Biology subjects

Huynh, C.

Publications and source records attributed to Huynh, C..

13 recordsLinked to original sources

bioRxiv2026

Multi-scale dissection, compaction and derivatization of mammalian developmental enhancers

Gene expression during mammalian development is orchestrated by non-coding cis-regulatory DNA elements (CREs) such as distal enhancers1-3. Despite their fundamental importance, and notwithstanding recent progress in predictive modeling4-9, many high-level properties of enhancer grammar remain unresolved. How does the length of an autonomously active CRE constrain its activity? How robust are CREs to mutations or rearrangements of transcription factor binding sites (TFBSs)? And how much epistasis exists among these sites? As predictive models solely trained on endogenous CREs are unlikely to resolve these questions10, we subjected several endogenous CREs to intensive sequence-level perturbation. Specifically, we assayed >35,000 variants of 5 parietal endoderm enhancers, with variants organized into four perturbation classes, designed to probe: (i) the functional sufficiency of sub-fragments via dense multi-size tiling, (ii) local epistasis via multi-hit saturation mutagenesis, (iii) activity-size tradeoffs via model-guided compaction, or (iv) functional resilience via sequence derivatization anchored on key TFBSs, including random deposition, reconstitution, and synthetic thripsis. This multi-scale dissection revealed rich phenomena. Sub-tiling uncovered sharp non-additivity between activity and fragment size, highlighting strongly synergistic TFBS clusters. Compaction showed that natural CREs lie far from the activity-size Pareto front, and that model-guided deletions can yield shorter yet stronger elements. Mutational scanning exposed a spectrum of CRE robustness, from tolerant to fragile, together with rare but consequential epistasis between individual TFBSs. Finally, TFBS-anchored derivatization demonstrated that background sequence can influence activity on par with TFBS arrangement. Strikingly, a substantial fraction of CRE derivatives exceeded the activity of their endogenous progenitors. Taken together, these results reveal both soft and stiff directions in regulatory sequence space, advancing a quantitative phenomenology of how enhancer sequences encode function and robustness.

genomics↗

bioRxiv2025

The Association Between SLIT2 in Human Vitreous Humor and Plasma and Neurocognitive Test Scores

BackgroundSlit Guidance Ligand 2 (SLIT2) binds Roundabout (ROBO) guidance receptors to direct axon pathfinding and neuron migration during nervous system development. SLIT2 expression has previously been linked to dementia risk. ObjectiveTo study the association between SLIT2 expression in human vitreous humor and plasma samples and neurocognitive test scores in a cross-sectional cohort study utilizing a novel, highly-sensitive Meso Scale Discovery (MSD) assay for SLIT2 detection. MethodsSeventy-nine individuals with a mean age of 55.79 {+/-} 12.03 years underwent eye surgery with collection of vitreous humor, blood (plasma) collection, and neurocognitive assessment. Vitreous humor and plasma samples were analyzed by SLIT2 MSD electrochemiluminescence immunoassay. Associations between SLIT2 levels in vitreous humor and plasma were analyzed using GraphPad Prism. ResultsWe found up to a 7-fold higher level of SLIT2 in human vitreous humor compared to plasma. Lower vitreous SLIT2 levels were associated with a lower Montreal Cognitive Assessment (MoCA) score and Immediate Recall Verbatim (IRV) z-score, and higher plasma SLIT2 was associated with a lower MoCA score. In multivariate analysis using single and multiple predictor models, the same significant associations were found when adjusted for age, sex, race, diabetic status, diabetic retinopathy status, glaucoma status, and Apolipoprotein E (APOE) genotype. ConclusionsSLIT2 protein levels are significantly associated with MoCA score and IRV z-score in middle-aged individuals. The relationship remained significant when adjusted for demographics, co-morbidity, and APOE genotype, suggesting SLIT2 may be a sensitive biomarker for detection of mild cognitive impairment and early dementia, and warrants further studies.

neuroscience↗

bioRxiv2025

Structural elucidation of the haptoglobin-hemoglobin clearance mechanism by macrophage scavenger receptor CD163

