title: PD0325901 Alleviates Thrombin-Inhibited Osteogenic Differentiation Through an IL-1β-Activated Feedback Loop between MEK-Erk1/2 and NF-κB Signal Pathways: Insights from Bioinformatics and Experimental Verification

city: Shanghai

first: Chunmeng

last: Huang

address: Changjiang Rd

institute: The Second Rehabilitation Hospital of Shanghai

summary: Thrombin transcriptionally induces inflammatory genes and suppresses osteogenic markers via RNA-seq identified pathways. The MEK inhibitor PD0325901 (PD03) is identified to be a key modulator targeting the IL-1β-MMP-9 axis. Our finding highlights that PD03 inhibits thrombin-induced suppression of osteoblast differentiation through repressing IL-1β-mediated MEK-Erk1/2 and NF-κB feedback loop and restoring osteogenic gene expression.

overall_design: The experimental design involved isolating primary osteoblasts from the calvaria of 24-hour-old male Sprague-Dawley rats using 0.1% collagenase type I digestion, culturing them in high-glucose DMEM with 10% fetal bovine serum and penicillin-streptomycin, and using first-passage cells for analyses; for osteogenic differentiation, cells at 75% confluence were induced in medium containing 10% fetal bovine serum, 10 mM β-glycerophosphate, 50 µg/mL L-ascorbic acid, and 10^{-7} M dexamethasone, with groups including control (induction medium alone), thrombin (20 U/mL thrombin), PD03 (20 U/mL thrombin + 0.1 µM PD0325901), C188 (20 U/mL thrombin + 5 µM C188-9), QNZ (20 U/mL thrombin + 0.1 µM QNZ), and Vorapaxar (20 U/mL thrombin + 0.1/0.5/1 µM Vorapaxar), inducing for 7 days to assess gene expression or 21 days for mineralization; transient transfection used Lipofectamine 2000 with pCDH-GFP-COX-2 plasmid or MMP-9 siRNA, verifying efficiency by western blot and evaluating osteogenic markers after 7 days; RNA-seq on 7-day induced cells (three replicates per group) involved library construction on Illumina HiSeq 4000.

first: Yangshuo

last: Ge

first: Jun

last: Shen

first: Tingting

last: Meng

first: Minjun

last: Zhao

first: Jianli

last: Yin

first: Xinhui

first: Liaolin

first: Jiahui

last: Luo

first: Yuqing

last: Zhai

first: Jiawei

last: Du

first: Yilin

last: Wang

first: Xuezong

first: Pingping

last: Sun

first: Daofang

last: Ding


title: Genetically targeted mTORC1 inhibitor reveals transcriptional control by nuclear mTORC1

status: Public on Feb 24 2026

city: La Jolla

first: Jin

last: Zhang

state: California

address: 9500 Gilman Dr.

department: Pharmacology

country: USA

institute: University of California San Diego

type: Expression profiling by high throughput sequencing

summary: mTORC1 is a nutrient sensor which integrates diverse inputs to regulate protein translation and cell growth. While mTORC1 is activated on the lysosome in the classical model, it has become increasingly clear that this multifaceted signaling complex is active at various subcellular locations, such as the nucleus. However, what specific functions mTORC1 serves at these locations and how its signaling is compartmentalized are not unclear. To interrogate subcellular pools of mTORC1, we developed TerminaTOR, a genetically encodable inhibitor of mTORC1 that can be targeted to specific subcellular locations. When TerminaTOR is directed to the lysosome, it inhibits canonical lysosomal mTORC1 and induces autophagy. Furthermore, TerminaTOR targeted to the nucleus specifically inhibits nuclear mTORC1, uncovering non-canonical roles of nuclear mTORC1 in regulating the transcription of CCAAT motif-containing genes. Thus, mTORC1 exhibits functional spatial compartmentalization, and TerminaTOR serves as a powerful tool for unraveling spatially regulated functions of mTORC1 across different scales

overall_design: We generated two doxycycline inducible stably NIH3T3 cell lines that express H2A-mCherry (Control) or H2A-TerminaTOR (Nuclear TerminaTOR), which specifically inhibits nuclear mTORC1 activity. We then starved and stimulated the cells with PDGF to induce nuclear mTORC1 activity. We collected RNA samples and conducted RNA-seq to understand the nuclear mTORC1-controlled transcription.

