Biology subjects

Park, K.

Publications and source records attributed to Park, K..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Human immunodeficiency virus-1 induces and targets host genomic R-loops for viral genome integration

Although HIV-1 integration sites favor active transcription units in the human genome, high-resolution analysis of individual HIV-1 integration sites has shown that the virus can integrate into a variety of host genomic locations, including non-genic regions. The invisible infection by HIV-1 integrating into non-genic regions, challenging the traditional understanding of HIV-1 integration site selection, is more problematic because they are selected for preservation in the host genome during prolonged antiretroviral therapies. Here, we showed that HIV-1 integrates its viral genome into the vicinity of R-loops, a genomic structure composed of DNA- RNA hybrids. VSV-G-pseudotyped HIV-1 infection initiates the formation of R-loops in both genic and non-genic regions of the host genome and preferentially integrates into R-loop-rich regions. Using a HeLa cell model that can independently control transcriptional activity and R-loop formation, we demonstrated that the exogenous formation of R-loops directs HIV-1 integration-targeting sites. We also found that HIV-1 integrase proteins physically bind to the host genomic R-loops. These findings provide novel insights into the mechanisms underlying retroviral integration and the new strategies for antiretroviral therapy against HIV-1 latent infection.

molecular biology↗

bioRxiv2024

Discovery of the First-in-class G9a/GLP PROTAC Degrader

Aberrantly expressed lysine methyltransferases G9a and GLP, which catalyze mono- and di-methylation of histone H3 lysine 9 (H3K9), have been implicated in numerous cancers. Recent studies have uncovered both catalytic and non-catalytic oncogenic functions of G9a/GLP. As such, G9a/GLP catalytic inhibitors have displayed limited anticancer activity. Here, we report the discovery of the first-in-class G9a/GLP proteolysis targeting chimera (PROTAC) degrader, 10 (MS8709), as a potential anticancer therapeutic. 10 induces G9a/GLP degradation in a concentration-, time, and ubiquitin-proteasome system (UPS)-dependent manner, does not alter the mRNA expression of G9a/GLP and is selective for G9a/GLP over other methyltransferases. Moreover, 10 displays superior cell growth inhibition to the parent G9a/GLP inhibitor UNC0642 in prostate, leukemia, and lung cancer cells and has suitable mouse pharmacokinetic properties for in vivo efficacy studies. Overall, 10 is a valuable chemical biology tool to further investigate the functions of G9a/GLP and a potential therapeutic for treating G9a/GLP-dependent cancers. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/582210v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1b3c923org.highwire.dtl.DTLVardef@5a22f0org.highwire.dtl.DTLVardef@1abbabeorg.highwire.dtl.DTLVardef@1711f29_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

p300 KAT regulates SOX10 stability and function in human melanoma

SOX10 is a lineage-specific transcription factor critical for melanoma tumor growth, while SOX10 loss-of-function drives the emergence of therapy-resistant, invasive melanoma phenotypes. A major challenge has been developing therapeutic strategies targeting SOX10s role in melanoma proliferation, while preventing a concomitant increase in tumor cell invasion. Here, we report that the lysine acetyltransferase (KAT) EP300 and SOX10 gene loci on Chromosome 22 are frequently co-amplified in melanomas, including UV-associated and acral tumors. We further show that p300 KAT activity mediates SOX10 protein stability and that the p300 inhibitor, A-485, downregulates SOX10 protein levels in melanoma cells via proteasome-mediated degradation. Additionally, A-485 potently inhibits proliferation of SOX10+ melanoma cells while decreasing invasion in AXLhigh/MITFlow melanoma cells through downregulation of metastasis-related genes. We conclude that the SOX10/p300 axis is critical to melanoma growth and invasion, and that inhibition of p300 KAT activity through A-485 may be a worthwhile therapeutic approach for SOX10-reliant tumors.

cancer biology↗

bioRxiv2024

Metabolic Quadrivalency in RSeT Human Embryonic Stem Cells

One of the most important properties of human embryonic stem cells (hESCs) is related to their pluripotent states. In our recent study, we identified a previously unrecognized pluripotent state induced by RSeT medium. This state makes primed hESCs resistant to conversion to naive pluripotent state. In this study, we have further characterized the metabolic features in these RSeT hESCs, including metabolic gene expression, metabolomic analysis, and various functional assays. The commonly reported metabolic modes include glycolysis or both glycolysis and oxidative phosphorylation (i.e., metabolic bivalency) in pluripotent stem cells. However, besides the presence of metabolic bivalency, RSeT hESCs exhibited a unique metabolome with additional fatty acid oxidation and imbalanced nucleotide metabolism. This metabolic quadrivalency is linked to hESC growth independent of oxygen tension and restricted capacity for naive reprogramming in these cells. Thus, this study provides new insights into pluripotent state transitions and metabolic stress-associated hPSC growth in vitro.

