Biology subjects

Lee, R.

Publications and source records attributed to Lee, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Transient Partial Inhibition of DNA Polymerase α Promotes Chromatin Plasticity and Enhances Human Cell Reprogramming

Somatic cell reprogramming requires extensive remodeling of epigenomic and transcriptional state, yet the mechanisms that enhance chromatin plasticity during cell-fate conversion remain incompletely understood. Beyond its essential role in genome duplication, DNA replication provides a dynamic window for chromatin remodeling and may therefore influence cellular plasticity during reprogramming. Here, we show that low-dose, transient inhibition of the lagging-strand DNA polymerase (Pol ) complex markedly enhances reprogramming of human peripheral blood mononuclear cells into induced pluripotent stem cells (iPSCs). Fine-tuned inhibition of the catalytic subunit PolA1 increases cell survival under reprogramming-associated stress and enhances cellular resilience to diverse external stresses, preserves genomic stability, and accelerates the acquisition of pluripotent features. PolA1-inhibited cells undergo rapid increases in chromatin accessibility following reprogramming factor delivery and display accelerated progression toward the iPSC state. Consistent with enhanced developmental plasticity, colonies derived from PolA1 inhibitor-treatment display dome-shaped morphology, increased expression of several naive pluripotency markers such as SUSD2. Integrated imaging and single-nucleus multiomic analyses establish transient PolA1 inhibition as a potent regulator of early chromatin remodeling during reprogramming. Functionally, PolA1 inhibitor-derived iPSC lines maintain stable pluripotency and exhibit enhanced differentiation potential, including increased embryoid body budding and accelerated cardiomyocyte differentiation and maturation. Notably, transient and low-dose PolA1 inhibition alters replication-coupled histone incorporation without reducing DNA replication speed or affecting cell cycle progression. This two-step regime primes human PBMCs for sustained gains in reprogramming efficiency with pre- and post-treatment. Together, these findings identify replication-coupled chromatin regulation as an underappreciated mechanism regulating human cell fate plasticity and demonstrate that transient modulation of Pol can simultaneously enhance epigenomic plasticity and preserve genome stability during reprogramming.

molecular biology↗

bioRxiv2026

Germline mitophagy selectively eliminates deleterious mitochondrial DNA across generations

Faithful transmission of genetic information through the immortal germline is essential for organismal health and species survival, yet how mutant mitochondrial genomes (mtDNAs) are selectively eliminated across generations remains unclear. Here, we show that germline mitophagy functions as a mutation-responsive surveillance system that selectively eliminates mutant mtDNAs and shapes inheritance across generations. In C. elegans, mitochondria enriched for mutant mtDNAs are selectively removed in the maternal germline prior to oocyte fertilization via PINK1/Parkin-dependent and BNIP3-mediated mitophagy pathways activated by mtDNA defects. Germline mitophagy declines with age, resulting in offspring that inherit increased burdens of mutant mtDNAs. Conversely, enhancing mitophagy within germ cells restricts the transmission of deleterious genomes in a compounding manner, ultimately driving their complete elimination from the matrilineal lineage. Together, our findings demonstrate that germline mitophagy is a critical determinant of intergenerational mitochondrial genome inheritance, establishing its role in restricting the transmission of defective genetic information.

Cell Biology↗

bioRxiv2026

Immunometabolic Remodeling of Perivascular Adipose Tissue in Murine Lupus: Implications for Lupus Vasculopathy