first: Yanghao

last: Zhong

first: Ayse

middle: Z

last: Sahan

first: Zhengyao

last: Shao

first: Qianyi

first: Xin

last: Zhou

first: Jack

middle: G

last: Haggett

first: Keiichi

last: Koshizuka

first: Samuel

middle: A

last: Myers

first: Jorge

middle: S

last: Gutkind


title: RNA-seq analysis of liquid-induced suspended animation (LISA) in C. elegans

city: San Francisco

first: Junqiang

last: Liu

state: CA - California

address: 555 Mission Bay Blvd South

institute: University of California, San Francisco

summary: To elucidate the molecular landscape associated with liquid-induced suspended animation (LISA) in C. elegans, we performed transcriptomic profiling of animals subjected to high-density isosmotic liquid conditions. Wild-type N2 animals were maintained at 20 °C. To induce LISA, synchronized L4 larvae were incubated under static high-density liquid conditions (“standing” treatment) for 12 hours, while control animals were maintained under standard plate culture conditions prior to collection.  At the time of sample collection, animals were washed off NGM plates using M9 buffer, collected into 1.5 mL microcentrifuge tubes, and homogenized using a tissue disruptor. Total RNA was extracted using the FastPure Cell/Tissue Total RNA Isolation Kit V2 (RC112-01, Vazyme) according to the manufacturer’s instructions. RNA concentration and purity were measured using a NanoDrop spectrophotometer, and RNA integrity was assessed by agarose gel electrophoresis. For each sample, 1 µg of total RNA was used for library preparation and sequencing. Three  biological replicates were included for each condition.  Transcriptomic analysis revealed robust induction of stress-responsive genes during LISA. Notably, members of the small heat shock protein family, particularly the hsp-16 gene cluster, were among the most strongly upregulated transcripts. This induction was sharply confined to the genomic hsp-16 cluster without affecting adjacent genes. These findings define a distinct LISA-associated transcriptional state characterized by coordinated stress-responsive gene expression, consistent with a dormant yet physiologically primed condition.

overall_design: For standing treatment, synchronized L4 larvae were either subjected to standing 12 hours or maintained under control conditions prior to collection.

first: Dengke

last: Ma


title: Nitrogen metabolism profiling reveals cell state-specific pyrimidine synthesis pathway choice

city: Dallas

middle: Kent

last: McBrayer

state: Texas

address: 6000 Harry Hines Boulevard, NL11.110K

department: Children's Research Institute

institute: UT Southwestern Medical Center

summary: Stable isotope tracing assays track few metabolites, yet cells use many nutrients to sustain nitrogen metabolism. Here, we create a platform for tracing 30 nitrogen isotope-labeled metabolites in parallel to enable a system-level understanding of cellular nitrogen metabolism. This platform reveals that while primitive cells engage both de novo and salvage pyrimidine synthesis pathways, differentiated cells nearly exclusively salvage uridine. This link between cell state and pyrimidine synthesis pathway preference persists in murine and human tissues. Mechanistically, we find that S1900 phosphorylation of CAD, the first enzyme of the de novo pathway, is induced by uridine deprivation in differentiated cells and constitutively enriched in primitive cells. Mimicking CAD S1900 phosphorylation in differentiated cells constitutively activates de novo pyrimidine synthesis, while blocking this modification impairs the cellular response to uridine starvation. Collectively, we establish a method for nitrogen metabolism profiling and define a mechanism of cell state-specific pyrimidine synthesis pathway choice.

overall_design: We performed RNA sequencing on HNF, NPC, neurons, and differentiating neurons under varying uridine concentrations.

