Biology subjects

Kim, S. H.

Publications and source records attributed to Kim, S. H..

At least 19 recordsLinked to original sources

bioRxiv2026

Microbead-Based Heterodimer Assay for Amplification-Free Detection of Single-Stranded DNA

Direct detection of rare nucleic-acid targets without molecular amplification requires specific recognition events to be made observable while remaining distinguishable from nonspecific interactions. Here, we introduce a microbead-based heterodimer assay (MBHA) that converts two-site recognition of single-stranded DNA into spatially resolved, two-color bead-pairing events for direct nucleic acid detection. Two spectrally distinct microbead populations are functionalized with partially double-stranded DNA probes exposing target-binding domains complementary to separate regions of the target. Target-mediated bridging produces heterotypic bead pairs that are classified by fluorescence identity and an interparticle-distance criterion, while residual background is evaluated relative to blank measurements. Under the tested conditions, 166 fM was the lowest target concentration exceeding the blank-derived detection threshold. A sequence-scrambled synthetic non-target DNA remained below threshold across the tested concentration range, and detection at 166 fM was retained in the presence of 1 uM non-target DNA, corresponding to an approximately six-million-fold molar excess. Probe redesign further extended the assay to a second 60-nucleotide synthetic DNA target derived from the SARS-CoV-2 ORF1ab sequence. This proof-of-concept study establishes heterodimer counting as an amplification-free measurement strategy in which rare molecular-recognition events are made directly observable and individually classifiable without increasing target copy number.

biophysics↗

bioRxiv2026

Breaking the link between demyelination and axon loss: SARM1 inhibition as a neuroprotective strategy for multiple sclerosis

Axonal degeneration is a principal driver of irreversible neurological disability in multiple sclerosis (MS), yet current treatments fail to target this neurodegenerative phase directly. Sterile alpha and TIR domain-containing protein 1 (SARM1) has emerged as an executioner of programmed axon destruction and is overexpressed in grey and white matter MS tissue, making its inhibition a promising therapeutic strategy. However, the effects of SARM1 inhibition on localized neurodegeneration versus systemic and central inflammation remain poorly understood in complex autoimmune environments like MS. Here, we investigated SARM1 pathology using global SARM1-/- knockout (SARM1-/-) mice, AAV-mediated CRISPR knockdown and overexpression of SARM1 in retinal ganglion cells (RGCs), and the small-molecule SARM1 inhibitor 5-iodoisoquinoline (5IIQ), across optic nerve crush (ONC) and experimental autoimmune encephalomyelitis (EAE) models. While global SARM1-/- did not alter the overall clinical course of EAE, it revealed a complex phenotype characterized by an altered peripheral inflammatory cytokine profile and persistent CNS immune infiltration, alongside preserved axonal and myelin integrity. RGC-restricted SARM1 knockdown partially preserved RGCs and axons during EAE, whereas SARM1 overexpression worsened both retinal function and axonal injury. Pharmacological inhibition with 5IIQ preserved axonal integrity and restored visual function in both the ONC and EAE models, as confirmed by electrophysiology, and reduced serum neurofilament light chain (NfL) in EAE, without affecting demyelination. Together, these findings demonstrate that SARM1 inhibition uncouples axonal self-destruction from demyelination and gross neuroinflammation while providing robust structural and functional neuroprotection. This study establishes SARM1 as a viable target for neuroprotective co-therapies designed to complement existing immunomodulatory and remyelinating regimens in MS and related neurodegenerative disorders.

neuroscience↗

bioRxiv2026

AI-driven framework modeling perturbation in brain organoids reveals candidate genes for autism

Autism gene discovery is constrained by the rarity and heterogeneity of damaging variants, requiring large cohorts to identify susceptibility genes. Neural organoids and single-cell foundation models enable perturbation modeling in neurodevelopmental contexts. Here, we show that perturbation-informed foundation modeling of neural organoids can provide functional context for prioritizing candidate genes with genomic and clinical support. We constructed a 3.6-million-cell organoid atlas and trained models to predict genome-wide perturbation responses. Benchmarking 17 models identified a telencephalic neuron-specific model best preserving autism-relevant perturbation structure. Genome-wide profiling revealed two clusters associated with mid-fetal synaptic neuronal processes and early radial glia ubiquitin signaling. These clusters were supported by damaging-variant enrichment and clinical phenotypes across 89,916 family-based samples. Logistic-regression prioritization identified 343 candidates, including 167 in the key clusters, with convergence across TADA signals and recurrent evidence for NBEA and KLHDC10. This framework integrates predicted perturbation effects with genomic evidence to support autism candidate prioritization.