biochemistry↗

bioRxiv2024

A ribosome-associating chaperone mediates GTP-driven vectorial folding of nascent eEF1A

Eukaryotic translation elongation factor 1A (eEF1A) is a highly abundant, multi-domain GTPase. Post-translational steps essential for eEF1A biogenesis are carried out by bespoke chaperones but co-translational mechanisms tailored to eEF1A folding remain unexplored. Here, we find that the N-terminal, GTP-binding domain of eEF1A is prone to co-translational misfolding and using computational approaches, yeast genetics, and microscopy analysis, we identify the conserved yet uncharacterized yeast protein Ypl225w as a chaperone dedicated to solving this problem. Proteomics and biochemical reconstitution reveal that Ypl225ws interaction with ribosomal eEF1A nascent chains depends on additional binding of Ypl225w to the UBA domain of nascent polypeptide-associated complex (NAC). Lastly, we show by orthogonal chemical genetics that Ypl225w primes eEF1A nascent chains for their subsequent binding to GTP and release from Ypl225w. Our work establishes eEF1A as a model system for chaperone-dependent co-translational folding and unveils a novel mechanism for GTP-driven folding on the ribosome.

biochemistry↗

bioRxiv2024

De novo lipid synthesis and polarized prenylation drives cell invasion through basement membrane

SummaryInvasive cells form large, specialized protrusions to break through basement membrane (BM) matrix barriers. Park et al., reveal a crucial requirement for de novo lipid synthesis and a dynamic polarizing prenylation system to rapidly construct invasive protrusions that breach BMs. To breach basement membrane (BM), cells in development and cancer use large, transient, specialized lipid-rich membrane protrusions. Using live imaging, endogenous protein tagging, and cell-specific RNAi during C. elegans anchor cell (AC) invasion, we demonstrate that the lipogenic SREBP transcription factor SBP-1 drives expression of the fatty acid synthesis enzymes POD-2 and FASN-1 prior to invasion. We show that phospholipid producing LPIN-1 and sphingomyelin synthase SMS-1, which use fatty acids as substrates, produce lysosome stores that build the ACs invasive protrusion, and that SMS-1 also promotes protrusion localization of the lipid raft partitioning ZMP-1 matrix metalloproteinase. Finally, we discover that the endoplasmic reticulum (ER)-associated HMG-CoA reductase HMGR-1, which generates isoprenoids for prenylation, enriches at the AC invasive front, and that the final ER prenylation enzyme, ICMT-1, localizes to ER exit sites that dynamically polarize to deliver prenylated GTPases for protrusion formation. Together, these results reveal a collaboration between lipogenesis and a polarized lipid prenylation system that drives invasive protrusion formation.

cell biology↗

bioRxiv2024

Resistance to Naive and Formative Pluripotency Conversion in RSeT Human Embryonic Stem Cells

One of the most important properties of human embryonic stem cells (hESCs) is related to their primed and naive pluripotent states. Our previous meta-analysis indicates the existence of heterogeneous pluripotent states derived from diverse naive protocols. In this study, we have characterized a commercial medium (RSeT)-based pluripotent state under various growth conditions. Notably, RSeT hESCs can circumvent hypoxic growth conditions as required by naive hESCs, in which some RSeT cells (e.g., H1 cells) exhibit much lower single cell plating efficiency, having altered or much retarded cell growth under both normoxia and hypoxia. Evidently, hPSCs lack many transcriptomic hallmarks of naive and formative pluripotency (a phase between naive and primed states). Integrative transcriptome analysis suggests our primed and RSeT hESCs are close to the early stage of post-implantation embryos, similar to the previously reported primary hESCs and early hESC cultures. Moreover, RSeT hESCs did not express naive surface markers such as CD75, SUSD2, and CD130 at a significant level. Biochemically, RSeT hESCs exhibit a differential dependency of FGF2 and co-independency of both Janus kinase (JAK) and TGF{beta} signaling in a cell-line-specific manner. Thus, RSeT hESCs represent a previously unrecognized pluripotent state downstream of formative pluripotency. Our data suggest that human naive pluripotent potentials may be restricted in RSeT medium. Hence, this study provides new insights into pluripotent state transitions in vitro.

cell biology↗

bioRxiv2023

Structural elucidation of how ARF small GTPases induce membrane tubulation for vesicle fission