Intravascular hemolysis releases hemoglobin into the bloodstream, which can damage vascular and renal tissues due to its oxidative nature. Circulating haptoglobin acts as a primary defense by binding to free hemoglobin, forming a haptoglobin-hemoglobin (HpHb) complex that is then recognized and cleared by the CD163 scavenger receptor on macrophages. While the function and structure of HpHb complex are mostly well-defined, the molecular mechanism underlying its interaction with CD163 remains unclear. Here we report the cryo-electron microscopy structures of CD163 in its unliganded state and in its complex with HpHb. These structures reveal that CD163 functions as a trimer, forming a composite binding site at its center for one protomer of the dimeric HpHb, resulting in a 3:1 binding stoichiometry. In the unliganded state, CD163 can also form a trimer, but in an autoinhibitory configuration that occludes the ligand binding site. Widespread electrostatic interactions mediated by calcium ions are pivotal in both pre-ligand and ligand-bound receptor assemblies. This calcium-dependent mechanism enables CD163/HpHb complexes to assemble and, once internalized, disassemble into individual components upon reaching the endosome, where low calcium and lower pH conditions prevail. Collectively, this study elucidates the molecular mechanism by which CD163-mediated endocytosis efficiently clears different isoforms of HpHb.

biophysics↗

bioRxiv2025

Extensive length and homology dependent chimerism in pool-packaged AAV libraries

Adeno-associated viruses (AAVs) have emerged as the foremost gene therapy delivery vehicles due to their versatility, durability, and safety profile. Here we demonstrate extensive chimerism, manifesting as pervasive barcode swapping, among complex AAV libraries that are packaged as a pool. The observed chimerism is length- and homology-dependent but capsid-independent, in some cases affecting the majority of packaged AAV genomes. These results have implications for the design and deployment of functional AAV libraries in both research and clinical settings.

genomics↗

bioRxiv2024

ZMYND11 Functions in Bimodal Regulation of Latent Genes and Brain-like Splicing to Safeguard Corticogenesis

Despite the litany of pathogenic variants linked to neurodevelopmental disorders (NDD) including autism (ASD) and intellectual disability1,2, our understanding of the underlying mechanisms caused by risk genes remain unclear. Here, we leveraged a human pluripotent stem cell model to uncover the neurodevelopmental consequences of mutations in ZMYND11, a newly implicated risk gene3,4. ZMYND11, known for its tumor suppressor function, encodes a histone-reader that recognizes sites of transcriptional elongation and acts as a co-repressor5,6. Our findings reveal that ZMYND11-deficient cortical neural stem cells showed upregulation of latent developmental pathways, impairing progenitor and neuron production. In addition to its role on histones, ZMYND11 controls a brain-specific isoform switch involving the splicing regulator RBFOX2. Extending our findings to other chromatin-related ASD risk factors revealed similar developmental pathway activation and splicing dysregulation, partially rescuable through ZMYND11s regulatory functions.

neuroscience↗

bioRxiv2024

Lineage-resolved analysis of embryonic gene expression evolution in C. elegans and C. briggsae

What constraints govern the evolution of gene expression patterns across development remains a fundamental question of evolutionary biology. The advent of single-cell sequencing opens the possibility of learning these constraints by systematically profiling homologous cells across different organisms. The nematode C. elegans is a well-studied model for embryonic development, and its invariant lineage that is conserved with other Caenorhabditis species makes it an ideal model to directly compare gene expression between homologous progenitor and terminal cell types across evolution. We have measured the spatiotemporal divergence of gene expression across embryogenesis by collecting, annotating, and comparing the transcriptomes of homologous embryonic progenitors and terminal cell types, using a dataset comprising >200,000 C. elegans cells and >190,000 C. briggsae cells. We find a high level of similarity in gene expression programs between the species despite tens of millions of years of evolutionary divergence, consistent with their conserved developmental lineages. Even still, thousands of genes show divergence in their cell-type specific expression patterns, and these are enriched for categories involved in environmental response and behavior. Comparing the degree of expression conservation across cell types reveals that certain cell types such as neurons, have diverged more than others such as the intestine and body wall muscle. Taken together, this work identifies likely constraints on the evolution of developmental gene expression and provides a powerful resource for addressing diverse evolutionary questions.

evolutionary biology↗

bioRxiv2024

Binding profiles for 954 Drosophila and C. elegans transcription factors revealtissue specific regulatory relationships

A catalog of transcription factor (TF) binding sites in the genome is critical for deciphering regulatory relationships. Here we present the culmination of the modERN (model organism Encyclopedia of Regulatory Networks) consortium that systematically assayed TF binding events in vivo in two major model organisms, Drosophila melanogaster (fly) and Caenorhabditis elegans (worm). We describe key features of these datasets, comprising 604 TFs identifying 3.6M sites in the fly and 350 TFs identifying 0.9 M sites in the worm. Applying a machine learning model to these data identifies sets of TFs with a prominent role in promoting target gene expression in specific cell types. TF binding data are available through the ENCODE Data Coordinating Center and at https://epic.gs.washington.edu/modERNresource, which provides access to processed and summary data, as well as widgets to probe cell type-specific TF-target relationships. These data are a rich resource that should fuel investigations into TF function during development.

