for over a century, but their molecular-level analysis is still lacking. Here we use single-cell Hi-C to study chromosome conformations in thousands of individual cells, and discover a continuum of cis-interaction profiles that finely position individual cells along the cell cycle. We show that chromosomal compartments, topological domains (TADs), contact insulation and long-range loops, all defined by ensemble Hi-C maps, are governed by distinct cell cycle dynamics. In particular, DNA replication correlates with build-up of compartments and reduction in TAD insulation, while loops are generally stable from G1 through S and G2. Analysing whole genome 3D structural models using haploid cell data, we discover a radial architecture of chromosomal compartments with distinct epigenomic signatures. Our single-cell data creates an essential new paradigm for the re-interpretation of chromosome conformation maps through the prism of the cell cycle.

genomics↗