The ADP-Ribosylation Factor (ARF) small GTPases have been found to act in vesicle fission through a direct ability to tubulate membrane. Here, we have used cryo-electron microscopy (EM) to solve the structure of an ARF6 protein lattice assembled on tubulated membrane to 3.9 [A] resolution. ARF6 forms tetramers that polymerize into helical arrays to form this lattice. We identify, and confirm functionally, protein contacts critical for this lattice formation. The solved structure also suggests how the ARF amphipathic helix is positioned in the lattice for membrane insertion, and how a GTPase-activating protein (GAP) docks onto the lattice to catalyze ARF-GTP hydrolysis in completing membrane fission. As ARF1 and ARF6 are structurally conserved, we have also modeled ARF1 onto the ARF6 lattice, which has allowed us to pursue the reconstitution of Coat Protein I (COPI) vesicles to confirm more definitively that the ARF lattice acts in vesicle fission. Our findings are notable for having achieved the first detailed glimpse of how a small GTPase bends membrane and having provided a molecular understanding of how an ARF protein acts in vesicle fission.

cell biology↗

bioRxiv2023

Operator model for evolutionary dynamics

Drift, selection, and mutation are integral evolutionary factors. In this article, operator model is newly suggested to intuitively represent those evolutionary factors into mathematical operators, and to ultimately offer unconventional methodology for understanding evolutionary dynamics. To be specific, each of the drift, selection, and mutation was respectively interpreted as operator which in essence is a random matrix that acts upon the vector which contains population distribution information. The simulation results from the operator model coincided with the previous theoretical results for beneficial mutation accumulation rate in concurrent and successional regimes for asexually reproducing case. Furthermore, beneficial mutation accumulation in strong drift regime for asexually reproducing case was observed from the simulation while allowing the interactions of mutations with diverse selection coefficients. Lastly, methods to justify, reinforce, apply, and expand the operator model were discussed to scrutinize the implications of the model. With its unique characteristics, the operator model is expected to broaden perspective and to offer effective methodology for understanding the evolutionary process.

evolutionary biology↗

bioRxiv2023

Decoding spatial organization maps and context-specific landscapes of breast cancer and its microenvironment via high-resolution spatial transcriptomic analysis

Single-cell RNA transcriptomics has revealed the intricate heterogeneity of both tumors and their microenvironment. However, a notable limitation is its inability to retain spatial context, a crucial aspect of understanding cell identity and function. In this study, we employed imaging-based single-cell spatial transcriptomics to elucidate the tumor and immunological landscapes of two breast cancer samples. By resolving over 400 000 cells per slide, we demonstrated that transcriptional differences lead to structural disparities within and between tumors. Additionally, we observed that the composition of the tumor microenvironment varies depending on its spatial location. Notably, we detected immune cell gradients transitioning from the tumor periphery to its core regions and from tertiary lymphoid structure to immune inflamed regions, in alignment with the specific function of each cell type. This finding facilitated a more precise classification of the tumor immune microenvironment. This study provides a comprehensive dataset for breast cancer researchers and underscores the significance of spatial context in understanding the multifaceted heterogeneity of cancer and its environment.

cancer biology↗

bioRxiv2023

Break-induced replication underlies formation of inverted triplications and generates unexpected diversity in haplotype structures

BackgroundThe duplication-triplication/inverted-duplication (DUP-TRP/INV-DUP) structure is a type of complex genomic rearrangement (CGR) hypothesized to result from replicative repair of DNA due to replication fork collapse. It is often mediated by a pair of inverted low-copy repeats (LCR) followed by iterative template switches resulting in at least two breakpoint junctions in cis. Although it has been identified as an important mutation signature of pathogenicity for genomic disorders and cancer genomes, its architecture remains unresolved and is predicted to display at least four structural variation (SV) haplotypes. ResultsHere we studied the genomic architecture of DUP-TRP/INV-DUP by investigating the genomic DNA of 24 patients with neurodevelopmental disorders identified by array comparative genomic hybridization (aCGH) on whom we found evidence for the existence of 4 out of 4 predicted SV haplotypes. Using a combination of short-read genome sequencing (GS), long- read GS, optical genome mapping and StrandSeq the haplotype structure was resolved in 18 samples. This approach refined the point of template switching between inverted LCRs in 4 samples revealing a DNA segment of [~]2.2-5.5 kb of 100% nucleotide similarity. A prediction model was developed to infer the LCR used to mediate the non-allelic homology repair. ConclusionsThese data provide experimental evidence supporting the hypothesis that inverted LCRs act as a recombinant substrate in replication-based repair mechanisms. Such inverted repeats are particularly relevant for formation of copy-number associated inversions, including the DUP-TRP/INV-DUP structures. Moreover, this type of CGR can result in multiple conformers which contributes to generate diverse SV haplotypes in susceptible loci.