first: Milan

middle: R

last: Savani

first: Bingbing

last: Li

first: Tracey

last: Shipman

middle: K


title: Selective targeting of a histone-like silencer Sfx to the R6K conjugal transfer operon [ChIP-Seq]

city: Columbus

first: Irina

last: Artsimovitch

state: Ohio

address: 318 W 12th Ave

department: Microbiology

institute: The Ohio State University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Conjugative plasmids drive bacterial evolution and the spread of antibiotic resistance, yet their gene expression must be silenced to protect the host. A histone-like protein H-NS represses many mobile and sedentary xenogenes but fails to silence the conjugal transfer vir operon of R6K, a prototype IncX plasmid. Instead, R6K encodes its own H-NS homolog, Sfx, to repress the vir operon. Here, we show that, unlike other plasmid silencers that target promoters, Sfx cooperates with Rho factor to arrest transcription elongation. ChIP-seq reveals that despite sharing similar DNA motifs and a preference for negative supercoiling, Sfx and H-NS occupy distinct niches: Sfx binds weakly to the chromosome but is enriched on the R6K vir operon while H-NS is excluded. We hypothesize that this selective targeting is mediated by Sfx-vir DNA interactions and phase separation. We show that Sfx binding to vir DNA critically depends on DNA topology but not on the target location. Our results suggest that Sfx forms stable nucleoprotein filaments that are impermeable to competitors and phase separates with R6K DNA to exclude H-NS. Our results suggest how functionally distinct histone-like proteins may achieve target specificity despite broad recognition motifs.

overall_design:
ChIP-seq assays for comparing the binding sites of H-NS and its homolog Sfx encoded on R6K plasmid in Escherichia coli. The sequences of all plasmids used in this study can be found in the accompanying manuscript.
ChIP-seq of RNA polymerase in E. coli carrying the Sfx deletion R6K plasmid to identify potential promoters in R6K.
RNA ChIP-seq assay to identify RNAs ChIPed by FLAG-tagged Sfx.

first: Bing

last: Wang


title: Selective targeting of a histone-like silencer Sfx to the R6K conjugal transfer operon [RNA-Seq]

summary: Conjugative DNA transfer is mediated by large membrane-spanning type IV secretion system (T4SS) complexes encoded by long plasmid operons (commonly called tra or vir). Conjugative IncX plasmids encode histone-like protein H-NS homolog as core gene. R6K, a prototype IncX plasmid, encodes its own H-NS homolog, Sfx, to repress the vir operon. Here, we show that, unlike other plasmid silencers that target promoters, Sfx cooperates with Rho factor to arrest transcription elongation. Our results show that Sfx mainly targets the R6K vir operon.

overall_design: RNA-seq profiling of E. coli harboring plasmid R6K derivatives to detect the targets of histone-like silencer Sfx.


title: Metformin reduces RAN proteins and rescues molecular and behavioral phenotypes in SCA8 mice

city: Gainesvillle

first: Lisa

last: Romano

state: Florida

address: 2033 Mowry Road

department: Molecular genetics and microbiology

institute: University of Florida

summary: Spinocerebellar ataxia type 8 (SCA8) is a member of a group of dominantly inherited, debilitating neurological diseases caused by CAG•CTG expansions for which there are no effective disease-targeting treatments. RAN translation, which was discovered in SCA8, has previously been shown to occur across CAG and CUG expansion transcripts, making treatments that work for SCA8 potentially relevant to a broader group of diseases, including SCA1, 2, 3, 6, 7, 12, Huntington’s Disease (HD), Fuch’s endothelial corneal dystrophy (FECD), and myotonic dystrophy type 1 (DM1). Additionally, CUG and CAG expansion transcripts have been reported to cause RNA gain-of-function effects. In C9orf72 ALS/FTD mice, we previously showed that metformin reduced RAN protein levels and improved behavior and neurological phenotypes. Using SCA8 BAC transgenic mice as a model for CAG•CTG expansion diseases, we now show that metformin improves ambulatory performance using rotarod, DigiGait, and open-field measures. At the molecular level, metformin-treated mice showed reduced RAN protein levels and improved alternative splicing abnormalities without changing the levels of the expanded RNAs. Metformin-treated mice also showed decreased neuroinflammation, with reduced astrogliosis and fewer activated microglia. These data provide strong preclinical support for testing metformin in clinical trials for SCA8 and potentially the broader group of CAG•CTG repeat expansion disorders.