BackgroundPatients with systemic lupus erythematosus (SLE) face markedly increased cardiovascular disease (CVD) risk driven by mechanisms beyond traditional risk factors. Thoracic aortic perivascular adipose tissue (tPVAT) is dysfunctional in lupus and exacerbates endothelial dysfunction, yet the molecular basis of this dysfunction remains poorly defined. MethodsIntegrated multi-omics profiling, including bulk RNA-seq, untargeted proteomics, lipidomics, and high-dimensional spectral flow cytometry, was performed on tPVAT from 15-week-old MRL/lpr mice (active lupus, n = 4-6) and MRL control mice (n = 5-6). Adipogenic differentiation capacity of tPVAT adipose stromal and progenitor cells (ASPCs) from MRL/lpr was assessed by Oil Red O staining at 5 (pre-dieasea) and 15 weeks (active disease), with subcutaneous ASPCs used as depot controls. ResultsTranscriptomic profiling of tPVAT from MRL/lpr mice identified 2,742 upregulated and 1,494 downregulated genes (adjusted p < 0.001, |log2FC| > 1), with strong activation of interferon, IL6-JAK-STAT3, and TNFA signaling pathways together with suppression of fatty acid metabolism, oxidative phosphorylation, and adipogenic pathways. Proteomic and lipidomic analyses were concordant, revealing broad downregulation of mitochondrial bioenergetic machinery, depletion of cardiolipin and acylcarnitines, and enrichment of ceramide phosphoinositols and lysophosphatidylcholines. Cardiolipin strongly correlated with the mitochondrial/metabolic protein module (r = 0.95) and inversely with the immune/inflammatory protein module (r = -0.92). Spectral flow cytometry confirmed marked CD45+ leukocyte infiltration dominated by T cells, together with a significantly reduced Treg/CD4+ ratio indicating loss of local immunoregulatory balance. ASPCs derived from PVAT of 15-week-old MRL/lpr mice exhibited impaired white and beige adipogenic differentiation, while APCs from PVAT of 5-week-old MRL/lpr mice, and from subcutaneous adipose tissues of 15-week-old MRL/lpr mice, had normal white and beige differentiation, consistent with an acquired, depot-specific, disease-stage-dependent progenitor defect in PVAT of MRL/lpr mice. ConclusionsLupus tPVAT undergoes a concordant cross-platform molecular reprogramming of mitochondrial bioenergetic genes coupled with establishment of an interferon-dominant immune niche and acquired loss of ASPC adipogenic capacity. These findings provide a molecular framework for lupus PVAT dysfunction and identify restoration of mitochondrial function, suppression of interferon-driven inflammation, and renewal of progenitor differentiation as potential therapeutic strategies for lupus vasculopathy.

molecular biology↗

bioRxiv2026

Structural Basis of Polypurine Track Strand Displacement by HIV-1 Reverse Transcriptase

To complete reverse transcription, HIV-1 reverse transcriptase (RT) must displace the RNase H-resistant polypurine tract (PPT) primers. This enables synthesis of the long terminal repeats and formation of the central cDNA flap. However, the molecular mechanism of this PPT strand displacement (SD) has remained unknown, and no structural data exist on how a retroviral polymerase execute these reactions. We report the first cryo-EM structures of HIV-1 RT bound to nucleic acid substrates containing either a PPTRNA or PPTDNA displacement strand, with incoming dATP positioned at the polymerase catalytic site. These structures reveal key features of the PPT displacement mechanism by RT. Specifically, we observed a binding mode where the template nucleotide (T1) base-paired to the first displacement nucleotide (D1) undergoes a 90{degrees} rotation relative to the preceding template base (T0). This sharp template flip positions D1 [~]30 [A] away from the primers 3-end and is coordinated by RTp66 residues at the SD interface: F61 and R78 contact T1/T0 to drive template translocation, while W24 engages both T1 and D1 to stabilize the displacement strand. Biochemical and virological mutagenesis experiments confirm that interactions with F61 and R78 are essential for both canonical cDNA polymerization and SD, whereas the W24-nucleotide interactions are required exclusively for SD but are dispensable for standard cDNA synthesis. These results contribute to the structural and functional understanding of PPT strand displacement by HIV-1 RT and reveal a distinct mechanistic vulnerability for the design of next-generation antiretrovirals.

biochemistry↗

bioRxiv2026

Complementary constraints in germ and immune cells shape evolution of gene regulation and phenotype

New mutations arise in germ cells and must preserve fertility while altering somatic phenotype to confer a fitness advantage. To understand how whole organisms balance these constraints, we generated mice with humanized noncoding promoter sequence at Traf6, an NF-{kappa}B pathway component with recently diverged chromatin state in mouse germ cells. The humanized allele (Traf6h) reinstated human-like expression in germ and immune cells, which we traced to a 13 base pair deletion that impairs CTCF binding. Traf6h had no effect on fertility but induced a sensitized innate immune response to endotoxin. Other NF-{kappa}B pathway genes followed a similar pattern, suggesting that their expression has evolved in tandem and consistent with known mouse-human differences in inflammatory response. Our results reveal distinct roles for germ and immune cells in shaping gene regulatory evolution, where immune cells quickly adjust phenotype, enabling selection, while germ cells robustly maintain fertility despite changing expression levels, enabling inheritance.

