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Lee, C.

Publications and source records attributed to Lee, C..

At least 199 records · Page 11Linked to original sources
bioRxiv2022

Activity-based CRISPR Scanning Uncovers Allostery in DNA Methylation Maintenance Machinery

Allostery enables dynamic control of protein function. A paradigmatic example is the tightly orchestrated process of DNA methylation maintenance. Despite their fundamental importance, systematic identification of allosteric sites remains highly challenging. Here we perform CRISPR scanning on the essential maintenance methylation machinery--DNMT1 and its partner UHRF1--with the activity-based inhibitor decitabine to uncover allosteric mechanisms regulating DNMT1. Through computational analyses, we identify putative mutational hotspots in DNMT1 distal from the active site that encompass mutations spanning a multi-domain autoinhibitory interface and the uncharacterized BAH2 domain. We biochemically characterize these mutations as gain-of-function mutations that increase DNMT1 activity. Extrapolating our analysis to UHRF1, we discern putative gain-of-function mutations in multiple domains, including key residues across the autoinhibitory TTD-PBR interface. Collectively, our findings highlight the utility of activity-based CRISPR scanning for nominating candidate allosteric sites, even beyond the direct drug target.

genomics↗
bioRxiv2022

Base editor scanning charts the DNMT3A activity landscape

DNA methylation is critical for regulating gene expression, necessitating its accurate placement by enzymes such as the DNA methyltransferase DNMT3A. Dysregulation of this process is known to cause aberrant development and oncogenesis, yet how DNMT3A is regulated holistically by its three domains remains challenging to study. Here we integrate base editing with a DNA methylation reporter to perform in situ mutational scanning of DNMT3A in cells. We identify mutations throughout the protein that perturb function, including ones at an interdomain interface that block allosteric activation. Unexpectedly, we also find mutations in the PWWP domain, a histone reader, that modulate enzyme activity despite preserving histone recognition and protein stability. These effects arise from altered PWWP domain DNA affinity, which we show is a noncanonical function required for full activity in cells. Our findings highlight mechanisms of interdomain crosstalk and demonstrate a generalizable strategy to probe sequence-activity relationships of nonessential chromatin regulators.

biochemistry↗
bioRxiv2022

Little skate genome exposes the gene regulatory mechanisms underlying the evolution of vertebrate locomotion

The little skate Leucoraja erinacea, a cartilaginous fish, displays pelvic fin driven walking-like behaviors using genetic programs and neuronal subtypes similar to those of land vertebrates. However, mechanistic studies on little skate motor circuit development have been limited, due to a lack of high-quality reference genome. Here, we generated an assembly of the little skate genome, containing precise gene annotation and structures, which allowed post-genome analysis of spinal motor neurons (MNs) essential for locomotion. Through interspecies comparison of mouse, skate and chicken MN transcriptomes, shared and divergent MN expression profiles were identified. Conserved MN genes were enriched for early-stage nervous system development. Comparison of accessible chromatin regions between mouse and skate MNs revealed conservation of the potential regulators with divergent transcription factor (TF) networks through which expression of MN genes is differentially regulated. TF networks in little skate MNs are much simpler than those in mouse MNs, suggesting a more fine-grained control of gene expression operates in mouse MNs. These findings suggest conserved and divergent mechanisms controlling MN development system of vertebrates during evolution and the contribution of intricate gene regulatory networks in the emergence of sophisticated motor system in tetrapods.

evolutionary biology↗
bioRxiv2022

Large haploblocks underlie rapid adaptation in an invasive weed

Adaptation is the central feature and leading explanation for the evolutionary diversification of life. Adaptation is also notoriously difficult to study in nature, owing to its complexity and logistically prohibitive timescale. We leverage extensive contemporary and historical collections of Ambrosia artemisiifolia--an aggressively invasive weed and primary cause of pollen-induced hayfever--to track the phenotypic and genetic causes of recent local adaptation across its native and invasive ranges in North America and Europe, respectively. Large haploblocks-- indicative of chromosomal inversions--contain a disproportionate share (26%) of genomic regions conferring parallel adaptation to local climates between ranges, are associated with rapidly adapting traits, and exhibit dramatic frequency shifts over space and time. These results highlight the importance of large-effect standing variants in rapid adaptation, which have been critical to A. artemisiifolias global spread across vast climatic gradients.

