Biology subjects

Chen, K. Y.

Publications and source records attributed to Chen, K. Y..

17 recordsLinked to original sources

bioRxiv2026

Palmitate-activated IRE1-XBP1 signaling limits DNA damage and is associated with H2A.X-linked DNA damage response transcripts in triple-negative breast cancer cells

Palmitate-associated lipotoxic stress activates the endoplasmic-reticulum stress sensor protein IRE1, but how IRE1 signaling influences genotoxic-stress responses in triple-negative breast cancer remains unclear. Here, we investigated IRE1 and XBP1 signaling in MDA-MB-231 cells exposed to palmitate and etoposide. Under combined lipotoxic and genotoxic stress, XBP1 depletion increased DNA-damage accumulation in IRE1 wild-type cells but not in IRE1-deficient cells, whereas IRE1 re-expression reduced DNA damage. Pharmacologic IRE1 RNase inhibition also increased etoposide-associated DNA damage, supporting a protective role for IRE1 RNase-dependent signaling. IRE1 status and XBP1 manipulation differentially affected H2A.X and {gamma}H2A.X abundance, indicating context-dependent regulation of chromatin-associated DNA-damage signaling. At the RNA level, H2A.X was enriched in IRE1 immunoprecipitates, and the H2A.X-linked DNA-damage-response transcripts HUWE1 and BRCA2 also associated with IRE1 under combined stress. These findings link palmitate-activated IRE1-XBP1 signaling to genome-protective responses during genotoxic stress and identify a network of IRE1-associated DNA-damage-response transcripts in triple-negative breast cancer cells. Direct IRE1-mediated cleavage of these transcripts remains to be established.

cancer biology↗

bioRxiv2026

Massively multiplex multimodal chemical screens at single-cell resolution

Recent applications of scRNA-seq for massively multiplexed chemical screens have enabled comprehensive profiling of drug responses at unprecedented scale and resolution. However, current assays remain limited to RNA readouts, lacking information on other phenotypic and mechanistic layers such as chromatin accessibility, protein abundance and post-translational modifications. Here, we introduce a scalable framework for multimodal chemical screens, combining parallel small-molecule perturbations with multimodal readouts. We extend existing experimental platforms into icCITE-plex and DOGMA-plex, enabling joint profiling of RNA, protein, and epigenomic responses to thousands of chemical perturbations in parallel. To systematically decode the regulatory circuitry underlying these responses, we develop MoCAVI, a contrastive analysis framework that disentangles the effect of small molecules from control variation in multimodal measurements, and PERCISTRA, a pipeline that infers causal links between chromatin accessibility and gene expression. Applied across ~410,000 primary T cells under ~2,800 conditions, our approach resolves compound-specific mechanisms, highlights off-target effects, and links chromatin accessibility changes to transcription factor networks in primary T cells. Our results establish a generalizable platform for profiling and analyzing cellular responses to chemical perturbations across multiple modalities.

genomics↗

bioRxiv2025

Acute cold exposure in humans shifts the circulating proteome to a cardioprotective and anti-aging profile

Cold exposure has been proposed to provide a constellation of salutary effects, yet its molecular correlates remain largely unknown. Brown adipose tissue (BAT) is the main site of adaptive thermogenesis, and its prevalence is linked with cardiometabolic health. Since the benefits of BAT activation and cold exposure more generally may be mediated through blood-borne factors, we conducted an extensive analysis of the circulating proteome linked with an acute cold challenge in healthy adults. Our goal was to uncover early molecular changes triggered by cooling and establish their specific relationships with the human brown adipocyte secretome as well as various phenotypic traits. Based on comprehensive inter-cohort validations, we provide the first reproducible proteomic signature of cold exposure in humans. Our data demonstrate that cooling favorably modulates circulating mediators linked with chronological aging, as well as metabolic and cardiovascular diseases, providing new potential biochemical transducers of the benefits associated with cold therapy. HighlightsO_LICooling alters the plasma proteome with striking concordance in independent human cohorts. C_LIO_LICooling represses circulating proteins linked with type 2 diabetes, hypercholesterolemia, hypertension, coronary heart disease and heart failure. C_LIO_LIThe circulating signature of cooling resembles a cardioprotective and anti-aging profile. C_LI

physiology↗

bioRxiv2025

Single Nuclei-Derived Molecular Subtypes of Gastrointestinal Stromal Tumors Correlate with Clinicopathologic Features and Predict Clinical Outcomes

