Biology subjects

Chan, K.

Publications and source records attributed to Chan, K..

At least 19 recordsLinked to original sources

bioRxiv2026

GAT1508, a selective neuronal GIRK1/2 activator, exhibits superior antiepileptic efficacy and synergizes with cannabidiol

G protein-gated inwardly rectifying potassium (GIRK; Kir3) channels are key regulators of neuronal excitability and represent promising therapeutic targets for epilepsy. Here, we characterize GAT1508, a urea-based small-molecule activator selectively targeting neuronal GIRK1/2 heterotetramers, and compare its efficacy with previously described urea compounds ML297 and GiGA1. In a pentylenetetrazole (PTZ)-induced seizure model, GAT1508 demonstrated robust antiseizure activity, outperforming both ML297 and GiGA1 and achieving efficacy comparable to valproic acid. Electrophysiological studies using two-electrode voltage clamp in Xenopus oocytes revealed that GAT1508, unlike ML297 and GiGA1, selectively activates GIRK1/2 channels without significant activation of cardiac GIRK1/4 channels, suggesting reduced risk of cardiovascular side effects. Structural and computational analyses indicate that enhanced stabilization of GAT1508 compared to ML297 within the GIRK1/2 binding pocket promotes both ion permeation and channel gating through coordinated interactions with key residues. Furthermore, GAT1508, like the two other urea drugs, exhibited synergistic activation of GIRK channels when combined with G{beta}{gamma}-mediated pathways and significantly potentiated the antiseizure effects of cannabidiol (CBD) in both hippocampal slice recordings and in vivo seizure models. These findings establish GAT1508 as a potent and selective GIRK1/2 activator and support a novel combination therapeutic strategy for epilepsy based on convergent GIRK channel modulation.

pharmacology and toxicology↗

bioRxiv2026

Characterization of artificial riboswitches for Coxsackievirus B3 detection using machine learning

With the increase of recycled water to offset water demand, the potential possibility to spread contagious RNA viruses, such as Coxsackievirus B3, increases. However, detection of viral particles remains challenging because of low viral concentrations in wastewater and high mutation rates of the RNA virus. Robust monitoring is needed with low cost and low infrastructure technologies to increase accessibility of monitoring technologies worldwide. Novel viral detection methods have been developed, based on synthetic riboswitches that bind to the target virus and trigger a reporter gene, thus amplifying the detection signals. Such monitoring technologies can leverage machine learning to optimize the candidate nucleic acid sequences used for detection. To support the design of effective riboswitches, we present a machine learning model for classifying riboswitch performance, integrating RNA sequence data with secondary structural features based on free energy calculations and parameters that represent single strandedness. This model uses a sparsely gated Mixture of Experts (MoE) architecture to route sequence and thermodynamic features to specialized experts, achieving strong generalization performance across cross-validation and held-out testing. When evaluated against baseline Decision Tree, Random Forest, Gaussian Naive Bayes, and Dense Neural Network classifiers, the MoE model demonstrated near-zero classification errors. Furthermore, post-hoc feature importance and k-mer analyzes reveal that near-perfect predictive performance in silico is strongly driven by sequence constructs in addition to generalizable folding mechanics. An ablation study of numerical feature and sequence inputs showed that while the Dense Neural Network also achieved high accuracy across all ablation conditions, the MoE architecture was retained as the primary model because its specialized subnetworks do not require all model parameters to be activated offering a potential computational advantage.

bioinformatics↗

bioRxiv2026

Proteomic Characterization of Prostate Cancer Bone Metastasis Subtypes Defines Progressive Remodeling States of the Bone-Tumor Microenvironment

