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Lee, K.

Publications and source records attributed to Lee, K..

At least 235 records · Page 13Linked to original sources
bioRxiv2020

Host infection by the grass-symbiotic fungus Epichloë festucaerequires catalytically active H3K9 and H3K36 methyl transferases

Recent studies have identified key genes in Epichloe festucae that control the symbiotic interaction of this filamentous fungus with its grass host. Here we report on the identification of specific fungal genes that determine its ability to infect and colonize the host. Deletion of setB, which encodes a homolog of the H3K36 histone methyltransferase Set2/KMT3, specifically reduced histone H3K36 trimethylation and led to severe defects in colony growth and hyphal development. The E. festucae {Delta}clrD mutant, which lacks the gene encoding the homolog of the H3K9 methyltransferase KMT1, displays similar developmental defects. Both mutants are completely defective in their ability to infect the host grass, and mutational studies of key residues in the catalytic SET domains from these proteins show that these phenotypes are dependent on the methyltransferase activities of SetB and ClrD. A comparison of the differences in the host transcriptome between seedlings inoculated with wild-type versus mutants suggests that the inability of these mutants to infect the host was not due to an aberrant host defense response. Co-inoculation of either {Delta}setB or {Delta}clrD with the wild-type strain enables these mutants to colonize the host. However, successful colonization by the mutants resulted in death or stunting of the host plant. Transcriptome analysis at the early infection stage identified four fungal candidate genes, three of which encode small-secreted proteins, that are differentially regulated in these mutants compared to wild-type. Deletion of crbA, which encodes a putative carbohydrate binding protein, resulted in significantly reduced host infection rates by E. festucae. Author SummaryThe filamentous fungus Epichloe festucae is an endophyte that forms highly regulated symbiotic interactions with the perennial ryegrass. Proper maintenance of such interactions is known to involve several signalling pathways, but much less is understood about the infection capability of this fungus in the host. In this study, we uncovered two epigenetic marks and their respective histone methyltransferases that are required for E. festucae to infect perennial ryegrass. Null mutants of the histone H3 lysine 9 and lysine 36 methyltransferases are completely defective in colonizing the host intercellular space, and these defects are dependent on the methyltransferase activities of these enzymes. Importantly, we observed no evidence for increased host defense response to these mutants that can account for their non-infection. Rather, these infection defects can be rescued by the wild-type strain in co-inoculation experiments, suggesting that failure of the mutants to infect is due to altered expression of genes encoding infection factors that are under the control of the above epigenetic marks that can be supplied by the wild-type strain. Among genes differentially expressed in the mutants at the early infection stage is a putative small-secreted protein with a carbohydrate binding function, which deletion in E. festucae severely reduced infection efficiency.

genetics↗
bioRxiv2020

FlyWire: Online community for whole-brain connectomics

Due to advances in automated image acquisition and analysis, new whole-brain connectomes beyond C. elegans are finally on the horizon. Proofreading of whole-brain automated reconstructions will require many person-years of effort, due to the huge volumes of data involved. Here we present FlyWire, an online community for proofreading neural circuits in a fly brain, and explain how its computational and social structures are organized to scale up to whole-brain connectomics. Browser-based 3D interactive segmentation by collaborative editing of a spatially chunked supervoxel graph makes it possible to distribute proofreading to individuals located virtually anywhere in the world. Information in the edit history is programmatically accessible for a variety of uses such as estimating proofreading accuracy or building incentive systems. An open community accelerates proofreading by recruiting more participants, and accelerates scientific discovery by requiring information sharing. We demonstrate how FlyWire enables circuit analysis by reconstructing and analysing the connectome of mechanosensory neurons.

neuroscience↗
bioRxiv2020

Generation of thymidine auxotrophic Agrobacterium tumefaciens strains for plant transformation