genetics↗

bioRxiv2023

Unveiling the enigma of Brain-resident immune cells

The immune system has been extensively studied in traditional immune hubs like the spleen and lymph nodes. However, recent advances in immunology highlight unique immune cell characteristics across anatomical compartments. In this study, we challenged conventional thinking by uncovering distinct immune cell populations within the brain parenchyma, separate from those in the blood, meninges, and choroid plexus, with unique transcriptional profiles. Brain-resident immune cells are not derived from maternal immune cells, and age-related changes, with an increase in CD8+ T cells in aged mice, are noted. Alzheimers disease (AD) alters microglias interaction with brain-resident immune cells, emphasizing immune-brain dynamics. Furthermore, we reveal dynamic immune cell interactions and essential cytokine roles in brain homeostasis, with stable cytokine expression but emerging signaling pathways in AD. In summary, this study advances our understanding of brain-resident immune cells in both normal and pathological conditions.

immunology↗

bioRxiv2023

Memory encoding and retrieval by retrosplenial parvalbumin interneurons is impaired in Alzheimer's disease model mice

Memory deficits in Alzheimers disease (AD) show a strong link with GABAergic interneuron dysfunctions1-7. Ensemble dynamics of GABAergic interneurons are critical in memory encoding and retrieval8-12 but how GABAergic interneuron dysfunction affects inhibitory ensemble dynamics in AD is unknown. As retrosplenial cortex (RSC) is a brain area critical for episodic memory13-16 and affected by beta-amyloid accumulation in early AD17-21, we address this question by performing Ca2+ imaging in RSC parvalbumin-expressing (PV) interneurons during a contextual fear memory (CFM) task in healthy control mice and the 5XFAD mouse model of AD. We found that subpopulations of PV interneurons that were responsive to aversive electric foot shocks during contextual fear conditioning (CFC) in the control mice significantly decreased in the 5XFAD mice, indicating a dysfunction in the recruitment of CFM-encoding PV interneurons. In control mice, CFC-responsive PV interneuron ensemble activities were selectively upregulated during the freezing epoch of the CFM retrieval, manifested by CFC-induced synaptic potentiation of PV interneuron-mediated inhibition. However, CFC-induced changes in PV interneuron ensemble dynamics during CFM retrieval and synaptic plasticity were both absent in the 5XFAD mice. Optogenetic silencing of PV interneurons during CFC in control mice mimicked the CFM deficits in 5XFAD mice, while optogenetic activation of PV interneurons during CFC in the 5XFAD mice restored CFM retrieval. These results demonstrate the necessity and sufficiency of CFC-responsive PV interneurons for CFM retrieval and suggest that synaptic dysfunction in CFM-encoding PV interneurons disrupt the inhibitory ensemble dynamics underlying CFM retrieval, subsequently leading to memory deficits in AD.

neuroscience↗

bioRxiv2023

Egocentric vector coding of geometric vertex in the retrosplenial cortex

Neural representation of the environmental features in a self-centered, egocentric manner is important in constructing an egocentric cognitive map that is critical for goal-directed navigation and episodic memory formation1. To create a geometrically detailed egocentric cognitive map, neural representations of edges and vertices of environmental features are needed. While egocentric neural representations of edges, like egocentric boundary vector cells2-6 and border cells exist7, those of vertices are unknown. Here we report that single neurons in the granular retrosplenial cortex (RSC) generate spatial receptive fields exclusively near the vertices of environmental geometries during free exploration, which we termed vertex cells. Each spatial receptive field of vertex cells occurred at a specific orientation and distance relative to the animal, tuned by head direction, indicating an egocentric vector coding of the vertex. The removal of physical boundaries that define the environmental geometry abolished egocentric vector coding vertex cells. Moreover, goal-directed navigation selectively strengthened the egocentric vertex vector coding at the vertex near the goal location. Overall, our results suggest that egocentric vector coding of vertex by granular RSC neurons help to construct a geometrically detailed egocentric cognitive map that guides goal-directed navigation.

neuroscience↗

bioRxiv2023

Impaired extinction of cocaine seeking in HIV-infected mice is accompanied by peripheral and central immune dysregulation