Prostate cancer (PC) bone metastases (PCBM) are a debilitating disease morbidity that primarily affects the axial skeleton. PCBM are predominantly osteosclerotic with mixed osteosclerotic/osteolytic features, including irregular bone deposition, loss of collagen alignment and increased porosity that mimic features of woven bone formed during development and repair. We hypothesize that progression of PCBM is driven by interactions between tumor cells and the bone microenvironment and is associated with changes in the stromal compartment. We performed tandem mass tag mass spectrometry proteomics on lumbar vertebral specimens from 32 cadaveric PCBM specimens and 3 age-matched controls. Unsupervised clustering segregated specimens into three clusters associated with benign, mixed, and osteosclerotic lesion states, characterized by distinct immune-, tumor-, and osteogenic-associated protein signatures. Histological analysis showed tumor infiltration was accompanied by marrow fibrosis and increased collagen III deposition. We identified a marker signature associated with osteosclerotic remodeling and mapped this signature to independent metastatic PC transcriptomic datasets. The proteomics-derived markers reproduced similar molecular groupings at RNA level, and robustly segregated bone PCBMs from soft tissue metastases PC in RNA data. Together, these findings define molecularly distinct states of tumor-driven changes in PCBM that extend beyond histological classification and may inform biomarker development and therapeutic stratification.

cancer biology↗

bioRxiv2026

Multi-omic characterization of axolotl perilymph-cerebrospinal fluid reveals shifts in composition during limb regeneration

The axolotl salamander can fully regenerate amputated limbs, yet the systemic consequences underlying this process remain largely understudied. Cerebrospinal fluid is an emerging signaling medium capable of communicating with both the central and peripheral nervous systems, but its composition and potential role in salamander limb regeneration have not yet been examined using modern multi-omics techniques. Here, we developed a protocol for extracting mixed perilymph-cerebrospinal fluid (P-CSF) from axolotl and provided the first proteomic and metabolomic characterization of this biological fluid. We identified 2,626 unique proteins and 173 high-confidence metabolites and quantified them across four time points of early limb regeneration. We demonstrated that limb amputation drives progressive shifts in P-CSF proteins, including an elevation of sarcomeric muscle proteins, regeneration-associated factors, and protease/extracellular matrix proteins. We observed shifts in metabolites involved in oxidative stress, polyunsaturated fatty acid oxidation, and histamine metabolism. Injury-comparison experiments revealed that the observed proteomic changes as a result of limb amputation are different than crush injury, denervation, or tail amputation. This study proposes axolotl P-CSF as a reservoir for limb amputation-associated systemic signaling and as a potential conduit of signals involved in limb regeneration.

systems biology↗

bioRxiv2026

TR-107, a novel mitochondrial ClpP agonist, induces robust antitumor activity against preclinical models of adrenocortical carcinoma

AbstractAdrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine malignancy originating from the adrenal cortex with limited effective treatment options. The underlying pathophysiology of ACC is uniquely characterized by abnormal steroid production and increased metabolic activity, highlighting the critical role of mitochondria in adrenal steroid hormone biosynthesis and tumor metabolism. In this study, we investigated the therapeutic potential of TR-107, a novel and highly selective small-molecule agonist targeting the mitochondrial protease ClpP. Pharmacologic hyperactivation of ClpP disrupts mitochondrial proteostasis and bioenergetics and has shown promising antitumor activity in various preclinical models. Our results demonstrated that TR-107 induces potent dose-dependent cytotoxic effects at nanomolar concentrations in ACC cell lines NCI-H295R and mACC3 as well as short-term ACC patient-derived organoid (PDO) models, markedly reducing cell viability and confluency in vitro. Metabolic analyses revealed that TR-107 significantly impaired oxygen consumption, indicating a disruption of oxidative phosphorylation and substantial attenuation of basal cellular respiration. Mechanistic studies showed dose-dependent increases in reactive oxygen species (ROS) levels and upregulation of proteins involved in mediating the ferroptotic rheostat. Pharmacokinetic assessment uncovered that TR-107 was not a substrate of the ABCB1 (MDR1/P-glycoprotein) efflux transporter, suggesting potential to overcome common multidrug resistance mechanisms. Given the importance of IGF-2 signaling in ACC, we further explored the combinatorial effects of TR-107 with IGF-1 receptor (IGF-1R) inhibitors and discovered that co-treatment produced synergistic reductions in cell viability across NCI-H295R, mACC3, and ACC PDOs. Collectively, these findings support the potential of mitochondrial ClpP hyperactivation as a promising therapeutic strategy for ACC and demonstrate that TR-107 exhibits significant antitumor activity as a monotherapy or in combination with IGF-1R inhibitors. These findings provide a strong rationale for advancing ClpP agonists into clinical development for the management of ACC.

cancer biology↗

bioRxiv2026

LncRNA-mediated organization of oncogenic chromatin state is a targetable vulnerability in leukemia