genomics↗

bioRxiv2023

Advancement in Cellular Topographic and Nanoparticle Capture Imaging by High Resolution Microscopy Incorporating a Freeze-Drying and Gaseous Nitrogen-based Approach

Scanning electron microscopy (SEM) offers an unparalleled view of the membrane topography of mammalian cells by using a conventional osmium (OsO4) and ethanol-based tissue preparation. However, conventional SEM methods limit optimal resolution due to ethanol and lipid interactions and interfere with visualization of fluorescent reporter proteins. Therefore, SEM correlative light and electron microscopy (CLEM) has been hindered by the adverse effects of ethanol and OsO4 on retention of fluorescence signals. To overcome this technological gap in achieving high-resolution SEM and retain fluorescent reporter signals, we developed a freeze-drying method with gaseous nitrogen (FDGN). We demonstrate that FDGN preserves cyto-architecture to allow visualization of detailed membrane topography while retaining fluorescent signals and that FDGN processing can be used in conjunction with a variety of high-resolution imaging systems to enable collection and validation of unique, high-quality data from these approaches. In particular, we show that FDGN coupled with high resolution microscopy provided detailed insight into viral or tumor-derived extracellular vesicle (TEV)-host cell interactions and may aid in designing new approaches to intervene during viral infection or to harness TEVs as therapeutic agents.

cell biology↗

bioRxiv2022

Whole-body gene expression atlas of an adult metazoan

Animals are integrated organ systems composed of interacting cells whose structure and function are in turn defined by their active genes. Understanding what distinguishes physiological and disease states therefore requires systemic knowledge of the gene activities that define the distinct cells that make up an animal. Towards this goal, this study reports the first single-cell resolution transcriptional atlas of a fertile multicellular organism: Caenorhabditis elegans. The scRNA-Seq compendium of wild-type young adult C. elegans comprises 159 distinct cell types with 18,033 genes expressed across cell types. Fewer than 300 of these genes are housekeeping genes as evidenced by their consistent expression across cell types and conditions, and by their basic and essential functions; 170 of these housekeeping genes are conserved across phyla. The 362 transcription factors with available ChIP-Seq data are linked to patterns of gene expression of different cell types. To identify potential interactions between cell types, we used the in silico tool cell2cell to predict molecular patterns reflecting both known and uncharacterized intercellular interactions across the C. elegans body. Finally, we present WormSeq (wormseq.org), a web interface that, among other functions, enables users to query gene expression across cell types, identify cell-type specific and potential housekeeping genes, analyze candidate ligand-receptors mediating communication between cells, and study promiscuous and cell-specific transcription factors. The datasets, analyses, and tools presented here will enable the generation of testable hypotheses about the cell and organ-specific function of genes in diverse biological contexts.

genomics↗

bioRxiv2022

A Tm4sf1-Marked Subpopulation of Endothelial Stem/Progenitor Cells Identified by Lung Single-Cell Omics of Pulmonary Arterial Hypertension

RationaleThe identification and role of endothelial progenitor cells (EPCs) in pulmonary arterial hypertension (PAH) remains controversial. Single-cell omics analysis can shed light on EPCs and their potential contribution to PAH pathobiology. ObjectivesWe aim to identify EPCs in rat lungs and assess their relevance to preclinical and human PAH. MethodsDifferential expression, gene set enrichment, cell-cell communication, and trajectory reconstruction analyses were performed on lung endothelial cells from single-cell RNA-seq of Sugen-hypoxia, monocrotaline, and control rats. Relevance to human PAH was assessed in multiple independent blood and lung transcriptomic datasets. Measurements and Main ResultsA subpopulation of endothelial cells (EA2) marked by Tm4sf1, a gene strongly implicated in cancer, harbored a distinct transcriptomic signature including Bmpr2 downregulation that was enriched for pathways such as inflammation and angiogenesis. Cell-to-cell communication networks specific to EA2 were activated in PAH such as CXCL12 signaling. Trajectory analysis demonstrated EA2 has a stem/progenitor cell phenotype. Analysis of independent datasets revealed Tm4sf1 is a marker for hematopoietic stem cells and is upregulated in PAH peripheral blood, particularly in patients with worse WHO functional class. EA2 signature genes including Procr and Sulf1 were found to be differentially regulated in the lungs of PAH patients and in PAH models in vitro, such as BMPR2 knockdown. ConclusionsOur study uncovered a novel Tm4sf1-marked stem/progenitor subpopulation of rat lung endothelial cells and demonstrated its relevance to preclinical and human PAH. Future experimental studies are warranted to further elucidate the pathogenic role and therapeutic potential of targeting EA2 and Tm4sf1 in PAH.