bioinformatics↗

bioRxiv2026

Seven mutations unlock strict synthetic methylotrophy in engineered Pseudomonas putida

Methanol is a reduced, soluble one-carbon (C1) feedstock for sustainable bioproduction, but converting this potential into robust microbial growth remains difficult. Several synthetic C1 assimilation routes depend on autocatalytic cycles, whose operation requires coordinated control of redox balance, toxic intermediates, substrate regeneration, and host regulation. Here, we implemented the serine-threonine cycle (STC) in the soil bacterium Pseudomonas putida and used growth-coupled selection with adaptive laboratory evolution (ALE) to transition from mixotrophic C1 incorporation to strict methylotrophy. The evolved strain grew with methanol as the sole carbon and energy source under atmospheric CO2 with a doubling time of ca. 40 h. Whole-genome sequencing, reverse genetics, biosensors, isotope labelling, and comparative RNA sequencing showed that evolution repeatedly targeted native pyrroloquinoline quinone (PQQ)-dependent methanol oxidation, membrane-bound transhydrogenase activity, glycine regeneration, STC enzyme balance, and global regulatory nodes. Additional ALE under glycine-methanol co-feeding increased growth rates and exposed further targets for improving cycle flux. These results establish P. putida as a chassis for strict synthetic methylotrophy and define actionable engineering routes toward C1 biomanufacturing. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/739708v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1642d2dorg.highwire.dtl.DTLVardef@1d203d1org.highwire.dtl.DTLVardef@f8a5d7org.highwire.dtl.DTLVardef@4fe272_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2026

AI-Informed neoantigen prioritization enables a multi-epitope mRNA/LNP vaccine with antigen-specific immunogenicity and antitumor activity

Personalized neoantigen vaccines are an emerging strategy for cancer immunotherapy, but their effectiveness depends on selecting tumor-specific antigens capable of inducing functional T-cell responses. The VACINUS AI-informed neoantigen prioritization framework previously identified and peptide-validated three immunogenic Tier 1 neoantigens in the B16F10 melanoma model. In this study, we extended that framework by translating these validated neoantigens into a multi-epitope messenger RNA vaccine formulated with lipid nanoparticles and evaluating its preclinical immunogenicity and antitumor activity. The three VACINUS-prioritized B16F10 neoantigens were encoded within a single multi-epitope construct, BF-V1_27-Ser, and formulated to generate BF-RNA-P. In B16F10 tumor-bearing mice, BF-RNA-P induced neoantigen-specific CD8+T-cell responses, with the strongest response directed against the B16F10-1-4 epitope. Combination with anti-PD-1 further enhanced vaccine-induced CD44+IFN-{gamma}+ CD8+ T-cell activation, whereas anti-PD-1 alone did not induce detectable peptide-specific responses. BF-RNA-P also suppressed tumor growth in vivo, and combination treatment produced the strongest antitumor effect, reflected by reduced tumor volume and lower endpoint tumor burden. Together, these findings demonstrate that VACINUS-prioritized and peptide-validated neoantigens can be reformatted into a multi-epitope messenger RNA/lipid nanoparticle vaccine while retaining antigen-specific immunogenicity and antitumor activity. This study provides preclinical proof-of-concept for integrating AI-informed, TCR-aware neoantigen prioritization with messenger RNA/lipid nanoparticle delivery as a translational strategy for personalized cancer vaccine development.

immunology↗

bioRxiv2026

Cysteine-mediated bortezomib resistance is governed by α-ketoacid availability

Dissecting how metabolite availability impacts drug sensitivity is critical for understanding therapy resistance. While proteasome inhibitors like the boronic acid proteasome inhibitor bortezomib are cornerstones of therapy for multiple myeloma, the clinical utility of these drugs is often limited by development of resistance, the metabolic drivers of which remain poorly understood. In this study, we find that -ketoacids, including pyruvate, increase sensitivity to boronic acid proteasome inhibitors independent of their conventional roles in metabolism. Instead, -ketoacids directly react with intracellular cysteine to form thiazolidines, sequestering cysteine away from forming conjugates with boronic acid proteasome inhibitors and detoxifying these drugs. Preventing cysteine-mediated detoxification through -ketoacid supplementation increases the efficacy of boronic acid proteasome inhibitors, leading to greater proteasome inhibition and cytotoxicity that is reversed by cysteine supplementation. These findings suggest that modulating available cysteine through -ketoacid interactions can impact effective levels of some drugs in cells, and represent a potential strategy to overcome resistance and maximize the efficacy of boronic acid proteasome inhibitors.