genetics↗

bioRxiv2026

Loss of Mitochondrial FMT Restores Chloroplast Proteostasis via Inter-organelle Compensation

Chloroplast proteostasis is vital for plant development, yet whether cells can actively reprogram organelle communication to restore plastid function when essential protease components fail remains unclear. Using a forward genetic suppressor screen in Arabidopsis, we identify loss of the mitochondria-associated protein FRIENDLY MITOCHONDRIA (FMT) as a strong suppressor of the virescent and growth-retarded clpc1 mutant, which lacks the major chloroplast Clp chaperone ClpC1. Suppression is highly specific and occurs independently of GUN1-mediated retrograde signaling. Integrated multi-omics analyses reveal that clpc1 and fmtclpc1 represent two distinct organelle signaling states. In clpc1, loss of ClpC1 triggers a plastid stress state characterized by repression of photosynthesis-associated transcription factors, induction of plastid metabolic stress markers, and impaired proteolytic activity. By contrast, loss of FMT shifts the system into a recovery state despite persistent mitochondrial clustering. Mechanistically, FMT negatively regulates CLPC2, a ClpC1 paralog, and fmt-mediated rescue results from CLPC2 derepression. Moderately elevated ClpC2 restores in vivo proteolysis, as evidenced by recovery of PAA2 substrate turnover, normalization of chloroplast ultrastructure, and reactivation of photosynthesis-related gene expression. Transcriptomic and proteomic profiling further reveal coordinated remodeling of nuclear gene expression and chloroplast protein investment in the recovery state, including reduced cytosolic folding stress and selective induction of jasmonic acid- and salicylic acid-associated signaling networks. Genetic analyses establish that REC1 and REC2 are required for full CLPC2 induction and phenotypic recovery. Together, our findings uncover a latent inter-organelle compensatory mechanism in which mitochondrial perturbation reprograms nuclear gene expression to restore chloroplast proteostasis when ClpC1 function is compromised.

plant biology↗

bioRxiv2026

Microbiome depletion rejuvenates the aging brain

Aging is associated with cognitive decline and increased vulnerability to neurodegeneration driven by an array of molecular and cellular changes like impaired vascular integrity, demyelination, reduced neurogenesis, and chronic inflammation. Recent studies implicate the gut microbiome as a modulator of brain aging, but the underlying mechanisms remain elusive. Here, we show that depleting the gut microbiome by administering antibiotics to aged mice induces widespread molecular and structural rejuvenation in the brain. Our transcriptomic analyses by single-nucleus RNA sequencing revealed pronounced transcriptional shifts across multiple brain cell types. We confirmed that antibiotic treatment improves vascular density, promotes myelination, enhances neurogenesis, and reduces microglial reactivity. Functionally, microbiome-depleted mice showed improved hippocampal memory performance. Analyses of brain and plasma cytokine levels showed a decrease in several pro-inflammatory factors post-treatment and identified candidate factors, including the chemokine eotaxin-1. Inhibiting eotaxin-1 alone can reverse several aspects of brain aging. Our findings demonstrate that age-associated microbial inflammation contributes to brain aging and that its attenuation can restore youthful features at the molecular, cellular, and functional levels. Targeting the gut microbiome or its circulating mediators may therefore represent a non-invasive approach to promote brain health and cognitive resilience in aging.

neuroscience↗

bioRxiv2026

A regulatory role of novel long non-coding RNA, BAZ1A-AS1, in vascular smooth muscle cell functions during neointima formation in human saphenous veins