Agrobacterium-mediated transformation is a widely used gene delivery method for fundamental researches and crop trait improvement projects. Auxotrophic Agrobacterium tumefaciens strains are highly desirable for plant transformation because they can be easily removed from the explants after co-cultivation due to their dependence on essential nutrient supplementation. The thymidine auxotrophic A. tumefaciens strain LBA4404Thy- has been successfully used for plant transformation, however, auxotrophic version of other commonly used strains are not available yet to public laboratories. Here we report the generation of EHA101, EHA105 and EHA105D thymidine auxotrophic strains. These strains exhibited thymidine-dependent growth in the bacterial medium, and the transient GUS expression assay using Arabidopsis seedling showed that they retain the equivalent T-DNA transfer capability as the original strains thus are suitable for plant transformation.

plant biology↗
bioRxiv2020

Molecular Parallelism Underlies Convergent Highland Adaptation of Maize Landraces

Convergent phenotypic evolution provides some of the strongest evidence for adaptation. However, the extent to which recurrent phenotypic adaptation has arisen via parallelism at the molecular level remains unresolved, as does the evolutionary origin of alleles underlying such adaptation. Here, we investigate genetic mechanisms of convergent highland adaptation in maize landrace populations and evaluate the genetic sources of recurrently selected alleles. Population branch excess statistics reveal strong evidence of parallel adaptation at the level of individual SNPs, genes and pathways in four independent highland maize populations, even though most SNPs show unique patterns of local adaptation. The majority of selected SNPs originated via migration from a single population, most likely in the Mesoamerican highlands. Polygenic adaptation analyses of quantitative traits reveal that alleles affecting flowering time are significantly associated with elevation, indicating the flowering time pathway was targeted by highland adaptation. In addition, repeatedly selected genes were significantly enriched in the flowering time pathway, indicating their significance in adapting to highland conditions. Overall, our study system represents a promising model to study convergent evolution in plants with potential applications to crop adaptation across environmental gradients.

evolutionary biology↗
bioRxiv2020

Soluble corn fiber reduces ovalbumin-induced sinonasal inflammation via the gut microbiota-airway axis

Asthma is a chronic airway inflammatory disease that affects approximately 300 million people worldwide, causing a substantial healthcare burden. Although there is a large degree of heterogeneity in the inflammatory response of asthmatics, a subset of patients are characterized by type-2 inflammation, which is in part mediated by TH2 cells in both the upper and lower airways. Asthma prevalence is increased in low-socioeconomic-status populations, where disparities in health behavior exist, including a shift toward a western diet characterized by low dietary fiber. Gut microbes metabolize fiber into short chain fatty acids that can reduce type-2 inflammation in peripheral organs, such as the airways. We hypothesized that soluble fiber can reduce ovalbumin (OVA)-induced upper airway inflammation in the context of the unified airway hypothesis, in mice maintained on ingredient-matched western (WD) and control diets (CD) through production of short chain fatty acids. Our results show that soluble fiber reduces IL-4 and IL-13 gene expression (p<0.05, Mann Whitney) in the sinonasal cavity of CD-fed mice, but this effect was lost in WD-fed mice. This loss of protection in WD-fed mice parallels compositional changes of the cecal and fecal microbiota. Mice fed a soluble fiber supplement while being maintained on a WD had altered microbial communities characterized by lower abundance of fiber fermentering bacteria. This work can be used to develop effective microbiome-based therapeutics as a low-cost method to reduce asthma morbidity. IMPORTANCEPrevious research has supported that western-style diets, typically high-fat and low-fiber, are associated with changes in the gut microbiome and increased inflammation. Western diets are accessible and prominent in low-socioeconomic-status populations, where asthma rates are highest; however, there has yet to be a low-cost asthma therapeutic. For the first time, we investigated whether supplementation with a physiologically relevant quantity of soluble corn fiber can reduce allergic airway inflammation. Our study supports that soluble corn fiber supplementation is associated with compositional shifts of the gut microbiota and reduced airway inflammation, promoting the use of fiber as a low-cost microbiome modifying therapy to reduce asthma-associated inflammation. However, soluble corn fiber in conjunction with a western diet resulted in an alternate gut microbiome composition and loss of protection against allergic airway inflammation. These findings further support the importance of the gut microbiota in host health.