Long non-coding RNAs (lncRNAs) are increasingly recognized as critical regulators of gene expression underlying various cellular functions, however, the functional and mechanistic contributions of most lncRNAs to tumorigenesis remain poorly defined, and targeting of lncRNAs is challenging with conventional therapeutic approaches. Here, we uncover human PAN3-AS1 and its murine ortholog Lnc35682, previously uncharacterized lncRNAs embedded within a conserved syntenic genomic locus, as highly expressed in acute myeloid leukemia (AML). Using genetic mouse models, human cell lines and primary patient samples, we show that PAN3-AS1 is essential for leukemia maintenance but dispensable for normal hematopoiesis. Mechanistically, we find that elevated PAN3-AS1 influences chromatin accessibility, thus promoting leukemia gene expression programs. This is mediated, at least in part, by PAN3-AS1s association with the nuclear lamina through a defined functional region that is required for its leukemogenic function. We further characterize a feed-forward regulatory circuit between PAN3-AS1 and its neighboring gene FLT3 that directly links the aberrant lncRNA functions to the FLT3-mutant AML subtype. To therapeutically exploit the regulatory node, we engineer a myeloid leukemia-preferentially targeted lipid nanoparticle (LNP) formulation and demonstrate effective delivery of siRNAs against endogenous targets into leukemia cells in experimental animals. LNP-siPAN3-AS1 alone or in combination with a clinically used FLT3 inhibitor, Gilteritinib, reduces leukemia burden and significantly delay leukemogenesis in vivo. Overall, our study uncovers a therapeutic vulnerable lncRNA-centric circuitry and provides compelling preclinical evidence for the development and application of a novel RNA targeting-LNP based therapy for treatment of myeloid leukemia. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/744058v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@10c9d42org.highwire.dtl.DTLVardef@12864e7org.highwire.dtl.DTLVardef@f87f6aorg.highwire.dtl.DTLVardef@750cbf_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2026

History of Traumatic Brain Injury with Loss of Consciousness and APOE ϵ4 Carriers Synergistically Increase Late-Life Amyloid PET Burden

BackgroundTraumatic brain injury with loss of consciousness (TBI-LOC) is an established risk factor for dementia, yet the pathways linking remote TBI to Alzheimers disease (AD) biology remain incompletely defined. APOE {varepsilon}4 is the strongest genetic predictor of amyloid accumulation in late-onset AD, it may moderate the long-term consequences of head injury. This study investigates whether history TBI-LOC independently contributes or synergistically interacts with APOE {varepsilon}4 to amplify late-life amyloid and tau burden. Methods429 participants completed the Ohio State University TBI screening tool and an amyloid PET scan (centiloids). A subcohort (n=352) also underwent tau PET. TBI history was classified by recency (<10 vs >10 years) and severity (no TBI, dazing/confusion [TBI-DZ], TBI-LOC). Analyses were stratified by degree of clinical impairment as assessed by Clinical Dementia Rating (CDR=0 vs CDR>0). Logistic and linear regression models examined associations between TBI and amyloid, adjusting for age, sex, education, and APOE {varepsilon}4, including an APOE*TBI-LOC status interaction term, while Fishers exact tests evaluated TBI recency and biomarker positivity. ResultsIn CDR=0 participants (n=365), 119 reported a history of TBI, comprising 56 TBI-DZ and 63 TBI-LOC. TBI-LOC but not TBI-DZ, correlated with elevated amyloid PET levels (p<0.001; [4.6-17]). Furthermore, an interaction between APOE {varepsilon}4 and TBI-LOC indicated that TBI-LOC augmented the amyloid-related risk associated with the APOE {varepsilon}4 allele (p=0.003; [4.3-21]). The interaction persisted when stratified by TBI recency with only remote TBI-LOC (occurring more than 10 years prior) associated with increased amyloid PET (p=0.003 [5.2-25]). No association between TBI and tau was identified in a subset with tau PET, and no TBI-amyloid correlations were observed among symptomatic participants (CDR>0; n=64) suggesting a ceiling effect of pathology once clinical dementia is present. ConclusionsHistory of remote TBI-LOC is linked to elevated amyloid PET levels in later life, particularly among APOE {varepsilon}4 carriers with a CDR=0. The robust findings for amyloid, contrasted with null tau results and the reduced association in symptomatic cases underscore the importance of considering TBI history when screening for preclinical AD and assessing early-stage risk.