genomics↗

bioRxiv2021

Maternal diet disrupts the placenta-brain axis in a sex-specific manner

High maternal weight is associated with a number of detrimental outcomes in offspring, including increased susceptibility to neurological disorders such as anxiety, depression, and communicative disorders (e.g. autism spectrum disorders)1-8. Despite widespread acknowledgement of sex-biases in the prevalence, incidence, and age of onset of these disorders, few studies have investigated potential sex-biased mechanisms underlying disorder susceptibility. Here, we use a mouse model to demonstrate how maternal high-fat diet, one contributor to overweight, causes endotoxin accumulation in fetal tissue, and subsequent perinatal inflammation influences sex-specific behavioral outcomes in offspring. In male high-fat diet offspring, increased macrophage toll like receptor 4 signaling results in excess phagocytosis of serotonin neurons in the developing dorsal raphe nucleus, decreasing serotonin bioavailability in the fetal and adult brain. Bulk sequencing from a large cohort of matched first trimester human fetal brain, placenta, and maternal decidua samples reveals sex-specific transcriptome-wide changes in placenta and brain tissue in response to maternal triglyceride accumulation (a proxy for dietary fat content). Further, we find that fetal brain serotonin levels decrease as maternal dietary fat intake increases in males only. These findings uncover a microglia-dependent mechanism through which maternal diet may impact offspring susceptibility for neuropsychiatric disorder development in a sex-specific manner.

neuroscience↗

bioRxiv2019

A lineage-resolved molecular atlas of C. elegans embryogenesis at single cell resolution

C. elegans is an animal with few cells, but a striking diversity of cell types. Here, we characterize the molecular basis for their specification by profiling the transcriptomes of 84,625 single embryonic cells. We identify 284 terminal and pre-terminal cell types, mapping most single cell transcriptomes to their exact position in C. elegans invariant lineage. We use these annotations to perform the first quantitative analysis of the relationship between lineage and the transcriptome for a whole organism. We find that a strong lineage-transcriptome correlation in the early embryo breaks down in the final two cell divisions as cells adopt their terminal fates and that most distinct lineages that produce the same anatomical cell type converge to a homogenous transcriptomic state. Users can explore our data with a graphical application "VisCello".

genomics↗

bioRxiv2017

Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing

Conventional methods for profiling the molecular content of biological samples fail to resolve heterogeneity that is present at the level of single cells. In the past few years, single cell RNA sequencing has emerged as a powerful strategy for overcoming this challenge. However, its adoption has been limited by a paucity of methods that are at once simple to implement and cost effective to scale massively. Here, we describe a combinatorial indexing strategy to profile the transcriptomes of large numbers of single cells or single nuclei without requiring the physical isolation of each cell (Single cell Combinatorial Indexing RNA-seq or sci-RNA-seq). We show that sci-RNA-seq can be used to efficiently profile the transcriptomes of tens-of-thousands of single cells per experiment, and demonstrate that we can stratify cell types from these data. Key advantages of sci-RNA-seq over contemporary alternatives such as droplet-based single cell RNA-seq include sublinear cost scaling, a reliance on widely available reagents and equipment, the ability to concurrently process many samples within a single workflow, compatibility with methanol fixation of cells, cell capture based on DNA content rather than cell size, and the flexibility to profile either cells or nuclei. As a demonstration of sci-RNA-seq, we profile the transcriptomes of 42,035 single cells from C. elegans at the L2 stage, effectively 50-fold \"shotgun cellular coverage\" of the somatic cell composition of this organism at this stage. We identify 27 distinct cell types, including rare cell types such as the two distal tip cells of the developing gonad, estimate consensus expression profiles and define cell-type specific and selective genes. Given that C. elegans is the only organism with a fully mapped cellular lineage, these data represent a rich resource for future methods aimed at defining cell types and states. They will advance our understanding of developmental biology, and constitute a major step towards a comprehensive, single-cell molecular atlas of a whole animal.

genomics↗