microbiology↗
bioRxiv2020

Atlas and developmental dynamics of mouse DNase I hypersensitive sites

Early mammalian development is orchestrated by genome-encoded regulatory elements populated by a changing complement of regulatory factors, creating a dynamic chromatin landscape. To define the spatiotemporal organization of regulatory DNA landscapes during mouse development and maturation, we generated nucleotide-resolution DNA accessibility maps from 15 tissues sampled at 9 intervals spanning post-conception day 9.5 through early adult, and integrated these with 41 adult-stage DNase-seq profiles to create a global atlas of mouse regulatory DNA. Collectively, we delineated >1.8 million DNase I hypersensitive sites (DHSs), with the vast majority displaying temporal and tissue-selective patterning. Here we show that tissue regulatory DNA compartments show sharp embryonic-to-fetal transitions characterized by wholesale turnover of DHSs and progressive domination by a diminishing number of transcription factors. We show further that aligning mouse and human fetal development on a regulatory axis exposes disease-associated variation enriched in early intervals lacking human samples. Our results provide an expansive new resource for decoding mammalian developmental regulatory programs.

genomics↗
bioRxiv2020

Mitochondrial dsRNAs activate PKR and TLR3 to promote chondrocyte degeneration in osteoarthritis

Protein kinase R (PKR) is an immune response protein that becomes activated by long double-stranded RNAs (dsRNAs). Several studies reported the misactivation of PKR in patients of degenerative diseases including primary osteoarthritis (OA). However, the molecular identity of PKR-activating dsRNAs remains unknown. Here, we investigate the role of mitochondrial dsRNAs (mt-dsRNAs) in the development of OA. We find that in response to OA-mimicking stressors, cytosolic efflux of mt-dsRNAs is increased, leading to PKR activation and subsequent induction of inflammatory cytokines and apoptosis. Moreover, mt-dsRNAs are exported to the extracellular space where they activate toll-like receptor 3. Elevated expression of mt-dsRNAs in the synovial fluids of OA patients further supports our data. Lastly, we show that autophagy protects chondrocytes from mitochondrial dysfunction partly by removing cytosolic mt-dsRNAs. Together, these findings establish the PKR-mt-dsRNA as a critical regulatory axis in OA development and suggest mt-dsRNAs as a potential target in fighting OA.

molecular biology↗
bioRxiv2020

An image resource of subdivided Drosophila GAL4-driver expression patterns for neuron-level searches

Precise, repeatable genetic access to specific neurons via GAL4/UAS and related methods is a key advantage of Drosophila neuroscience. Neuronal targeting is typically documented using light microscopy of full GAL4 expression patterns, which generally lack the single-cell resolution required for reliable cell type identification. Here we use stochastic GAL4 labeling with the MultiColor FlpOut approach to generate cellular resolution confocal images at large scale. We are releasing aligned images of 74,000 such adult central nervous systems. An anticipated use of this resource is to bridge the gap between neurons identified by electron or light microscopy. Identifying individual neurons that make up each GAL4 expression pattern improves the prediction of split-GAL4 combinations targeting particular neurons. To this end we have made the images searchable on the NeuronBridge website. We demonstrate the potential of NeuronBridge to rapidly and effectively identify neuron matches based on morphology across imaging modalities and datasets.

neuroscience↗
bioRxiv2020

Pseudomonas aeruginosa biofilms display carbohydrate ligands for CD206 and CD209 that interfere with their receptor function