neuroscience↗

bioRxiv2026

Midazolam suppresses glioma progression by attenuating neuronal activity and downregulating IGF1 signaling

Glioma is the most common primary malignant tumor of the brain, and accumulating evidence indicates that neuronal activity plays a pivotal role in tumor progression. In this study, neuronal activity is modulated in vitro using potassium chloride (KCl)-induced depolarization and midazolam (MDZ)-mediated suppression. MDZ is a neuronal activity modulation medication, commonly used for sedation, anxiolysis, and amnesia in clinics. After treatment, conditioned media derived from these neuronal cultures are subsequently co-cultured with glioma cells. EdU incorporation assays demonstrate that MDZ significantly inhibits glioma cell proliferation in vitro. Furthermore, an orthotopic xenograft glioma model is established to assess the anti-tumor efficacy of MDZ in vivo, as evaluated by tumor volume and Ki-67 immunostaining. Mechanistically, insulin-like growth factor 1 (IGF1) is identified as the neuronal-activity-regulated factor that promotes glioma growth through activation of the PI3K/AKT signaling pathway. Moreover, transcriptomic profiling of brain tissues reveals that MDZ attenuates neuronal activity and downregulates neuron-derived growth factors in both glioma and non-tumor regions, thereby exerting anti-tumor effects in vivo. Collectively, these findings demonstrate that MDZ suppresses glioma progression by suppressing neuronal activity and inhibiting neuron-derived trophic factors, providing new insights into the development of therapeutic strategies for glioma.

neuroscience↗

bioRxiv2026

Ion Channel Nano-Diagnostics for ER+ Breast Cancer

Ion channels are pore-forming transmembrane proteins that allow ions to move down an electrochemical gradient and across the channel pore and regulate many cell functions. Among them, are the G-protein-gated inwardly-rectifying K+ channels 1 (GIRK1) that are ubiquitously expressed with major functions in the brain and heart. Interestingly, significantly higher GIRK1 expression has been found in estrogen receptor positive (ER+) breast cancer patients compared to patients with HER2+ tumors or normal patients, and that was statistically correlated with shorter survival times and metastatic potential. Herein, we report the preparation of [~]4 nm GAT1508-coated poly(ethylene glycol) gold nanoparticle (PEGylated AuNP) biomarker for ER+ breast cancer cell screening through an optical microscope. A urea-based small molecule, GAT1508, with an N-methylpyrazole benzyl group on one side and a bromo-thiophene tail on the other side, has been shown to predominantly bind GIRK1 subunits and specifically activate GIRK1/2 channels. Two derivatives of GAT1508were synthesized and characterized: an ethylamine derivative (GAT1508-EA) with a chain extension from the benzyl ring, and a propylamine derivative (GAT1508-PA) with a chain extension from the pyrazole ring. Electrophysiology (TEVC and whole-cell patch-clump) experiments as well as fluorescence studies (Thallium assay) showed that only GAT1508-PA inhibited GIRK1/2-mediated K+ currents in transfected HEK293GIRK1 cells. Docking studies showed strong binding for the propylamine GAT1508 derivative, both in the amine form (GAT1508-PA) as well as in the amide form (GAT1508-PA-EG2; coupled with PEG as in the AuNPs). GAT1508-PEG-AuNPs (GAT1508-NPs) were synthesized subsequently with [~]65 wt% metal loading. UV-Vis studies revealed the presence of the conjugated ligand at 260 nm. Flow cytometry studies showed binding of Alexa 594-labeled GAT1508-NPs in ER+ MCF-7 breast cancer cells with a strong interaction, while incubation of fixed MCF-7 cells with a GAT1508-NP solution led to optical detection of ER+ breast cancer cells, without the need of fluorescent dyes and additional amplification steps. Detection was not feasible in MDA-MB-231 cells, a triple (-) breast cell line that does not express GIRK1. This is the first study, to our knowledge, that couples nanotechnology with small molecule drug design and electrophysiology to develop ion channel-tracing molecular probes for the detection/screening of ER+ breast cancer.