cancer biology↗

bioRxiv2026

Annual life-history strategy hitchhikes low-light adaptation in a clonal seagrass

While life-history strategies are typically fixed within species, evolutionary transitions between perenniality and annuality can occur. In clonal seagrasses, annual and perennial plants often coexist in the same population, providing a unique model for studying the genetic basis of this transition. Two seagrass Zostera marina populations in South Korea display a striking dichotomy: shallow-water sub-populations follow a typical perennial strategy, whereas their deep-water counterparts are annual. Here we show that this shift from perenniality to annuality, potentially caused by the SAPK7 gene, is genetically coupled with the CAO gene, which is under strong positive selection for low-light adaptation. The up-regulation of the SAPK7 gene triggers early flowering in seedlings, before the formation of any lateral shoots via asexual reproduction. In this special case where the genet contains only one ramet, the post-reproductive death of the ramet is equivalent to the death of the whole genet, which explains the annual phenotype. Our findings reveal a mechanistic example where annuality overcomes perenniality by hitchhiking on a positively selected gene. Given that the ancestral state of plants is perennial, this coupling of annuality with beneficial alleles may represent one of the pathways for the repeated evolution of annual life histories across flowering plants.

evolutionary biology↗

bioRxiv2026

Variation in infant subcortical brain development from 6 to 12 months in Down syndrome

IntroductionDown syndrome (DS), arising from Trisomy 21, is the most common genetic condition associated with intellectual disability. While smaller total brain volumes have been consistently observed in DS, no longitudinal neuroimaging studies have examined volumetric brain development in DS during infancy, a period of rapid neural growth when interventions may have the greatest impact. MethodHigh-resolution T1- and T2-weighted images were acquired during natural sleep in a multisite longitudinal cohort of 44 infants with DS and 39 control infants without DS at ages 6 and 12 months. Neuroimaging data were harmonized to reduce batch effects, and a novel deep-learning, repeated-measures segmentation approach was applied to optimize neuroanatomical segmentations. Total intracranial volume (ICV) and bilateral absolute subcortical volumes (amygdala, caudate, hippocampus, pallidum, putamen, thalamus) were first directly compared in infants with and without DS at 6 and 12 months. Hierarchical linear modeling (HLM) evaluated longitudinal group differences for each structure, accounting for sex, gestational age, and laterality. Subcortical group differences estimated by HLM were also compared to group differences in total ICV. ResultsICV in infants with DS was lower than controls at 6 months (12.6%; p<.001) and 12 months (16.3%; p<.001). Subcortical structures displayed a range of lower volumes (6.9%-13.1%; ps[≤].003) in infants with DS, although the caudate and putamen were exceptions. Caudate volumes were on average lower in DS but not significantly different from controls, while putamen volumes were on average higher in DS but not significantly different from controls, except for the right putamen, which was significantly larger (5.3%; p=.018) at 6 months. In HLM, ICV and all subcortical structures showed slower growth in DS from 6 to 12 months, except for the amygdala and putamen, which displayed similar growth rates to controls. DS-associated reductions in subcortical volumes were similar in magnitude to ICV, although 12-month caudate and 6- and 12-month putamen volumes were enlarged relative to ICV. ConclusionInfants with DS exhibited substantially reduced ICV and widespread reductions in subcortical volumes and growth from 6-12 months. Across a range of volumetric differences, findings were most distinct in the basal ganglia, for which volume reductions were attenuated in the caudate, while the putamen was uniquely enlarged with comparable growth to controls. These observations support early regional specificity in the neural impact of Trisomy 21 and underscore the utility of infant neuroimaging to inform biologically based interventions and clinical trial readiness in DS.