Bacterial biofilms represent a challenge to the healthcare system because of their resilience against antimicrobials and immune attack. Biofilms consist of bacterial aggregates embedded in an extracellular polymeric substance (EPS) composed of carbohydrate polymers, nucleic acids and proteins. Carbohydrates within P. aeruginosa biofilms include neutral and mannose-rich Psl, and cationic Pel composed of N-acetyl-galactosamine and N-acetyl-glucosamine. Here we show that P. aeruginosa biofilms display ligands for the C-type lectin receptors mannose receptor (MR, CD206) and Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209). Binding of MR and DC-SIGN to P. aeruginosa biofilms is carbohydrate-and calcium-dependent and extends to biofilms formed by clinical isolates. Confocal analysis of P. aeruginosa biofilms shows abundant DC-SIGN ligands among bacteria aggregates while MR ligands concentrate into discrete clusters. DC-SIGN ligands are also detected in planktonic P. aeruginosa cultures and depend on the presence of the common polysaccharide antigen. Carbohydrates purified from P. aeruginosa biofilms are recognised by DC-SIGN and MR; both receptors preferentially bind the high molecular weight fraction (HMW; >132,000Da) with KDs in the nM range. HMW preparations contain 74.9-80.9% mannose, display -mannan segments and alter the morphology of human dendritic cells without causing obvious changes in cytokine responses. Finally, HMW interferes with the endocytic activity of cell-associated MR and DC-SIGN. This work identifies MR and DC-SIGN as receptors for bacterial biofilms and highlights the potential for biofilm-associated carbohydrates as immunomodulators through engagement of C-type lectin receptors. Author SummarySelective engagement of pattern recognition receptors during infection guides the decision-making process during induction of immune responses. This work identifies mannose-rich carbohydrates within bacterial biofilms as novel molecular patterns associated with bacterial infections. P. aeruginosa biofilms and biofilm-derived carbohydrates bind two important lectin receptors, MR (CD206) and DC-SIGN (CD209), involved in recognition of self and immune evasion. Abundance of MR and DC-SIGN ligands in the context of P. aeruginosa biofilms could impact immune responses and promote chronic infection.

microbiology↗
bioRxiv2020

A Deep Learning Pipeline for Nucleus Segmentation

Deep learning is rapidly becoming the technique of choice for automated segmentation of nuclei in biological image analysis workflows. In order to evaluate the feasibility of training nuclear segmentation models on small, custom annotated image datasets that have been augmented, we have designed a computational pipeline to systematically compare different nuclear segmentation model architectures and model training strategies. Using this approach, we demonstrate that transfer learning and tuning of training parameters, such as the composition, size and pre-processing of the training image dataset, can lead to robust nuclear segmentation models, which match, and often exceed, the performance of existing, off-the-shelf deep learning models pre-trained on large image datasets. We envision a practical scenario where deep learning nuclear segmentation models trained in this way can be shared across a laboratory, facility, or institution, and continuously improved by training them on progressively larger and varied image datasets. Our work provides computational tools and a practical framework for deep learning-based biological image segmentation using small annotated image datasets.

cell biology↗
bioRxiv2020

ASpediaFI: Functional interaction analysis of alternative splicing events

Alternative splicing (AS) regulates biological process governing phenotype or disease. However, it is challenging to systemically analyze global regulation of AS events, their gene interactions, and functions. Here, we introduce a novel application, ASpediaFI for identifying AS events and co-regulated gene interactions implicated in pathways. Our method establishes an interaction network including AS events, performs random walk with restart, and finally identifies a functional subnetwork containing the AS event. We validated the capability of ASpediaFI to interpret biological relevance based on three case studies. Using simulation data, we achieved higher accuracy than with other methods and detected pathway-associated AS events.

bioinformatics↗
bioRxiv2020

Nuclear Import of the HIV-1 Core Precedes Reverse Transcription and Uncoating

HIV-1 particles contain a core formed by ~1500 capsid protein monomers housing viral RNA. HIV-1 core uncoating---disassembly---is required for infection. HIV-1 reverse transcription (RT) occurs before or during uncoating, but the cellular compartment where RT and uncoating occurs is unknown. Using imaging and biochemical assays to track HIV-1 capsids in nuclei during infection, we demonstrated that higher-order capsid complexes or complete cores containing viral genome are imported into nuclear compartments. Additionally, inhibition of RT that stabilizes the core during infection does not prevent capsid nuclear import; thus, RT may occur in nuclear compartments. We separated infected cells into cytosolic and nuclear fractions to measure RT during infection. Most observable RT intermediates were enriched in nuclear fractions, suggesting that most HIV-1 RT occurs in the nuclear compartment alongside uncoating. Thus, nuclear import precedes RT and uncoating, fundamentally changing our understanding of HIV-1 infection.

microbiology↗
bioRxiv2020

Chandelier cell anatomy and function reveal a variably distributed but common signal