bioengineering↗

bioRxiv2026

Protein-guided RNA barcoding links transcriptomes to synaptic architecture

Mammalian brain function relies on the precise synaptic architecture of diverse cell types, yet scalable methods for linking a neurons transcriptomic profile to its neuroanatomy remain limited. We present Synapse-seq, an in vivo strategy in which cell-identifying barcoded mRNAs are routed to subcellular compartments via targeting proteins and detected by single-cell and spatial genomics. Using AAV delivery for minimal perturbation of gene expression, we directed barcodes to presynaptic terminals (via synaptophysin) in four distinct circuits, or to postsynaptic sites (via nanobodies to endogenous PSD95) of hippocampal excitatory neurons. In the mouse primary visual cortex, presynaptic Synapse-seq recovered known long-range projections and discovered cortical layer subtypes with distinct thalamic innervation. In the anterior cortex, we elucidated simple topographic rules of corticostriatal innervation: intratelencephalic neurons followed a continuous depth-to-target gradient, while extratelencephalic neurons exhibited striatal collaterals that spatially correlated with medullary innervation. Finally, postsynaptic barcoding of excitatory neurons revealed cell type-specific variation in dendritic architectures across and within hippocampal subfields. These data establish Synapse-seq as a versatile, genomics-based approach for the integrated definition of molecular identity and synaptic organization across mammalian brains.

neuroscience↗

bioRxiv2025

OmniShim - a vendor-independent B0 Shimming software toolbox

PurposeTo introduce the OmniShim Toolbox, a software package designed to calibrate and perform B0 shimming in user-defined regions of interest across systems from different vendors, supporting various shim orders. MethodsIn this study, we systematically compared the performance of vendor-implemented B0 shim routines with our custom developed OmniShim toolbox, designed to improve static magnetic field homogeneity. Single-voxel magnetic resonance spectroscopy (MRS) data were acquired from the prefrontal cortex and occipital lobe, while magnetic resonance spectroscopic imaging (MRSI) data were collected from regions above and below the corpus callosum in healthy volunteers. Measurements were conducted on both 3T and 7T MR systems to evaluate the robustness and scalability of each B0 shimming strategy across different field strengths. Additionally, functional MRI (fMRI) data were acquired at 7T to assess the impact of improved shimming on EPI data quality and BOLD contrast. ResultsThe OmniShim Toolbox demonstrated superior B0 homogeneity across all applications and field strength, leading to reduced signal dropout in fMRI and MRSI data, significantly improved spectral linewidths in SV MRS and MRSI data as well improved detection of neural networks by resting state fMRI. ConclusionThe proposed OmniShim Toolbox offers a robust and flexible approach to control B0 inhomogeneity, resulting in substantial improvements in image quality and spectral resolution. These enhancements benefit a broad range of MR applications.

bioengineering↗

bioRxiv2025

Dysregulation of placental mitochondrial structure dynamics and clearance in maternal obesity and gestational diabetes

IntroductionThe placenta is exposed to an altered metabolic environment in obesity and gestational diabetes (GDM) leading to disruption in placental function. Mitochondria are critical for energy production and cellular adaptation to stress. We previously reported reduced trophoblast mitochondrial respiration in GDM. Here we examine changes in mitochondrial structure dynamics, quality and protein homeostasis as well as clearance in both obese and GDM placentas of male and female fetuses. As obesity significantly increases the risk for GDM, our goal is to determine the distinct effects of each on placental mitochondria. MethodsWe collected placental villous tissue following elective cesarean section at term from lean (LN, pre-pregnancy BMI 18.5-24.9), obese (OB, BMI>30) or obese with type A2 GDM women. Expression of proteins involved in mitochondrial biogenesis, structure dynamics, quality control and clearance were assessed by Western blotting. Significant changes between groups were determined in fetal sex-dependent and independent manner. ResultsOnly placentas from obese women showed increase in proteins regulating mitochondrial biogenesis (PGC-1 and SIRT1). We report fetal sex-specific changes in mitochondrial fusion but an overall decline in fission in OB and GDM placentas. Both maternal obesity and GDM affected proteins involved in maintaining mitochondrial protein quality and genome stability. This was accompanied by a reduction in mitochondrial complexes, suggesting impaired mitochondrial function. Obesity led to partial activation of mitophagy pathways (e.g., increased PINK1 without PARKIN activation), GDM placentas failed to mount this response. DiscussionObesity and GDM affect placental mitochondria through distinct complex sex-specific mechanisms that may contribute to altered mitochondrial function.