BackgroundNeointimal proliferation (NP) is a major cause of stenosis and occlusion in arteries and veins. Human saphenous veins (HSV), often used for coronary artery bypass grafting, frequently fail in part due to NP. Long non-coding RNAs (lncRNAs) have emerged as critical regulators of vascular smooth muscle cell (VSMC) phenotype, yet lncRNAs governing NP in human veins remain largely uncharacterized. Methods and ResultsUsing an ex vivo model of NP in human SV, we performed bulk RNA sequencing on SV tissues with and without NP. Among differentially expressed lncRNAs, we identified a previously uncharacterized transcript, Bromodomain Adjacent to Zinc Finger A1 antisense 1 (BAZ1A-AS1), and its predicted cis-regulatory partner gene BAZ1A, as markedly upregulated during NP and predominantly enriched in VSMCs. Both transcripts were induced by genotoxic stimuli, with BAZ1A-AS1 showing transient induction preceding sustained BAZ1A upregulation, consistent with a priming role in the DNA damage response. Silencing of either BAZ1A-AS1 or BAZ1A attenuated VSMC proliferation and migration, accompanied by upregulation of contractile markers and suppression of proliferative and inflammatory transcriptional programs. ChIRP-qPCR demonstrated that BAZ1A-AS1 physically interacts with 3' distal exons of BAZ1A at the DNA level while selectively engaging CNN1, CCND1, and IL6 mRNAs, indicating mechanistically distinct chromatin- and RNA-level regulatory functions. Baz1a haploinsufficiency attenuated neointima formation and preserved VSMC contractile identity in a mouse carotid artery ligation model. ConclusionsWe identify BAZ1A-AS1 and BAZ1A as stress-responsive regulators of VSMC phenotype, operating through dual mechanisms of cis-regulatory chromatin interaction and selective mRNA engagement, and demonstrate a novel role for the BAZ1A-AS1/BAZ1A axis in promoting NP.

physiology↗

bioRxiv2026

Prostaglandin E2 Reverses Myofibroblast Differentiation in Eosinophilic Esophagitis

Background & AimsUnchecked inflammation in Eosinophilic esophagitis (EoE) leads to esophageal fibrosis and eventual stricture. Differentiated fibroblasts, termed myofibroblasts, are the main effector cells in fibrosis, responsible for secreting extracellular matrix proteins leading to tissue stiffness. Regulating myofibroblasts has not been explored as a therapeutic possibility in the fibrostenotic esophagus. Herein, we aim to investigate the efficacy of Prostaglandin E2 (PGE2) in dedifferentiation of the EoE myofibroblast. MethodsWe evaluated the efficacy and mechanism of myofibroblast dedifferentiation using fetal esophageal fibroblasts (FEF3), patient-derived fibroblasts, and a murine model of EoE. ResultsFibrosis markers (SMA, FN1, and COL1A1) and contractility of myofibroblasts were significantly decreased by PGE2 via the cAMP pathway. PGE2 treatment decreased nuclear accumulation of phospho-Smad2/3-YAP complex and induced phospho-YAP proteasomal degradation. Transcriptome analyses of FEF3 treated with TGF{beta} or PGE2 revealed that the Integrin1 pathway, and specifically thrombospondin 1 (THBS-1), was significantly upregulated by TGF{beta} and downregulated by PGE2, as supported by pseudo-bulk single-cell RNA-seq of EoE biopsies. THBS-1 was shown to be regulated by PGE2 via the cAMP/YAP pathway, and its knockdown induced myofibroblasts dedifferentiation. In a murine model of EoE, Butaprost, agonist of the E-prostanoid G protein-coupled receptor 2, treatment significantly reduced the expression of THBS-1, SMA, and FN1 along with a decrease in YAP nuclear translocation. Additionally, collagen fiber organization in the lamina propria was markedly reduced. ConclusionPGE2 promotes dedifferentiation of myofibroblasts in EoE via the cAMP/YAP/ THBS-1 pathway. Our data suggest that PGE2 is a promising treatment strategy for EoE with stenosis. What You Need to KnowO_ST_ABSBackground and ContextC_ST_ABSIn Eosinophilic esophagitis, unchecked inflammation and tissue stiffness drives fibroblast differentiation and fibrostenosis of the esophagus, yet targeting myofibroblasts as regulators of extracellular matrix deposition in fibrostenotic disease remains clinically unexplored. New FindingsProstaglandin E2 promotes dedifferentiation of myofibroblasts in eosinophilic esophagitis via the cAMP/YAP pathway, with Thrombospondin-1 identified as a critical YAP regulated target driving fibrostenosis. LimitationsThis study focused on fibroblast-specific mechanisms. The effects of PGE2 on esophageal epithelial differentiation, barrier function, and immune cell recruitment in EoE remain to be determined. Clinical Research RelevanceThis study demonstrates proof-of-concept that pharmacological reversal of established fibrosis is achievable in EoE. PGE2 and its EP2-selective agonists represent translatable therapeutic targets for fibrostenotic EoE--a patient population that remains treatment-refractory to current immunosuppressive approaches. Basic Research RelevanceThe cAMP/YAP/THBS-1 signaling in fibroblasts emerges as a critical therapeutic target for esophageal fibrosis. Importantly, this work demonstrates that terminally differentiated myofibroblasts retain remarkable plasticity and can dedifferentiate--challenging the paradigm that fibrosis is irreversible.