evolutionary biology↗
bioRxiv2022

Uncovering the hologenomic basis of an extraordinary plant invasion

While invasive species are a key driver of the global biodiversity crisis, the drivers of invasiveness remain debated. To investigate the genomic basis of invasiveness in plants, we use the invasive weed Ambrosia artemisiifolia, introduced to Europe in the late 19th century, resequencing 655 ragweed genomes, including 308 herbarium specimens collected up to 190 years ago. In introduced European populations, we report selection signatures in defense genes and lower prevalence of particular plant pathogens in the invasive range. Together with temporal changes in population structure associated with introgression from closely related Ambrosia species, escape from microbial enemies likely favoured the plants remarkable success as an invasive species. One-Sentence SummaryThe invasive success of European ragweed was facilitated by release from enemy microbes and inter-species hybridization.

genomics↗
bioRxiv2022

Exocyst stimulates each step of exocytic SNARE complex assembly and vesicle fusion

The exocyst is a large multisubunit tethering complex essential for targeting and fusion of secretory vesicles in eukaryotic cells. Although the assembled exocyst complex is proposed to tether vesicles to the plasma membrane and activate the SNARE proteins for membrane fusion, only little is known about the key biochemical steps that exocyst stimulates in the course of SNARE complex assembly, a critical question defining the essential molecular role of the exocyst complex. Here, we use a combination of single molecule and bulk fluorescence assays with purified octameric yeast exocyst complexes to examine the role of exocyst in a reconstituted SNARE assembly and fusion system. We show that the exocyst complex simulates multiple steps spanning from SNARE protein activation to ternary complex assembly, rather than affecting only a specific subset of steps. We also observed that the exocyst has important downstream roles in driving membrane fusion, up to full content mixing of vesicle lumens. Our results suggest that the exocyst complex provides extensive chaperoning functions for the entire process of SNARE complex assembly, presumably using its multi-faceted structure provided by the eight subunits.

molecular biology↗
bioRxiv2021

Haplotype-resolved inversion landscape reveals hotspots of mutational recurrence associated with genomic disorders

Unlike copy number variants (CNVs), inversions remain an underexplored genetic variation class. By integrating multiple genomic technologies, we discover 729 inversions in 41 human genomes. Approximately 85% of inversions <2 kbp form by twin-priming during L1-retrotransposition; 80% of the larger inversions are balanced and affect twice as many base pairs as CNVs. Balanced inversions show an excess of common variants, and 72% are flanked by segmental duplications (SDs) or mobile elements. Since this suggests recurrence due to non-allelic homologous recombination, we developed complementary approaches to identify recurrent inversion formation. We describe 40 recurrent inversions encompassing 0.6% of the genome, showing inversion rates up to 2.7x10-4 per locus and generation. Recurrent inversions exhibit a sex- chromosomal bias, and significantly co-localize to the critical regions of genomic disorders. We propose that inversion recurrence results in an elevated number of heterozygous carriers and structural SD diversity, which increases mutability in the population and predisposes to disease- causing CNVs.

genomics↗
bioRxiv2021

A computational study on the optimization of transcranial temporal interfering stimulation with high-definition electrode using unsupervised neural network

Transcranial temporal interfering stimulation (tTIS) can focally stimulate deep parts of the brain, which are related to specific functions, by using beats at two high AC frequencies that do not affect the human brain. However, it has limitations in terms of calculation time and precision for optimization because of its complexity and non-linearity. We aimed to propose a method using an unsupervised neural network (USNN) for tTIS to optimize quickly the interfering current value of high-definition electrodes, which can finely stimulate the deep part of the brain, and analyze the performance and characteristics of tTIS. A computational study was conducted using 16 realistic head models. This method generated the strongest stimulation on the target, even when targeting deep areas or multi-target stimulation. The tTIS was robust with target depth compared with transcranial alternating current stimulation, and mis-stimulation could be reduced compared with the case of using two-pair inferential stimulation. Optimization of a target could be performed in 3 min. By proposing the USNN for tTIS, we showed that the electrode currents of tTIS can be optimized quickly and accurately, and the possibility of stimulating the deep part of the brain precisely with transcranial electrical stimulation was confirmed.

bioengineering↗
bioRxiv2021

Notch-dependent DNA cis-regulatory elements and their dose-dependent control of C. elegans stem cell self-renewal