The activity and connectivity of inhibitory cells has a profound impact on the operation of neuronal networks. While the average connectivity of many inhibitory cell types has been characterized, we still lack an understanding of how individual interneurons distribute their synapses onto their targets and how heterogeneous the inhibition is onto different individual excitatory neurons. Here, we use large-scale volumetric electron microscopy (EM) and functional imaging to address this question for chandelier cells in layer 2/3 of mouse visual cortex. Using dense morphological reconstructions from EM, we mapped the complete chandelier input onto 153 pyramidal neurons. We find that the number of input synapses is highly variable across the population, but the variability is correlated with structural features of the target neuron: soma depth, soma size, and the number of perisomatic synapses received. Functionally, we found that chandelier cell activity in vivo was highly correlated and tracks pupil diameter, a proxy for arousal state. We propose that chandelier cells provide a global signal whose strength is individually adjusted for each target neuron. This approach, combining comprehensive structural analysis with functional recordings of identified cell types, will be a powerful tool to uncover the wiring rules across the diversity of cortical cell types.

neuroscience↗
bioRxiv2020

EASE: EM-Assisted Source Extraction from calcium imaging data

Combining two-photon calcium imaging (2PCI) and electron microscopy (EM) provides arguably the most powerful current approach for connecting function to structure in neural circuits. Recent years have seen dramatic advances in obtaining and processing CI and EM data separately. In addition, several joint CI-EM datasets (with CI performed in vivo, followed by EM reconstruction of the same volume) have been collected. However, no automated analysis tools yet exist that can match each signal extracted from the CI data to a cell segment extracted from EM; previous efforts have been largely manual and focused on analyzing calcium activity in cell bodies, neglecting potentially rich functional information from axons and dendrites. There are two major roadblocks to solving this matching problem: first, dense EM reconstruction extracts orders of magnitude more segments than are visible in the corresponding CI field of view, and second, due to optical constraints and non-uniform brightness of the calcium indicator in each cell, direct matching of EM and CI spatial components is nontrivial. In this work we develop a pipeline for fusing CI and densely-reconstructed EM data. We model the observed CI data using a constrained nonnegative matrix factorization (CNMF) framework, in which segments extracted from the EM reconstruction serve to initialize and constrain the spatial components of the matrix factorization. We develop an efficient iterative procedure for solving the resulting combined matching and matrix factorization problem and apply this procedure to joint CI-EM data from mouse visual cortex. The method recovers hundreds of dendritic components from the CI data, visible across multiple functional scans at different depths, matched with densely-reconstructed three-dimensional neural segments recovered from the EM volume. We publicly release the output of this analysis as a new gold standard dataset that can be used to score algorithms for demixing signals from 2PCI data. Finally, we show that this database can be exploited to (1) learn a mapping from 3d EM segmentations to predict the corresponding 2d spatial components estimated from CI data, and (2) train a neural network to denoise these estimated spatial components. This neural network denoiser is a stand-alone module that can be dropped in to enhance any existing 2PCI analysis pipeline.

neuroscience↗
bioRxiv2020

Implementation of a pre- and in-scan system to minimize head motion in pediatric participants undergoing fMRI scans

BackgroundPerforming fMRI scans of children can be a difficult task, as participants tend to move while being scanned. Head motion represents a significant confound in functional magnetic resonance imaging (fMRI) connectivity analyses, and methods to limit the impact of movement on data quality are needed. One approach has been to use shorter MRI protocols, though this potentially reduces the reliability of the results. ObjectiveHere we describe steps we have taken to limit head motion in an ongoing fMRI study of children undergoing a 60 minute MRI scan protocol. Specifically, we have used a mock scan protocol that trains participants to lie still while being scanned. We provide a detailed protocol and describe other in-scanner measures we have implemented, including an incentive system and the use of a weighted blanket. Materials and methodsParticipants who received a formal mock scan (n = 12) were compared to participants who had an informal mock scan (n = 7). A replication group of participants (n = 16), including five with autism spectrum disorder, who received a formal mock scan were also compared to the informal mock scan group. The primary measure of interest was the mean frame-to-frame displacement across eight functional runs during the fMRI protocol. ResultsParticipants in the formal mock scan and replication group tended to exhibit more low-motion functional scans than the informal mock scan group (P < 0.05). Across different functional scan conditions (i.e. while watching movie clips, performing an attention task, and during resting-state scans), effect sizes tended to be large (Hedges g > 0.8). ConclusionResults indicate that with appropriate measures, it is possible to achieve low-motion fMRI data in younger participants undergoing a long scan protocol.