Historically, gastrointestinal stromal tumor (GIST) has been subtyped by oncogenic driver mutations. However, tumors with the same mutational profile can have variable biology. To further explore the impact of molecular diversity on GIST biology, we performed single nucleus RNA sequencing on 16 primary GIST and utilized an integrated single cell atlas of the normal GI tract composed from multiple publicly available datasets to identify six distinct GIST cell states. We then statistically estimated the relative abundances of these profiles in bulk transcriptomic data. These were used to define six common GIST molecular subtypes based upon one or two predominant tumor cell states. We found that these molecular subtypes correlate with tumor locations, mutational profiles, and patient outcomes, and validated these subtypes in an independent international cohort. These molecular subtypes have the potential to be used for clinical prognostication for patients with GIST, identifying new therapeutic targets, and studying the cell of transformation of GIST.

cancer biology↗

bioRxiv2025

Joint single-cell measurements of surface proteins, intracellular proteins and gene expression with icCITE-seq

The development of single-cell RNA-sequencing assays has transformed our understanding of cellular and tissue heterogeneity, yielding significant insights into disease biology and its underlying mechanisms. In this work, we describe icCITE-seq (intracellular cellular indexing of transcriptomes and epitopes), a scalable method that simultaneously measures surface and intracellular protein levels alongside gene expression across thousands of cells. We validate the specificity of intracellular staining and demonstrate the utility of this multi-omic approach in interrogating phenotypic cellular states through targeted genetic perturbations in primary human T cells. icCITE-seq enables systematic profiling of gene expression, coupled with cytoplasmic, nuclear and PTM epitopes, providing an integrated approach towards understanding cellular identity, complexity and disease regulatory mechanisms.

immunology↗

bioRxiv2024

Age-Related Patterns of DNA Methylation Changes

Epigenetic clocks have achieved significant success in aging research, but they often assume linear methylation changes with age and lack biological interpretability. Using data from 4,641 samples across 23 GEO datasets, we analyzed 1,557 CpGs from nine widely used clocks with minimal overlap, and identified consistent age-associated methylation patterns. We then identified 19,432 age-associated CpGs (aaCpGs) that were strongly correlated with age and showed high consistency between sexes, with faster methylation changes observed in males. Most aaCpGs were identified during early and late life stages, indicating accelerated epigenetic changes during development and aging. No specific genomic enrichment was observed. Clustering analysis revealed four distinct, non-linear age-related methylation trajectories. These findings underscore the complexity of epigenetic aging and suggest that current clocks may overlook important dynamic patterns, particularly after age 65. Incorporating these insights could improve the accuracy and biological relevance of future epigenetic clocks, especially for use across diverse age ranges and populations.

molecular biology↗

bioRxiv2024

Decoding RNA Metabolism by RNA-linked CRISPR Screening in Human Cells

RNAs undergo a complex choreography of metabolic processes in human cells that are regulated by thousands of RNA-associated proteins. While the effects of individual RNA-associated proteins on RNA metabolism have been extensively characterized, the full complement of regulators for most RNA metabolic events remain unknown. Here we present a massively parallel RNA-linked CRISPR (ReLiC) screening approach to measure the responses of diverse RNA metabolic events to knockout of 2,092 human genes encoding all known RNA-associated proteins. ReLiC screens highlight modular interactions between gene networks regulating splicing, translation, and decay of mRNAs. When combined with biochemical fractionation of polysomes, ReLiC reveals striking pathway-specific coupling between growth fitness and mRNA translation. Perturbing different components of the translation and proteostasis machineries have distinct effects on ribosome occupancy, while perturbing mRNA transcription leaves ribosome occupancy largely intact. Isoform-selective ReLiC screens capture differential regulation of intron retention and exon skipping by SF3b complex subunits. Chemogenomic screens using ReLiC decipher translational regulators upstream of mRNA decay and uncover a role for the ribosome collision sensor GCN1 during treatment with the anti-leukemic drug homoharringtonine. Our work demonstrates ReLiC as a versatile platform for discovering and dissecting regulatory principles of human RNA metabolism.