A long-standing biological question is how DNA cis-regulatory elements shape transcriptional patterns during metazoan development. The use of reporter constructs, cell culture and computational modeling has made enormous contributions to understanding this fundamental question, but analysis of regulatory elements in their natural developmental context is an essential but rarely used complement. Here, we edited Notch-dependent cis-regulatory elements in the endogenous C. elegans sygl-1 gene, which encodes a key stem cell regulator. We then analyzed the in vivo consequences of those mutations - on both gene expression (nascent transcripts, mRNA, protein) and stem cell maintenance. Mutation of a single element in a three-element homotypic cluster reduced expression as well as stem cell pool size by about half, while mutation of two elements essentially abolished them. We find that LBS number and LBS neighborhood are both important to activity: elements on separate chromosomes function additively, while elements in the same cluster act synergistically. Our approach of precise CRISPR/Cas9 gene editing coupled with quantitation of both molecular and biological readouts establishes a powerful model for in vivo functional analyses of DNA cis-regulatory elements. Summary statementNotch-dependent DNA cis-regulatory elements work together in their developmental context to shape a transcriptional gradient, control stem cell pool size, and govern differentiation onset.

developmental biology↗
bioRxiv2021

A temporally resolved, multiplex molecular recorder based on sequential genome editing

DNA is naturally well-suited to serve as a digital medium for in vivo molecular recording. However, DNA-based memory devices described to date are constrained in terms of the number of distinct signals that can be concurrently recorded and/or by a failure to capture the precise order of recorded events1. Here we describe DNA Ticker Tape, a general system for in vivo molecular recording that largely overcomes these limitations. Blank DNA Ticker Tape consists of a tandem array of partial CRISPR-Cas9 target sites, with all but the first site truncated at their 5 ends, and therefore inactive. Signals of interest are coupled to the expression of specific prime editing guide RNAs2. Editing events are insertional, and record the identity of the guide RNA mediating the insertion while also shifting the position of the "write head" by one unit along the tandem array, i.e. sequential genome editing. In this proof-of-concept of DNA Ticker Tape, we demonstrate the recording and decoding of complex event histories or short text messages; evaluate the performance of dozens of orthogonal tapes; and construct "long tape" potentially capable of recording the order of as many as 20 serial events. Finally, we demonstrate how DNA Ticker Tape simplifies the decoding of cell lineage histories.

genomics↗
bioRxiv2021

Multiplex genomic recording of enhancer and signal transduction activity in mammalian cells

Measurements of gene expression and signal transduction activity are conventionally performed with methods that require either the destruction or live imaging of a biological sample within the timeframe of interest. Here we demonstrate an alternative paradigm, termed ENGRAM (ENhancer-driven Genomic Recording of transcriptional Activity in Multiplex), in which the activity and dynamics of multiple transcriptional reporters are stably recorded to DNA. ENGRAM is based on the prime editing-mediated insertion of signal- or enhancer-specific barcodes to a genomically encoded recording unit. We show how this strategy can be used to concurrently genomically record the relative activity of at least hundreds of enhancers with high fidelity, sensitivity and reproducibility. Leveraging synthetic enhancers that are responsive to specific signal transduction pathways, we further demonstrate time- and concentration-dependent genomic recording of Wnt, NF-{kappa}B, and Tet-On activity. Finally, by coupling ENGRAM to sequential genome editing, we show how serially occurring molecular events can potentially be ordered. Looking forward, we envision that multiplex, ENGRAM-based recording of the strength, duration and order of enhancer and signal transduction activities has broad potential for application in functional genomics, developmental biology and neuroscience.

genomics↗
bioRxiv2021

Longitudinal single-cell transcriptional dynamics throughout neurodegeneration in SCA1

Neurodegeneration is a protracted process involving progressive changes in myriad cell types that ultimately result in neuronal death. Changes in vulnerable neuronal populations are highly influenced by concomitant changes in surrounding cells, complicating experimental approaches to interrogate the simultaneous events that underlie neurodegeneration. To dissect how individual cell types within a heterogeneous tissue contribute to the pathogenesis and progression of a neurodegenerative disorder, we performed longitudinal single-nucleus RNA sequencing of the mouse and human spinocerebellar ataxia type 1 (SCA1) cerebellum, establishing continuous dynamic trajectories of each population. Furthermore, we defined the precise transcriptional changes that precede loss of Purkinje cells and identified early oligodendroglial impairments that can profoundly impact cerebellar function. Finally, we applied a deep learning method to accurately predict disease state and identify drivers of disease. Together, this work uncovers new roles for diverse cerebellar cell types in SCA1 and provides a generalizable analysis framework for studying neurodegeneration.