Substance use disorders (SUDs) are highly comorbid with HIV infection, necessitating an understanding of the interactive effects of drug exposure and HIV. The relationship between progressive HIV infection and cocaine use disorder is likely bidirectional, with cocaine use having direct effects on immune function while HIV infection can alter addiction-related behavior. To better characterized the neurobehavioral and immune consequences of HIV infection and cocaine exposure, this study utilized a humanized mouse model to investigate the outcomes of progressive HIV infection on cocaine-related behaviors in a cocaine conditioned place preference (CPP) model, and the interactive effects of cocaine and HIV infection on peripheral and central nervous system inflammation. HIV infection did not impact the formation of a cocaine CPP, but did result in resistance to extinction of the CPP. No effects of HIV on yohimbine-primed reinstatement or cocaine seeking under conflict were observed. These behavioral alterations were accompanied by immune changes in HIV infected mice, including increased prefrontal cortex astrocyte immunoreactivity and brain-region specific effects on microglia number and reactivity. Peripheral immune system changes were observed in both mouse and human markers. Among other targets, this included HIV-induced reductions in mouse IL-1 and G-CSF and human TNF and cocaine-induced alterations in human TNF and mouse GM-CSF such that cocaine exposure increases both cytokines only in the absence of HIV infection. Together these data provide new insights into the unique neurobehavioral processes underlying HIV infection and cocaine use disorders, and further how they interact to effect immune responses.

neuroscience↗

bioRxiv2023

Model-based compound hypothesis testing for snATAC-seq data with PACS

Single nucleus ATAC-seq (snATAC-seq) experimental designs have become increasingly complex with multiple factors that might affect chromatin accessibility, including genotype, cell type, tissue of origin, sample location, batch, etc., whose compound effects are difficult to test by existing methods. In addition, current snATAC-seq data present statistical difficulties due to their sparsity and variations in individual sequence capture. To address these problems, we present a zero-adjusted statistical model, Probability model of Accessible Chromatin of Single cells (PACS), that can allow complex hypothesis testing of factors that affect accessibility while accounting for sparse and incomplete data. For differential accessibility analysis, PACS controls the false positive rate and achieves on average a 17% to 122% higher power than existing tools. We demonstrate the effectiveness of PACS through several analysis tasks including supervised cell type annotation, compound hypothesis testing, batch effect correction, and spatiotemporal modeling. We apply PACS to several datasets from a variety of tissues and show its ability to reveal previously undiscovered insights in snATAC-seq data.

bioinformatics↗

bioRxiv2023

CRISPR/Cas9 gene editing to generate Drosophila LexA lines in secondary school classes

Genome editing in vivo with CRISPR/Cas9 generates powerful tools to study gene regulation and function. We developed CRISPR-based methods that permitted secondary school student scientists to convert Drosophila GAL4 lines to LexA lines. Our novel curricula implement a new donor strain optimizing Homology-assisted CRISPR knock-in (HACK) that simplifies screening using light microscopy. Successful curricula adoption by a consortium of schools led to the generation and characterization of 16 novel LexA lines. This includes extensive comparative tissue expression analysis between the parental Gal4 and derived LexA lines. From this collaboration, we established a workflow to systematically generate LexA lines from frequently-used GAL4 lines. Modular courses developed from this effort can be tailored to specific secondary school scheduling needs, and serve as a template for science educators to innovate courses and instructional goals. Our unique collaborations highlight that resources and expertise harnessed by university-based research laboratories can transform experiential science instruction in secondary schools while addressing research needs for the community of science.

genetics↗

bioRxiv2023

Non-coding sequence variation reveals fragility within interleukin 2 feedback circuitry and shapes autoimmune disease risk

Genetic variants associated with human autoimmune diseases commonly map to non-coding control regions, particularly enhancers that function selectively in immune cells and fine-tune gene expression within a relatively narrow range of values. How such modest, cell-type-selective changes can meaningfully shape organismal disease risk remains unclear. To explore this issue, we experimentally manipulated species-conserved enhancers within the disease-associated IL2RA locus and studied accompanying changes in the progression of autoimmunity. Perturbing distinct enhancers with restricted activity in conventional T cells (Tconvs) or regulatory T cells (Tregs)--two functionally antagonistic T cell subsets--caused only modest, cell-type-selective decreases in IL2ra expression parameters. However, these same perturbations had striking and opposing effects in vivo, completely preventing or severely accelerating disease in a murine model of type 1 diabetes. Quantitative tissue imaging and computational modelling revealed that each enhancer manipulation impinged on distinct IL-2-dependent feedback circuits. These imbalances altered the intracellular signaling and intercellular communication dynamics of activated Tregs and Tconvs, producing opposing spatial domains that amplified or constrained ongoing autoimmune responses. These findings demonstrate how subtle changes in gene regulation stemming from non-coding variation can propagate across biological scales due to non-linearities in intra- and intercellular feedback circuitry, dramatically shaping disease risk at the organismal level.

immunology↗