neuroscience↗

bioRxiv2026

Rank dependency of rescaled pruning in recurrent neural networks

Throughout development and maturity, neural circuits undergo massive synaptic pruning, yielding highly sparse connectivity while preserving robust population-level computations. These population dynamics are often low-dimensional, allowing task-related computations to be formalized as trajectories within latent subspaces. How such low-dimensional dynamics are preserved amid widespread network sparsification remains unclear. Here, we investigate how different synaptic pruning rules shape low-dimensional dynamics and task performance in recurrent neural networks (RNNs). Moving beyond previous approaches focused on random sparsification of low-rank networks or networks with strictly constrained structures, we systematically evaluate how biologically motivated pruning rules interact with a networks underlying rank. We show that post-pruning dynamics and task performance depend critically on the networks initial rank due to distinct eigenspectral characteristics across rank regimes. Combining mathematical analysis with simulations, we demonstrate that pruning with synaptic rescaling preserves low-dimensional dynamics with minimal distortion in low-rank RNNs, but degrades in the high-rank regime. Our findings suggest that low-rank structure, combined with homeostatic synaptic rescaling, is essential for maintaining stable, low-dimensional dynamics in sparse networks. Author summaryIn biological neural networks, population activity is fundamentally shaped by synaptic connection strengths. During development, many of these connections are eliminated through synaptic pruning--a process thought to minimize metabolic costs and optimize computational efficiency by promoting network sparsity. However, how networks preserve their core functionalities after losing a vast numbers of connections remains an open question. Here, we combine mathematical analysis and simulations to examine how various pruning strategies affect network dynamics and computation in both low- and high-rank recurrent neural networks (RNNs). We show that uncompensated connection removal drastically alters network activity. However, implementing a homeostatic rescaling that strengthens remaining connections preserves original dynamics exclusively in low-rank networks, where connectivity is governed by a few dominant structural patterns. In contrast, pruning with homeostatic rescaling fails to maintain dynamics in high-rank networks. Our findings suggest that low-rank connectivity coupled with network homeostasis is crucial for maintaining brain function throughout significant developmental pruning.

neuroscience↗

bioRxiv2026

ATR kinase inhibitors induce mitochondrial fission in CD8+ T cells and impair immune memory in vivo

The DNA damage response kinase ATR restrains CDK1 activity during S and G2 phases of the cell cycle, confining CDK1-driven processes to mitosis. ATR kinase inhibitors were originally developed to potentiate chemotherapy-induced DNA damage at stalled replication forks and to disrupt DNA damage-induced cell cycle checkpoints. Recent evidence, however, reveals that these inhibitors also disrupt cell cycle organization in cells that have not sustained any DNA damage. We show that ATR kinase inhibitors potently trigger unscheduled mitochondrial fission, causing loss of mitochondrial mass in actively dividing CD8+ T cells that persists in memory CD8+ T cells. Moreover, ATR inhibition during the peak of CD8+ T cell expansion in a mouse model of LCMV Armstrong infection impairs the formation of immune memory. These findings carry significant clinical implications. ATR kinase inhibitors are currently being evaluated in clinical trials in combination with chemotherapy, radiation, and immune checkpoint inhibitors in patients where anti-tumor immune responses are recognized as a determinant of durable response. Our results identify an unexpected consequence of ATR inhibition that disrupts cellular metabolism with broad implications for both preclinical research and clinical application.

cancer biology↗

bioRxiv2026

Automated extraction of primary cilia-based biomarkers reveals ageing of cells.

We present an automated image-analysis methodology for quantitative assessment of primary cilia in cultured human primary fibroblasts. The proposed approach implements a modular processing pipeline combining deep-learning based nuclear segmentation with intensity-driven segmentation of the ciliary axoneme and basal body. These partial segmentations are integrated using a distance-based association strategy, enabling automated reconstruction of individual cilia and subsequent extraction of geometrical features. Performance was evaluated against manually segmented ground truth. While manual annotations showed a systematic underestimation of cilia length, both automated and manual analyses produced consistent population-level trends and identical statistical discrimination across sample groups. Application of the pipeline revealed a progressive reduction in cilia length and ciliation frequency with increasing passage number, demonstrating the sensitivity of the method to subtle morphological changes. The proposed framework enables scalable, reproducible, and objective quantification of primary cilia morphology and provides a computational tool applicable to high-throughput studies of cellular ageing and related phenotypes. All the code related to this work is available through GitHub: https://github.com/reyesaldasoro/Cilia.

cell biology↗

bioRxiv2026

A sensor of oxidative stress confers virulence via response memory in Acinetobacter baumannii