overall_design: In this study, we sought to determine whether metformin can prevent and/or reverse the behavioral and pathological phenotypes of SCA8 transgenic mice. Because RAN proteins start to accumulate early in the disease, we designed our study by treating female and male SCA8 BAC transgenic mice beginning at the age of 4 weeks, well before the development of behavioral and pathological phenotypes. During the treatment period of 60 weeks, animals were assessed for behavioral defects at 8, 16, 32, and 52 weeks. Final take-downs were done at 60 weeks of age. RNA-seq was runned on brainstem and cerebellum from 4 animal per group, including 4 Non-Transgenic mice treated with water (NT_Water), 4 SCA8 mice treated with water (SCA8_Water) and 4 mice treted with metformin (SCA8_Met).

middle: E

first: Laura

middle: P

last: Ranum


title: Tissue resident memory CD4+ T cells after neonatal or adult HMPV infection

city: Pittsburgh

first: Taylor

last: Eddens

state: PA

address: 4401 Penn Avenue, Rangos Research Building 9128

institute: University of Pittsburgh

summary: Tissue resident memory cells are long-lived sentinels that are generated after exposures at mucosal sites. In this experiment, neonatal (day of life 4-6) or adult (6-8 week old) mice were infected with human metapneumovirus (HMPV). 5 weeks following infection, tissue resident cells were identified using i.v. exclusion of circulating cells and flow sorted. Cells were then multiplexed and processed using the 10X genomics platform.

overall_design: Experimental conditions include: NeoTRMs (TRMs isolated from the lung 5 weeks after neonatal mouse infection with HMPV) or AdultTRMs (TRMs isolated from the lung 5 weeks after adult mouse infection with HMPV)

first: Kong

last: Chen

first: Li

last: Fan


title: Early fibrotic niches establish tumour-permissive microenvironments [Experiment 3]

city: New York City

first: Joo-Hyeon

last: Lee

state: NY

address: 430 East 67th Street

department: Developmental Biology

institute: Memorial Sloan Kettering Cancer Center

summary: Pathologic transformation represents a pivotal yet poorly defined window during which early alterations in epithelial stem cells remodel their surrounding niche to prime tumour initiation. Here, we integrate single-cell, spatial, and functional analyses to map the dynamics of this early multicellular reorganisation. We show that KrasG12D-mutant alveolar type II cells undergo an early reprogramming transition that transforms them into signalling hubs, with the Amphiregulin (Areg)-EGFR axis emerging as a central driver of cross-compartmental cooperation. Mutant epithelial cells secrete Areg to activate EGFR in adjacent fibroblasts, eliciting a regenerative-like fibrotic programme. These reprogrammed fibroblasts, in turn, reshape the immune landscape by expanding and reprogramming alveolar macrophages, amplifying inflammatory signalling, immune recruitment, and epithelial plasticity. Together, these interactions establish a self-reinforcing multicellular circuit that generates and maintains a tumour permissive niche. Disrupting that circuit through genetic and pharmacological perturbation of the Areg-EGFR axis prevents both early niche cell reprogramming and tumour formation. Findings from KRASG12D-inducible human alveolar organoids and early-stage lung adenocarcinoma tissues confirm conservation of these interactions, identifying early mutant epithelial-stromal crosstalk as a targetable step in pathologic transformation with potential to avert establishment of treatment-resistant disease.

overall_design: At 2 weeks post two doses of tamoxifen induction (0.2 mg/gbw) every other day, lung tissues were collected from Sftpc-CreERT2; Red2Kras; Aregflox/+ and Sftpc-CreERT2; Red2Kras; Aregflox/flox animals. Lungs from three mice of the same genotype were dissociated and pooled into a single-cell suspension. Lineage-labelled epithelial cells (EpCAM+RFP+) and a mixed population of mesenchymal and immune cells (1:1 ratio of mesenchymal cells (CD45–CD31–EpCAM–) and immune cells (CD45+CD31–EpCAM–)) were then sorted. For each genotype, two separate libraries were generated, resulting in a total of four libraries (2 x Aregflox/+ and 2 x Aregflox/flox). Libraries were sequenced using Illumina Novaseq 6000.