cell biology↗

bioRxiv2025

A telomere-to-telomere map of somatic mutation burden and functional impact in cancer

Oncogenesis involves widespread genetic and epigenetic alterations, yet the full spectrum of somatic variation genome-wide remains unresolved. We generated a near-telomere-to-telomere (T2T) diploid assembly of a donor paired with deep short- and long-read sequencing of their melanoma. This revealed that 16% of somatic variants occur in sequences absent from GRCh38, with satellite repeats acting as hotspots for UV-induced damage due to sequence-intrinsic mutability and inefficient repair. Centromere kinetochore domains emerged as focal sites of structural, genetic, and epigenetic variation, leading to remodeling of centromere kinetochore binding domains during tumor evolution. Single-molecule telomere reconstructions uncovered cycles of attrition, deletion, and telomerase-mediated extension that shape cancer telomeres. Finally, diploid chromatin maps exposed that copy number alterations and epimutations, rather than point mutations, predominate in rewiring cancer regulatory programs. These findings define the full landscape of a cancers somatic variation and their functional impact, establishing a blueprint for T2T studies of mosaicism.

genomics↗

bioRxiv2025

Splenic granulopoiesis and S100A9 drive resistance to checkpoint inhibitors conferred by liver metastases

Here, we investigate why liver metastases reduce the efficacy of immune checkpoint inhibitors (CPI). The poor prognosis of patients with liver metastases is associated with a systemic increase in neutrophils. Using experimental models, we confirm that mice with liver metastases respond poorly to CPI, have elevated neutrophils and suppress the response of subcutaneous lesions to CPI. We demonstrate that liver metastases, acting partly via IL-6, boost granulopoiesis in the spleen and promote the generation of immature S100A9hi neutrophils that suppress T-cell proliferation. Human liver metastases exhibit a similar increase in S100A9hi neutrophils. Neutrophil depletion attenuates the growth of liver metastases, but not subcutaneous metastases. Moreover, genetic deletion of S100A9 enables liver metastases to be effectively treated with CPI, and prevents liver metastases from suppressing the response in subcutaneous metastases. Thus, we document how liver metastases specifically change splenic granulopoiesis leading to changes in the microenvironment of non-hepatic lesions, and how targeting a key neutrophil protein restores the efficacy of CPI.

immunology↗

bioRxiv2025

Engineering anaerobic fungal-bacterial consortia for medium-chain fatty acid production from lignocellulosic biomass

Lignocellulosic biomass is a renewable feedstock for sustainable fuels and chemicals, yet industrial conversion remains constrained by carbohydrate solubilization. Inspired by herbivore rumen microbiomes, we engineered an anaerobic fungal-bacterial consortium converting native lignocellulose into medium-chain fatty acids (MCFAs) without pretreatment. Systematic screening identified a newly isolated anaerobic fungus, Neocallimastix sp. FC1, in co-culture with Megasphaera hexanoica as a top-performing pair, achieving a lignocellulose-to-MCFA yield of 21.0 % (carbon-to-carbon basis) through tight lactate cross-feeding without competition for soluble sugars. Because fungal lactate production rate constrained the growth of M. hexanoica, the bacterium reallocated protein from growth toward chain elongation, resulting in increased MCFAs production over butyrate. These results demonstrate that high lignocellulose-to-MCFA conversion by the consortium requires high lactate-producing capability and operating regimes sustaining low lactate concentrations at high flux. Technoeconomic analysis further identifies the cost and yield thresholds required for economically viable deployment, establishing quantitative design targets for pretreatment-free fungal-bacterial lignocellulose upgrading.

bioengineering↗

bioRxiv2025

δ-catenin haploinsufficiency is sufficient to alter behaviors and glutamatergic synapses in mice