All living organisms must adapt to environmental stresses to survive. The two-component system (TCS) is a prevalent signal transduction mechanism to alter gene expression in response to stress in bacteria. We report that the PmrB sensor of the PmrA/PmrB TCS senses sublethal oxidative stress and encodes a response memory that promotes virulence in Acinetobacter baumannii. PmrB detects oxidative stress through histidine residues with a nickel (Ni2+) cofactor. Nickel oxidation (Ni2+) induces an allosteric effect by altering PmrB conformation, enabling PmrA activation. Activated PmrA induces antioxidant defense genes, including iron-sulfur cluster repair, ferritin, catalase, and peroxidase. Notably, PmrB remains activated after sublethal oxidative stress is removed, as pathogens encounter in the bloodstream and airways during infection. This response memory enables bacteria to respond strongly to subsequent lethal oxidative stress and antimicrobial peptides. In a murine model, disrupting oxidative sensing reduces virulence. In addition, PmrB-mediated response memory is essential for high virulence in clinically isolated multidrug-resistant A. baumannii. Our findings demonstrate how subinhibitory stress detection enables pathogens to achieve full virulence potential and resist stresses during infection.

microbiology↗

bioRxiv2026

AUTOTAC-mediated targeted degradation of transthyretin aggregates ameliorates hereditary transthyretin amyloidosis

Hereditary transthyretin amyloidosis (hATTR) is characterized by extracellular deposition of amyloidogenic transthyretin (TTR) aggregates, yet the mechanisms governing their clearance remains poorly understood. Here, we identify a key role for the N-degron pathway in lysosomal degradation of the pathogenic TTRV30M variant. Misfolded intracellular TTRV30M was rapidly secreted and subsequently re-entered within 24 hours during cell-to-cell trafficking. The molecular chaperone R-BiP--N-terminally (Nt) arginylated HSPA5/BiP/GRP78-- associated with intracellular TTRV30M, and its Nt-arginine functioned as an agonist for the N-recognin sequestosome 1 (SQSTM1/p62). This interaction facilitated p62-dependent autophagosomal sequestration and lysosomal degradation of TTRV30M. To pharmacologically exploit this mechanism, we applied the AUTOTAC (AUTOphagy-TArgeting Chimera) platform, which enables the targeting of substrates to p62 for autophagic clearance. We developed Autotac 201 (ATC201), an 876-Da chimera designed to bind both the T4 pocket of aggregated TTR and p62, thereby promoting selective autophagic degradation. In cultured cells, ATC201 potently reduced intracellular TTRV30M aggregates in a manner depending on p62-mediated autophagy, exhibiting a DC of low nM. In hATTR model mice, ATC201 markedly lowered tissue TTR aggregate burden and restored autophagy pathway flux impaired by aggregate accumulation. Treatment improved nerve conduction parameters and reduced peripheral neuropathy scores, indicating functional rescue. ATC201 also led to preservation of muscle strength and attenuation of systemic amyloid deposition. Our findings reveal that the N-degron pathway orchestrates autophagic removal of TTR aggregates and demonstrate the therapeutic potential of AUTOTAC-based degraders for hATTR and other proteinopathies characterized by pathogenic protein aggregation.

cell biology↗

bioRxiv2026

Sensitive and Selective Next-Generation FRET-based PKA Biosensors

The cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway regulates diverse cellular processes through precise spatiotemporal control across subcellular compartments. Forster resonance energy transfer (FRET)-based A-kinase activity reporters (AKARs) have enabled live-cell visualization of PKA activity, but their limited dynamic range constrains detection of subtle or compartment-specific signaling events. Here, we present a suite of sixth-generation cyan/yellow FRET-based PKA sensors (the AKAR6 series) with substantially enhanced sensitivity and improved selectivity. Systematic optimization of the FRET donor-acceptor pair and sensor backbone yields superior performance versus previous best-in-class FRET-based AKARs, enabling robust detection of subtle PKA activity changes across diverse experimental modalities, including flow cytometry, fluorescence lifetime-based FRET, and two-photon imaging in brain slices. We further leverage kinome atlas data to engineer a variant with improved selectivity for more accurate visualization of nuclear PKA activity. Using the AKAR6 toolkit, we showed that in contrast to strong GPCR-induced PKA activities across all tested compartments in PC12 cells, growth factors stimulated a significant PKA activity at the trans-Golgi network but no detectable activity at cis-Golgi, signifying highly compartmentalized PKA signaling at the sub-organelle level. Furthermore, NGF and EGF induced sustained and transient PKA activity, respectively, across various intracellular compartments, including the nucleus, suggesting that growth factor-specific temporal controls are maintained across subcellular compartments. Together, the AKAR6 toolkit provides a sensitive, selective, and versatile platform for dissecting compartmentalized PKA signaling across cells and tissues.