molecular biology↗

bioRxiv2025

Quantitative analysis of genetic interactions in human cells from genome-wide CRISPR-Cas9 screens

Genetic interaction (GI) networks in model organisms have revealed how combinations of genome variants can impact phenotypes. To advance efforts toward a reference human GI network, we developed the quantitative Genetic Interaction (qGI) score, a method for precise GI measurement from genome-wide CRISPR-Cas9 screens in different query mutants constructed in a single human cell line. We found surprising prevalent systematic variation unrelated to GIs in CRISPR screen data, including both genomically linked effects and functionally coherent covariation. Leveraging [~]40 control screens in wild-type cells and half a billion differential fitness effect measurements, we developed a pipeline for CRISPR screen data processing and normalization to correct these artifacts and measure accurate, quantitative GIs. We also comprehensively characterized GI reproducibility by characterizing 4 - 5 biological replicates for [~]125,000 unique gene pairs. The qGI framework enables systematic identification of human GIs and provides broadly applicable strategies for analyzing context-specific CRISPR screen data.

bioinformatics↗

bioRxiv2025

CRISPR-Based Gene Dependency Screens reveal Mechanism of BRAF Inhibitor Resistance in Anaplastic Thyroid Cancer

Anaplastic thyroid cancer (ATC) is the most aggressive form of thyroid cancer. Despite recent advances in treating BRAFV600E-driven ATC, therapy resistance remains a significant challenge, often resulting in disease progression and death. Leveraging a focused CRISPR/KO screen in parallel with a CRISPR/activation screen, both tailored on response to BRAFV600E inhibitor treatment, we identified TAZ (encoded by the WWTR1 gene) deficiency as synthetically lethal with BRAF inhibitor in ATC. TAZ is overexpressed in ATC compared to well-differentiated thyroid tumors. We demonstrate that TAZ-deficient ATC cells display heightened sensitivity to BRAF inhibitors both in vitro and in vivo. Using gene essentiality score across a large panel of cancer cell lines, we found that BRAFV600E-driven cancers are highly sensitive to TAZ loss, unlike their counterparts with wild-type BRAF and non-BRAFV600E. Mechanistically, we demonstrate that dabrafenib triggers the Unfolded Protein Response (UPR) under ER stress and suppresses protein synthesis. TAZ loss represses the UPR, reverses the inhibition of protein synthesis, and triggers increased cell death by ferroptosis in dabrafenib-treated ATC. Collectively, our findings unveil TAZ as a new target to overcome resistance to BRAF inhibitors in undifferentiated thyroid cancer.

cancer biology↗

bioRxiv2025

Differential activation of p53-Lamin A/C and p16-RB mediated senescence pathways in trophoblast from pregnancies complicated by type A2 Gestational Diabetes Mellitus

Gestational diabetes mellitus (GDM) increases maternal risks such as hypertension and future type 2 diabetes while also contributing to fetal complications such as large-for-gestational-age infants and stillbirth. The placenta which is crucial for fetal development, exhibits structural and functional changes in GDM, but the impact of these alterations on placental trophoblast function remains unclear. During their differentiation villous cytotrophoblast display several characteristics of senescent cells however the role of senescence pathways in placental function remains unexplored in GDM. Here we investigate whether placental senescence pathways are altered in GDM, utilizing term villous tissue and primary trophoblasts to assess molecular changes, and determined fetal sex-based differences. Our data suggest that both p21 and p16 mediated senescence pathways are activated during trophoblast differentiation and are dysregulated in GDM placenta in a sexually dimorphic manner. We also provide evidence for increased activation of p53-Lamin A/C and p16-RB pathways in trophoblast from GDM placentas. Reduced expression of p21 and its downstream effects on GCM1 expression and {beta}hCG secretion outline how altered physiological senescence can affect trophoblast differentiation and function. This is a seminal study highlighting how placental senescence pathways are altered in pregnancies complicated by GDM.