molecular biology↗

bioRxiv2024

Specific oncogene activation of the cell of origin in mucosal melanoma

Mucosal melanoma (MM) is a deadly cancer derived from mucosal melanocytes. To test the consequences of MM genetics, we developed a zebrafish model in which all melanocytes experienced CCND1 expression and loss of PTEN and TP53. Surprisingly, melanoma only developed from melanocytes lining internal organs, analogous to the location of patient MM. We found that zebrafish MMs had a unique chromatin landscape from cutaneous melanoma. Internal melanocytes could be labeled using a MM-specific transcriptional enhancer. Normal zebrafish internal melanocytes shared a gene expression signature with MMs. Patient and zebrafish MMs have increased migratory neural crest gene and decreased antigen presentation gene expression, consistent with the increased metastatic behavior and decreased immunotherapy sensitivity of MM. Our work suggests the cell state of the originating melanocyte influences the behavior of derived melanomas. Our animal model phenotypically and transcriptionally mimics patient tumors, allowing this model to be used for MM therapeutic discovery.

cancer biology↗

bioRxiv2023

Massively parallel identification of sequence motifs triggering ribosome-associated mRNA quality control

Decay of mRNAs can be triggered by ribosome slowdown at stretches of rare codons or positively charged amino acids. However, the full diversity of sequences that trigger co-translational mRNA decay is poorly understood. To comprehensively identify sequence motifs that trigger mRNA decay, we use a massively parallel reporter assay to measure the effect of all possible combinations of codon pairs on mRNA levels in S. cerevisiae. In addition to known mRNA-destabilizing sequences, we identify several dipeptide repeats whose translation reduces mRNA levels. These include combinations of positively charged and bulky residues, as well as proline-glycine and proline-aspartate dipeptide repeats. Genetic deletion of the ribosome collision sensor Hel2 rescues the mRNA effects of these motifs, suggesting that they trigger ribosome slowdown and activate the ribosome-associated quality control (RQC) pathway. Deep mutational scanning of an mRNA-destabilizing dipeptide repeat reveals a complex interplay between the charge, bulkiness, and location of amino acid residues in conferring mRNA instability. Finally, we show that the mRNA effects of codon pairs are predictive of the effects of endogenous sequences. Our work highlights the complexity of sequence motifs driving co-translational mRNA decay in eukaryotes, and presents a high throughput approach to dissect their requirements at the codon level.

molecular biology↗

bioRxiv2020

Scalable, multimodal profiling of chromatin accessibility and protein levels in single cells

Recent technological advances have enabled massively parallel chromatin profiling with single-cell Assay for Transposase Accessible Chromatin by sequencing (scATAC-seq) in thousands of individual cells. Here, we extend these approaches and present ATAC with Select Antigen Profiling by sequencing, ASAP-seq, a tool to simultaneously profile accessible chromatin and protein levels in thousands of single cells. Our approach pairs sparse scATAC-seq data with robust detection of hundreds of cell surface and intracellular protein markers and optional capture of mitochondrial DNA (mtDNA) for clonal tracking, thus concomitantly capturing three distinct modalities in single cells. Importantly, ASAP-seq uses a novel bridging approach that repurposes antibody:oligo conjugates designed for existing technologies that pair protein measurements with single cell RNA-seq. We demonstrate the utility of ASAP-seq by revealing coordinated and distinct changes in chromatin, RNA, and surface proteins during native hematopoietic differentiation, peripheral blood mononuclear cell stimulation, and as a combinatorial decoder and reporter of multiplexed perturbations in primary T cells.

genomics↗

bioRxiv2020

Robust T-cell signaling triggered on soft polydimethylsiloxane-supported lipid-bilayers