bioengineering↗

bioRxiv2026

Deep learning-guided design of cell type-specific AAV promoters

Precise cell type targeting is critical for both clinical and experimental applications of adeno-associated viral (AAV) vectors, yet engineering vectors with cell type-specific activity remains a challenge. Here, we compared three strategies leveraging single-cell chromatin accessibility data to design cell type-specific AAV promoters, including a deep learning-based method to generate de novo regulatory sequences. When applied to target retinal ganglion cells or horizontal cells in mouse retina, deep learning-guided design consistently outperformed rational approaches, yielding synthetic promoters with stronger and more specific expression in vivo. Synthetic AAV promoters supported diverse transgenes, enabling the recording and ablation of targeted cells. Promoter activity was also maintained in human retinal organoids, suggesting that deep learning-designed sequences may be suitable for translation. Our findings highlight the potential of deep learning to synthesize cell type-specific AAV promoters and establish a versatile platform for cell type targeting with broad implications for gene therapy and basic research.

synthetic biology↗

bioRxiv2025

Emergence of a new material through ligation of DNA nanostar hydrogels

DNA nanostar hydrogels hold great promise as biomaterials, but their mechanical properties and nuclease susceptibility limit their use. Here, we propose ligation of nanostar sticky ends as a strategy to fundamentally transform the mechanical and dynamic properties of DNA nanostar hydrogels and enhance their nuclease resistance. Ligation eliminates free ends of DNA nanostars, conferring strong exonuclease resistance; suppresses strand rearrangement, converting liquid-like into gel-like assemblies; and prevents macroscopic reversibility despite preserving microscopic motif hybridization. Ligation also stiffens the material, enabling facile handling and 3D printing, and markedly slows digestion in serum-supplemented medium. In the presence of low concentrations of the DNase I inhibitor actin, ligated hydrogels remain stable for at least two weeks. As a proof-of-concept application, we captured and maintained cells within ligated hydrogels for one week in culture. By overcoming the limitations of DNA nanostar hydrogels, this work enables their use as programmable biomaterials that fully leverage the DNA nanotechnology toolbox.

bioengineering↗

bioRxiv2025

Living Neurosheets: Engineering readily deployable neural architectures

Inspired by the ability of brain structures to support cognitive processing, the need for reliable queryable neural models, and emerging biohybrid applications, we present Living Neurosheets: practical, malleable, modular, and scalable systems for realizing living neural architectures, bio-inspired or beyond evolutionary precedent. By innervating porous sheets, paper-like substrates are endowed with neural function while retaining exceptional versatility. Pre-programmed porosity anchors deposited neurons for reliable spatial control. Cutting, folding, and stacking allow intuitive design, modular composition, and batch fabrication (by the hundreds) of complex neural motifs that preserve geometry and function for months. Reversible integration with tissue-matched optoelectronics enables both sample reuse across hardware and hardware multiplexing across samples, for high-throughput, multimodal investigations. We illustrate these aspects across tissue topologies, gyri-inspired manifolds, and laminar cortical mimics, attaining control over entrainment, synchronization, propagation, suppression, and augmentation dynamics--key features of in vivo neural activity. Finally, via cross-instrument portability, we fuse spatial transcriptomics and electrophysiology, linking gene programs to emergent dynamics.

neuroscience↗

bioRxiv2025

Decoding Gene Responsiveness to Synthetic Chromatin Reader-Actuators with Multi-Modal Epigenomic Profiling

Cell identity is regulated by chromatin states that encode gene regulatory memory and shape responses to new inputs. To investigate how chromatin context influences inducibility in differentiated cells, we employed engineered synthetic reader-actuators (SRAs), fusion proteins containing the polycomb chromodomain (PCD) that binds H3K27me3. In MCF7 breast cancer cells, we mapped PCD-fusion occupancy by ChIP-seq and used RNA-seq to identify temporally resolved gene activation patterns. ChIP-seq profiling and machine learning models (MLM) demonstrated that PCD-fusion binding was predicted by the presence of H3K27me3, H4K20me1, or H3K36me3 enrichment, suggesting selective accessibility at enhancers and chromatin transition zones. Among genes with PCD-fusion enriched enhancers, the SRA-induced subset was distinguished by promoter features including bivalent histone modifications and enrichment of transcriptional repressors REST and MTA1. Collectively, our results demonstrate that SRA responsiveness depends on a specific chromatin signature beyond H3K27me3 alone. This study demonstrates the power of SRAs to dissect inducible chromatin features in their native genomic context, and suggests that epigenetically repressed regions in differentiated cells can retain regulatory plasticity.

synthetic biology↗