{delta}-catenin (also known as CTNND2) functions as an anchor for the glutamatergic AMPA receptor (AMPARs) to regulate synaptic activity in excitatory synapses. Alteration in the gene coding {delta}-catenin has been implicated in many neurological disorders. Some of these genetic alterations exhibit a profound loss of {delta}-catenin functions in excitatory synapses. We have shown that {delta}-catenin deficiency induced by the homozygous {delta}-catenin knockout (KO) and autism-associated missense glycine 34 to serine (G34S) mutation significantly alters AMPAR-mediated synaptic activity in cortical neurons and disrupts social behavior in mice. Importantly, many genetic disorders are caused by haploinsufficiency. Indeed, {delta}-catenin haploinsufficiency contributes to severe autism and learning disabilities in humans. However, previous studies have used only homozygous {delta}-catenin deficiency models. Therefore, it is important to examine the effects of {delta}-catenin haploinsufficiency on animals behaviors and excitatory synapses. Here, we use heterozygous {delta}-catenin KO and G34S mice as a {delta}-catenin haploinsufficiency model to examine this idea. Multiple behavioral assays, a social behavior test, contextual fear conditioning, and an open field test, reveal that both {delta}-catenin KO and G34S haploinsufficiency significantly disrupt animals social behavior and fear learning and memory. Interestingly, only KO haploinsufficiency mice show anxiety-like behavior. A biochemical assay using brain extracts demonstrates that {delta}-catenin haploinsufficiency significantly affects the levels of synaptic {delta}-catenin and AMPARs. Our findings thus suggest that {delta}-catenin haploinsufficiency affects animals behaviors via altering glutamatergic synaptic activity.

neuroscience↗

bioRxiv2025

Sterilizing activity of spectinamide MBX-4888A when replacing linezolid in the Nix-TB regimen in the relapsing BALB/c mouse model of tuberculosis

Spectinamides have garnered interest as experimental tuberculosis therapeutics owing to their safety profile and efficacy as partner agents when used in conjunction with established regimens in mice. The Nix-TB regimen of bedaquiline, pretomanid, and linezolid represents a short, effective regimen recommended for treatment of pre-extensively drug-resistant tuberculosis. However, linezolid administration is associated with severe adverse events which limits its use. Here we present preclinical data that spectinamide MBX-4888A can replace linezolid in Nix-TB.

microbiology↗

bioRxiv2025

Alteration of Water Exchange Rates Following Focused Ultrasound-Mediated BBB Opening in the Dorsal Striatum of Non-Human Primates: A Diffusion-Prepared pCASL Study

This study applied diffusion-prepared pseudo-continuous arterial spin labeling (DP-pCASL) to quantify cerebral blood flow (CBF), arterial transit time (ATT), and blood-brain barrier (BBB) water exchange rate (Kw) before and after focused ultrasound (FUS)-mediated blood-brain barrier opening (BBBO) in the dorsal striatum of four non-human primates. Six baseline and seven BBBO sessions were performed. DP-pCASL was acquired approximately 45 minutes after FUS sonication combined with intravenous microbubbles, and contrast-enhanced T1-weighted imaging was subsequently used to confirm the BBBO region. Whole-brain analyses revealed no significant changes in CBF or ATT following BBBO (permutation p > 0.05). Region-of-interest analysis within the sonicated caudate demonstrated a significant localized decrease in Kw, with median (IQR) values of 45.0 (40.6 - 55.6) min-1 at the BBBO site versus 61.6 (58.3 - 70.4) min-1 in the contralateral control region (p < 0.05), confirming spatially specific suppression of transendothelial water flux. In contrast, whole-brain Kw increased significantly following BBBO, with median (IQR) values of 49.8 (46.3 - 55.9) min-1 in non-BBBO sessions versus 59.4 (56.6 - 66.3) min-1 in BBBO sessions (p < 0.01), indicating a diffuse enhancement of water exchange across the brain. These findings establish DP-pCASL-derived Kw as a sensitive, non-contrast biomarker for both local and global BBB permeability changes induced by focused ultrasound, supporting its potential for longitudinal monitoring in preclinical and clinical neurotherapeutic applications.

neuroscience↗

bioRxiv2025

Modulation of huntingtin S421 phosphorylation in a Huntington's disease mouse model and its detection in nonhuman primate cerebrospinal fluid