The T-cell receptor (TCR) is thought to be triggered either by mechano-transduction or local tyrosine phosphatase exclusion at cell-cell contacts. However, the effects of the mechanical properties of activating surfaces have only been tested for late-stage T-cell activation, and phosphatase segregation has mostly been studied on glass-supported lipid bilayers that favor imaging but are orders-of-magnitude stiffer than typical cells. We developed a method for attaching lipid bilayers to polydimethylsiloxane polymer supports, producing soft bilayers with physiological levels of mechanical resistance (Youngs modulus of 4 kPa). Comparisons of T-cell behavior on soft and glass-supported bilayers revealed that early calcium signaling is unaffected by substrate rigidity, implying that early steps in TCR triggering are not mechanosensitive. Robust phosphatase exclusion was observed on the soft bilayers, however, suggesting it likely occurs at cell-cell contacts. This work sets the stage for an imaging-based exploration of receptor signaling under conditions closely mimicking physiological cell-cell contact.

biophysics↗

bioRxiv2020

Neural correlates of newsvendor-based decision making in the human brain: An exploratory study to link neuroeconomics with neuroimaging using fNIRS

Neuroeconomics with neuroimaging is a novel approach involving economics and neuroscience. The newsvendor problem (NP) is a prevalent economics concept that may be used to map brain activations during NP-evoked risky decision making. In this study, we hypothesized that key brain regions responsible for NP are dorsolateral prefrontal cortex (DLPFC) and orbitofrontal cortex (OFC). Twenty-seven human subjects participated in the study using 40 NP trials; the participants were randomly assigned to a group with a low-profit margin (LM) or high-profit margin (HM) treatment. Cerebral hemodynamic responses were recorded simultaneously during the NP experiments from all participants with a 77-channel functional Near-infrared Spectroscopy (fNIRS) system. After data preprocessing, general linear model was applied to generate brain activation maps, followed by statistical t-tests. The results showed that: (a) DLPFC and OFC were significantly evoked by NP versus baseline regardless of treatment types; (b) DLPFC and OFC were activated by HM versus baseline; and (c) DLPFC was activated during LM versus baseline. Furthermore, significant deactivation in right DLPFC was shown due to LM with respect to HM. This study affirms that DLPFC and OFC are two key cortical regions when solving NP. In particular, right DLPFC was found to be more deactivated under challenging risk decision making.

neuroscience↗

bioRxiv2019

CDK13 Mutations Drive Melanoma via Accumulation of Prematurely Terminated Transcripts

Transcriptional Cyclin Dependent Kinases modulate RNA Polymerase II function to impact gene expression. Here, we show that CDK13 is mutated in 4% of patient melanomas and mutation or downregulation is associated with poor overall survival. Mutant CDK13 lacks kinase activity and overexpression in zebrafish leads to accelerated melanoma. CDK13 mutant fish and human melanomas accumulate prematurely terminated RNAs that are translated into truncated proteins. CDK13 binds to and regulates the phosphorylation of ZC3H14, a member of the PolyA eXosome Targeting (PAXT) RNA degradation complex. ZC3H14 phosphorylation recruits the PAXT complex to degrade prematurely terminated polyadenylated transcripts in the nucleus. In the presence of mutant CDK13, ZC3H14 phosphorylation is compromised and consequently fails to recruit the PAXT complex, leading to truncated transcript stabilization. This work establishes a role for CDK13 and the PAXT nuclear RNA degradation complex in cancer and has prognostic significance for melanoma patients with mutated or downregulated CDK13.

biophysics↗

bioRxiv2019

Nanoaperture fabrication via colloidal lithography for single molecule fluorescence imaging

In single molecule fluorescence studies, background emission from labeled substrates often restricts their concentrations to non-physiological nanomolar values. One approach to address this challenge is the use of zero-mode waveguides (ZMWs), nanoscale holes in a thin metal film that physically and optically confine the observation volume allowing much higher concentrations of fluorescent substrates. Standard fabrication of ZMWs utilizes slow and costly E-beam nano-lithography. Herein, ZMWs are made using a self-assembled mask of polystyrene microspheres, enabling fabrication of thousands of ZMWs in parallel without sophisticated equipment. Polystyrene 1 m dia. microbeads self-assemble on a glass slide into a hexagonal array, forming a mask for the deposition of metallic posts in the inter-bead interstices. The width of those interstices (and subsequent posts) is adjusted within 100-300 nm by partially fusing the beads at the polystyrene glass transition temperature. The beads are dissolved in toluene, aluminum or gold cladding is deposited around the posts, and those are dissolved, leaving behind an array ZMWs. Parameter optimization and the performance of the ZMWs are presented. By using colloidal self-assembly, typical laboratories can make use of sub-wavelength ZMW technology avoiding the availability and expense of sophisticated clean-room environments and equipment.