cell biology↗

bioRxiv2025

Local nucleolar surveillance mechanisms maintain rDNA stability

The nucleoli are subdomains of the nucleus that form around actively transcribed ribosomal RNA (rRNA) genes. The highly repetitive and transcribed nature of the rRNA genes (rDNA) by RNA polymerase I (Pol I) poses a challenge for DNA repair and replication machineries. Here, we profile the nucleolar proteome and the chromatin landscape of stalled replication sites upon rDNA damage to characterize the early steps of nucleolar DNA damage response (nDDR). We observed early dynamics in nucleolar-nucleoplasmic proteome localization and identified nucleolar replication stress signatures involving chromatin remodeling networks, transcription-replication conflicts and DNA repair. Our findings define localized surveillance mechanisms that activate the nDDR. Further, we identified that upon rDNA damage, nucleolar RNA Polymerase (Pol) II binds to intergenic rDNA sequences and generates R-loops (DNA:RNA hybrid structures) that are essential for recruiting nDDR factors. Using a boutique CRISPR-Cas9 synthetic lethal screen of DNA repair factors with inhibitors of RNA Pol I transcription, we identified an unexpected protective role for the DNA translocase RAD54L in nDDR. Loss of Rad54L increases nucleolar R-loops and rDNA damage leading to defects in nucleolar structure and enhanced sensitivity to PARP and RNA Pol I inhibitors. Altogether, our study uncovers localized surveillance networks within the nucleolus that respond to rDNA damage. These insights expand our understanding of the molecular mechanisms governing nDDR and opens new avenues for developing nDDR-targeting therapies.

molecular biology↗

bioRxiv2024

Chromatin Remodelling in Damaged Intestinal Crypts Orchestrates Redundant TGFβ and Hippo Signalling to Drive Regeneration

Cell state dynamics underlying regeneration are under-characterized. Intestinal damage prompts reprogramming into revival stem cells (revSCs) that reconstitute Lgr5+ intestinal stem cells (ISCs). Single nuclei multiomics of chromatin accessibility and transcriptomes during regeneration from irradition showed revSCs display epigenetic profiles shared with ISCs and differentiated lineages. Furthermore, while revSC genes are accessible throughout homeostatic epithelia, damage-induced global alterations in crypt and revSC chromatin converge on TGF{beta}, as well as Hippo pathways. We show TGF{beta} directly induces functional revSCs and demonstrate individual revSCs form organoids with reconstituted Lgr5+ ISCs. Despite this, loss of TGF{beta} signalling yielded mild regenerative defects. In contrast, interference in both Hippo and TGF{beta} abolished revSCs, precluded generation of new ISCs and led to rapid intestinal collapse. Thus, the epithelium is poised to engage the revSC regenerative program that relies on crypt-localized, transient morphogen cues that function in a compensatory manner to support intestinal regeneration.

molecular biology↗

bioRxiv2024

Genome Instability Precedes Viral Integration in Human Papilloma Virus Transformed Tonsillar Keratinocytes

Approximately 70% of oropharyngeal squamous carcinomas (OPSCC) are associated with human papillomavirus (HPV). While HPV-positive (HPV+) OPSCC responds better to standard therapies and patients with HPV+ tumor generally have better outcomes than those with HPV-negative (HPV-) tumors, a subset of HPV+ patients do have poor outcomes. Our previous work suggested that tumors with integrated virus exhibit significantly greater genome wide genomic instability than those with only episomal viral genomes and patients with HPV+ OPSCC with episomal viral genomes had better outcomes. To explore the causal relation between viral integration and genomic instability, we have examined the time course of viral integration and genetic instability in tonsillar keratinocytes transformed with HPV16. HPV-infected human tonsil keratinocyte cell lines were continuously passaged and every fifth passage some cells were retained for genomic analysis. Whole genome sequencing and optical genomic mapping confirmed that virus integrated in five of six cell lines while remaining episomal in the sixth. In all lines genome instability occurred during early passages, but essentially ceased following viral integration but continued to occur later passages in the episomal line. To test tumorigenicity of the cell lines, cells were injected subcutaneously into the flanks of nude mice. A cell line with the integrated virus induced tumors following injection in the nude mouse while that with the episomal virus did not. We conclude that genomic instability is not the result of viral integration but likely promotes integration. Moreover, those transformants with episomal virus appear to be less tumorigenic than those with integrated virus.

cancer biology↗