neuroscience↗
bioRxiv2021

Versatile Encapsulation and Synthesis of Potent Therapeutic Liposomes by Thermal Equilibration

The wide-scale use of liposomal delivery systems is hampered by difficulties in obtaining potent liposomal suspensions. Passive and active loading strategies have been proposed to formulate drug encapsulated liposomes, but are limited by low efficiencies (passive) or high drug specificities (active). Here, we present an efficient and universal loading strategy for synthesizing therapeutic liposomes. Integrating a thermal equilibration technique with our unique liposome synthesis approach, co-loaded targeting liposomes can be engineered in an efficient and scalable manner with potencies 200-fold higher than typical passive encapsulation techniques. We demonstrate this capability through simultaneous co-loading of hydrophilic and hydrophobic small molecules and through targeted delivery of liposomal Doxorubicin to a metastatic breast cancer cell line MDA-MB-231. Molecular dynamic simulations are used to explain interactions between Doxorubicin and liposome membrane during thermal equilibration. By addressing the existing challenges, we have developed an unparalleled approach that will facilitate the formulation of novel theranostic and pharmaceutical strategies.

bioengineering↗
bioRxiv2021

Dual TBX5-Lineage and MYL2 Reporter System For Identification of Left Ventricular Cardiomyocytes During Human Induced Pluripotent Stem Cell Differentiation

During mammalian development, the left and right ventricles arise from early populations of cardiac progenitors known as the first and second heart fields, respectively. While these populations have been extensively studied in non-human model systems, their identification and study in vivo human tissues have been limited due to the ethical and technical limitations of accessing gastrulation stage human embryos. Human induced pluripotent stem cells (hiPSCs) present an exciting alternative for modeling early human embryogenesis due to their well-established ability to differentiate into all embryonic germ layers. Here, we describe the development of a TBX5/MYL2 lineage tracing reporter system that allows for the identification of FHF-progenitors and their descendants including left ventricular cardiomyocytes. Furthermore, using single cell RNA sequencing (scRNA-seq) with oligonucleotide-based sample multiplexing, we extensively profiled differentiating hiPSCs across 12 timepoints in two independent iPSC lines. Surprisingly, our reporter system and scRNA-seq analysis revealed a predominance of FHF differentiation using the small molecule Wnt-based 2D differentiation protocol. We compared this data with existing murine and 3D cardiac organoid scRNA-seq data and confirmed the dominance of left ventricular cardiomyocytes (>90%) in our hiPSC-derived progeny. Together, our work provides the scientific community with a powerful new genetic lineage tracing approach as well as a single cell transcriptomic atlas of hiPSCs undergoing cardiac differentiation.

developmental biology↗
bioRxiv2021

Direct Reprogramming of Non-limb Fibroblasts toCells with Properties of Limb Progenitors

The early limb bud consists of mesenchymal progenitors (limb progenitors) derived from the lateral plate mesoderm (LPM) that produce most of the tissues of the mature limb bud. The LPM also gives rise to the mesodermal components of the trunk, flank and neck. However, the mesenchymal cells generated at these other axial levels cannot produce the variety of cell types found in the limb bud, nor can they be directed to form a patterned appendage-like structure, even when placed in the context of the signals responsible for organizing the limb bud. Here, by taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28) normally expressed in the early limb bud, that are capable of imparting limb progenitor-like properties to non-limb fibroblasts. Cells reprogrammed by these factors show similar gene expression profiles, and can differentiate into similar cell types, as endogenous limb progenitors. The further addition of Lin41 potentiates proliferation of the reprogrammed cells while suppressing differentiation. These results suggest that these same four key factors may play pivotal roles in the specification of endogenous limb progenitors.

developmental biology↗
bioRxiv2021

Identification of the safe harbor locus, AAVS1, from porcine genome and site-specific integration of recombinase-mediated cassette exchange system in porcine fibroblasts using CRISPR/Cas9