biophysics↗

bioRxiv2018

Stochastic Simulation to Visualize Gene Expression and Error Correction in Living Cells

Stochastic simulation can make the molecular processes of cellular control more vivid than the traditional differential-equation approach by generating typical system histories instead of just statistical measures such as the mean and variance of a population. Simple simulations are now easy for students to construct from scratch, that is, without recourse to black-box packages. In some cases, their results can also be compared directly to single-molecule experimental data. After introducing the stochastic simulation algorithm, this article gives two case studies, involving gene expression and error correction, respectively. Code samples and resulting animations showing results are given in the online supplement.

biophysics↗

bioRxiv2018

Potential Bias of Doubly Labeled Water for Measuring Energy Expenditure Differences Between Diets Varying in Carbohydrate

BackgroundVery low-carbohydrate diets have been reported to substantially increase human energy expenditure as measured by doubly labeled water (DLW) but not by respiratory chambers. Do the DLW data reflect true physiological differences that are undetected by respiratory chambers? Alternatively, are the apparent DLW energy expenditure a consequence of failure to fully account for respiratory quotient (RQ) differences between diets?\n\nObjectiveTo examine energy expenditure differences between diets varying drastically in carbohydrate and to quantitatively compare DLW data with respiratory chamber and body composition measurements within an energy balance framework.\n\nDesignDLW measurements were obtained during the final two weeks of month-long baseline (BD; 50% carbohydrate, 35% fat, 15% protein) and isocaloric ketogenic diets (KD; 5% carbohydrate, 80% fat, 15% protein) in 17 men with BMI 25-35 kg/m2. Subjects resided 2d/week in respiratory chambers to measure energy expenditure (EEchamber). DLW expenditure was calculated using chamber-determined respiratory quotients (RQ) either unadjusted (EEDLW) or adjusted (EEDLW{Delta}RQ) for net energy imbalance using diet-specific coefficients. Accelerometers measured physical activity. Body composition changes were measured by dual-energy X-ray absorptiometry which were combined with energy intake measurements to calculate energy expenditure by balance (EEbal).\n\nResultsAfter transitioning from BD to KD, neither EEchamber nor EEbal were significantly changed ({triangleup}EEchamber=24{+/-}30 kcal/d; p=0.43 and {triangleup}EEbal=-141{+/-}118 kcal/d; p=0.25). Similarly, physical activity (-5.1{+/-}4.8%; p=0.3) and exercise efficiency (-1.6{+/-}2.4%; p=0.52) were not significantly changed. However, EEDLW was 209{+/-}83 kcal/d higher during the KD (p=0.023) but was not significantly increased when adjusted for energy balance (EEDLW{Delta}RQ =139{+/-}89 kcal/d; p=0.14). After removing 2 outliers whose EEDLW were incompatible with other data, EEDLW and EEDLW{triangleup}RQ were marginally increased during the KD by 126{+/-}62 kcal/d (p=0.063) and 46{+/-}65 kcal/d (p=0.49), respectively.\n\nConclusionsDLW calculations failing to account for diet-specific energy imbalance effects on RQ erroneously suggest that very low carbohydrate diets substantially increase energy expenditure.

physiology↗

bioRxiv2016

Millstone: Software for Multiplex Microbial Genome Analysis and Engineering

Inexpensive DNA sequencing and advances in genome editing have made computational analysis a major rate-limiting step in adaptive laboratory evolution and microbial genome engineering. We describe Millstone, a web-based platform which automates genotype comparison and visualization for projects with up to hundreds of genomic samples. To enable iterative genome engineering, Millstone allows users to design oligonucleotide libraries and create successive versions of reference genomes. Millstone is open source and easily deployable to a cloud platform, local cluster, or desktop, making it a scalable solution for any lab.

synthetic biology↗