Huntingtons disease (HD) is a progressive neurodegenerative disease caused by the pathologic expansion of a CAG repeat in the first exon of the huntingtin (HTT) gene, resulting in a huntingtin (HTT) protein with an expanded polyglutamine (polyQ) tract. Phosphorylation at residue S421 (pS421) is one of the post-translational modifications proposed to influence the biology of wild-type and mutant (m)HTT, such as HTT stability and clearance, HTT subcellular localization, mHTT toxicity, and regulation of HTT function in axonal transport. However, the detection and quantification of S421-HTT phosphorylation in relevant biological contexts have remained challenging and the consequences of pS421 in HD pathogenesis remains unclear. Here we report the development of a novel ultrasensitive immunoassay enabling the specific and sensitive detection of pS421-HTT in a variety of biologically relevant contexts. With this assay we conducted a longitudinal assessment of pS421 levels in tissues from a mouse model of HD to investigate the relationship between S421 phosphorylation and phenotypic progression. We also identified PRKACA, the cAMP-regulated catalytic subunit of PKA, as a kinase capable of phosphorylating S421-HTT, demonstrating its ability to regulate endogenous pS421 in human cells. Finally, we exploited the sensitivity of the assay to detect endogenous pS421-HTT in cerebrospinal fluid (CSF) from nonhuman primates, showing for the first time that phosphorylation at S421-HTT can be detected in this bio-fluid. These reagents and assay will enable investigation of the biological consequence and the relevance of pS421 in the natural history of HD.

neuroscience↗

bioRxiv2025

Huntington's disease LIG1 modifier variant increases ligase fidelity and suppresses somatic CAG repeat expansion

Huntingtons disease (HD) is a fatal neurodegenerative disorder caused by inheriting an expanded CAG repeat tract in the huntingtin gene (HTT) that further expands in somatic cells over an individuals lifetime. Genome-wide association studies have provided critical insight into factors that modify the course of disease. These include DNA repair genes that alter the rate of somatic expansion and other genes that do not appear to directly influence this process. One modifier gene is DNA ligase 1 (LIG1), in which a variant specifying a lysine to asparagine substitution (K845N) is associated with a profound (7-8 year) delay in the onset of motor signs. Here, we have taken a multifaceted approach to gain insight into the protective nature of this variant in HD. We demonstrate using in vitro ligase assays and enzyme kinetics that K845N enhances discrimination towards mismatched substrates and increases repair fidelity. Consistent with increased ligation fidelity, K845N confers protection against oxidative stress in cell-based assays. Finally, we demonstrate that the mouse LIG1 K843N orthologue suppresses somatic CAG expansion in HD knock-in mice. Overall, our data provide evidence that altered LIG1 function due to the K845N substitution may contribute to HD clinical delay by slowing somatic expansion in the brain and protecting the genome globally against damage. Significantly, our results provide a mechanistic foundation for considering DNA ligase fidelity as a therapeutic target in HD and potentially in other trinucleotide repeat disorders. Significance StatementWe analyzed a missense variant in DNA Ligase 1 (K845N) that is associated with a profound delay in the onset of Huntingtons disease (HD). We find that K845N enhances substrate discrimination towards mismatched substrates, thus increasing repair fidelity, conferring protection against oxidative stress and slows somatic expansion of the HD CAG repeat. Our observations provide insight into underlying mechanisms of disease modification and suggest avenues that can be harnessed for disease-modifying therapeutic intervention. Classification: Biological Sciences, Genetics

genetics↗

bioRxiv2025

Hierarchical Coordination of Polymerase Theta and RAD51 Resolves Clustered Replication Fork Collapse

The essential role of polymerase theta (Pol{theta})-mediated end joining (TMEJ), an alternative double strand break repair pathway, has been primarily studied in homologous recombination (HR)-deficient contexts(1, 2). Here, we uncover an indispensable role for TMEJ in HR-proficient mammalian cells during the repair of interstrand crosslinks (ICLs). We show that Pol{theta} is recruited downstream of canonical ICL repair steps--including ICL unhooking, RAD51 loading, and RAD51 ubiquitylation--and localizes to sites of unresolved HR through interactions with ubiquitylated RAD51 filaments. Using genomic scar profiling and targeted ICL repair assays, we find that TMEJ resolves a minor subset of lesions that are not amenable to HR repair, such as clustered ICLs that can induce two-ended replication fork collapse. These findings reveal a RAD51 ubiquitylation-dependent mechanism for Pol{theta} recruitment and establish TMEJ as a hierarchically deployed DNA repair pathway that safeguards genome stability when HR is insufficient to resolve replication-associated DNA damage. Short SummaryPol{theta} is recruited via RAD51 ubiquitylation to resolve clustered ICLs that generate HR-refractory replication fork collapse.

cell biology↗