Gene integration at site-specific loci, such as safe harbor regions for stable expression via transgenesis, is a critical approach for understanding the function of a gene in cells or animals. The AAVS1 locus is a well-known safe harbor site for human and mouse studies. In the present study, we found an AAVS1-like sequence in the porcine genome using the UCSC Genome Browser and designed TALEN and CRISPR/Cas9 to target AAVS1. The efficiency of CRISPR/Cas9 for targeting the AAVS1 locus in porcine cells was superior to that of TALEN. An AAVS1-targeting donor vector containing GFP was designed and cloned. We added a loxP-lox2272 cassette sequence to the donor vector for further exchange of various transgenes in the AAVS1-targeted cell line. The donor vector and CRISPR/Cas9 components targeting AAVS1 were transfected into a porcine fibroblast cell line. Targeted cells of CRISPR/Cas9-mediated homologous recombination were identified by antibiotic selection. Gene knock-in at the AAVS1 locus was confirmed by PCR analysis. To induce recombinase-mediated cassette exchange (RMCE), another donor vector containing the loxP-lox2272 cassette and inducible (Tet-on) Cre recombinase was cloned. The Cre-donor vector was transfected into the AAVS1-targeted cell line, and RMCE was induced by adding doxycycline to the culture medium. RMCE in porcine fibroblasts was confirmed using PCR analysis. In conclusion, gene targeting at the AAVS1 locus and RMCE in porcine fibroblasts was successful. This technology will be useful for future porcine transgenesis studies and the generation of stable transgenic pigs.

molecular biology↗
bioRxiv2021

Amelioration of L-DOPA-induced dyskinesia with vitamin D3 in Parkinsonian mice model

L-DOPA Induced Dyskinesia (LID) is associated with prolonged L-DOPA therapy. Vitamin-D receptor modulation improves motor-cognitive deficit in experimental LID Parkinsonism. Therefore, in this study, we investigated the mechanism underlying the anti-dyskinetic potential of Vitamin D3 (VD3). Dyskinesia was induced by chronic L-DOPA administration in 6-OHDA lesioned male C57BL6 mice. The experimental groups (Dyskinesia, Dyskinesia/VD3, and Dyskinesia/Amantadine) and controls were challenged with L-DOPA to determine the abnormal involuntary movements (AIMs) score during 14 days of VD3 (30 mg/kg) or Amantadine (40 mg/kg) treatment. Global behavioral Axial, Limb & Orolingual (ALO) AIMs were scored for 1 min at every 20 mins interval, over a duration of 100 mins on days 1,3,7,11 and 14 of treatment. Thereafter, brain samples were collected and processed for immunoblotting to assess striatal expression of tyrosine hydroxylase (TH), monoamine oxidase (MAO), cathecol-o-methyl transferase (COMT), dopamine decarboxylase (DDC), CD11b, BAX, P47phox, and IL-1{beta}. VD3 significantly attenuated ALO AIMs only on days 11 & 14, with maximal reduction of 32.7% compared with dyskinetic mice but had no effect on days 1, 3 & 7, while amantadine decreased AIMs all through days 1 to 14 with maximal reduction of 64.5%. TH and MAO-B expression were not significantly different across the groups. DDC was significantly suppressed in dyskinetic mice vs control (p<0.001) but remained unchanged in VD3 mice vs dyskinetic mice. COMT was upregulated in the dyskinetic group vs control (p<0.01) and attenuated in VD3 mice (p<0.05) compared to the dyskinetic group. Interestingly, VD3 inhibited significantly (p<0.01) oxidative stress (p47phox), apoptosis (BAX), inflammation (IL-1{beta}), and microglial activation (CD11b) in dyskinetic mice. Overall, we find that the anti-dyskinetic effects of VD3 is associated with modulation of striatal oxidative stress, microglial responses, inflammation, and apoptotic signaling. Impact statementThere are evidences showing that VD3 supplementation improves motor disorders, including Parkinsons disease. We hypothesized that VD3 could improve LID, an abnormal involuntary movement that results from prolonged L-DOPA therapy in the management of PD. We have demonstrated the novel anti-dyskinetic effect of VD3 and associated mechanistic factors in a mouse model of L-DOPA Induced Dyskinesia (LID), which identifies promising targets for dyskinesia therapy.

neuroscience↗