neuroscience↗
bioRxiv2020

An alternative miRISC targeting a coding mutation site in FOXL2 links to granulosa cell tumor

Recent evidence suggests that animal microRNAs (miRNAs) can target coding sequences (CDSs); however, the pathophysiological importance of such targeting remains unknown. Here, we show that a somatic heterozygous missense mutation (c.402C>G; p.C134W) in FOXL2, a feature shared by virtually all adult-type granulosa cell tumors (AGCTs), introduces a target site for miR-1236, which induces haploinsufficiency of the tumor-suppressor FOXL2. This miR-1236-mediated selective degradation of the variant FOXL2 mRNA is preferentially conducted by a distinct miRNA-loaded RNA-induced silencing complex (miRISC) directed by the Argonaute3 (AGO3) and DHX9 proteins. In both patients and mouse model of AGCT, the inversely regulated variant FOXL2 abundance with the miR-1236 levels was highly correlated with malignant features of AGCT. Our study provides a molecular basis for understanding the conserved FOXL2 CDS mutation-mediated etiology of AGCT, revealing the existence of a previously unidentified mechanism of miRNA-targeting disease-associated mutations in the CDS by forming a non-canonical miRISC.

cancer biology↗
bioRxiv2020

The Neural Basis for a Persistent Internal State in Drosophila Females

Sustained changes in mood or action require persistent changes in neural activity, but it has been difficult to identify and characterize the neural circuit mechanisms that underlie persistent activity and contribute to long-lasting changes in behavior. Here, we focus on changes in the behavioral state of Drosophila females that persist for minutes following optogenetic activation of a single class of central brain neurons termed pC1. We find that female pC1 neurons drive a variety of persistent behaviors in the presence of males, including increased receptivity, shoving, and chasing. By reconstructing cells in a volume electron microscopic image of the female brain, we classify 7 different pC1 cell types and, using cell type specific driver lines, determine that one of these, pC1-Alpha, is responsible for driving persistent female shoving and chasing. Using calcium imaging, we locate sites of minutes-long persistent neural activity in the brain, which include pC1 neurons themselves. Finally, we exhaustively reconstruct all synaptic partners of a single pC1-Alpha neuron, and find recurrent connectivity that could support the persistent neural activity. Our work thus links minutes-long persistent changes in behavior with persistent neural activity and recurrent circuit architecture in the female brain.

neuroscience↗
bioRxiv2020

Global reference mapping and dynamics of human transcription factor footprints

Combinatorial binding of transcription factors to regulatory DNA underpins gene regulation in all organisms. Genetic variation in regulatory regions has been connected with diseases and diverse phenotypic traits1, yet it remains challenging to distinguish variants that impact regulatory function2. Genomic DNase I footprinting enables quantitative, nucleotide-resolution delineation of sites of transcription factor occupancy within native chromatin3-5. However, to date only a small fraction of such sites have been precisely resolved on the human genome sequence5. To enable comprehensive mapping of transcription factor footprints, we produced high-density DNase I cleavage maps from 243 human cell and tissue types and states and integrated these data to delineate at nucleotide resolution ~4.5 million compact genomic elements encoding transcription factor occupancy. We map the fine-scale structure of ~1.6 million DHS and show that the overwhelming majority is populated by well-spaced sites of single transcription factor:DNA interaction. Cell context-dependent cis-regulation is chiefly executed by wholesale actuation of accessibility at regulatory DNA versus by differential transcription factor occupancy within accessible elements. We show further that the well-described enrichment of disease- and phenotypic trait-associated genetic variants in regulatory regions1,6 is almost entirely attributable to variants localizing within footprints, and that functional variants impacting transcription factor occupancy are nearly evenly partitioned between loss- and gain-of-function alleles. Unexpectedly, we find that the global density of human genetic variation is markedly increased within transcription factor footprints, revealing an unappreciated driver of cis-regulatory evolution. Our results provide a new framework for both global and nucleotide-precision analyses of gene regulatory mechanisms and functional genetic variation.

genomics↗