first: Erik

last: Cardoso

first: Jinwook

last: Choi


title: Targeting systemic and tumor metabolic balances with ketogenic diets enhances efficacy of therapy in FLT3-ITD acute myeloid leukemia

city: Strassen

first: Tony

last: KAOMA

address: 1 A-B Rue Thomas Edison

department: Quantitative Biology Unit

country: Luxembourg

institute: Luxembourg Institute of Health

summary: FMS-like tyrosine kinase 3 (FLT3) mutations in acute myeloid leukemia (AML) are associated with adverse prognosis. FLT3 inhibitors (FLT3i) improve therapeutic response, however diverse resistance mechanisms such as adaptations in lipid metabolism have been identified. We hypothesized that a lipid-rich ketogenic diet (KD) might alter both host and tumoral lipid metabolism, enhancing responses to FLT3i. In FLT3-mutated AML mouse models, three weeks of lard- or plant-based KD improved efficacy of FLT3i by two-fold reduction of engraftment and tumor burden. KD increased ketone bodies and lipid accumulation in plasma, liver and AML cells, and also induced a PUFA:MUFA imbalance. KD impacted pentoses, hexoses and amino acid metabolism, enhancing sugar phosphates and vitamins in host. Mechanistically, KD rewired anabolism towards fatty acid oxidation and glycine-utilizing pathways, modulated the expression of FLT3 signaling pathways and lipid biosynthesis, and promoted tumor cell differentiation. In conclusion, this study shows that KD reduces FLT3i-resistance, offering a promising therapeutic solution.

overall_design: CLDX model with MOLM14 cell engraftment treated with ketogenic diet versus standard diet (3 weeks) and gilteritinib versus vehicle (1 week); Dissection liver and bone marrow.

first: Jean-Emmanuel

last: Sarry

first: Fanny

last: Granat

first: Léa

last: Goupille

first: Ambrine

last: Sahal

last: Kaoma


title: RNA-seq analysis of mouse spinal cord after contusion injury with Syringin treatment

city: Dongying

first: Dejing

last: Zhang

state: Shandong

address: NO.31 Jinan Road

country: China

institute: Shengli Oilfield Central Hospital

summary: We performed RNA-seq to investigate the transcriptomic changes in mouse spinal cord at 3 days post-contusion injury, and to evaluate the effects of Syringin treatment (50 mg/kg daily for 3 days) compared to PBS control.

overall_design: Spinal cord contusion model was established in mice. Animals were divided into two groups: SCI group (subjected to contusion and treated with PBS) and SCI+Syr group (subjected to contusion and treated with Syringin at 50 mg/kg daily for 3 days). Spinal cord tissues were harvested at day 3 post-injury for RNA-seq. Three biological replicates per group.

first: Tao


title: Early fibrotic niches establish tumour-permissive microenvironments [Experiment 1]

overall_design: At 2 weeks post three doses of tamoxifen induction (0.2 mg/gbw) every other day, lung tissues were collected and mesenchymal cells (CD45–CD31–EpCAM–) were isolated from SftpcCreERT2; Confetti and Sftpc-CreERT2; Red2Kras mice. Cells from three mice of the same genotype were pooled into a single cell suspension to generate two separate libraries (1 x Confetti and 1 x Red2Kras), which were sequenced via 10x Genomics using Illumina Novaseq X 1.5B.


title: Facile Induction of Immune Tolerance by an Interleukin-2-TGF-β Surrogate Agonist

city: Palo alto

first: Qinli

last: Sun

address: 279 Campus Drive

institute: Stanford University

summary: CD4⁺ regulatory T (Treg) cells are essential for establishing and maintaining immune tolerance. Peripherally induced Tregs (pTregs), which differentiate from mature conventional CD4⁺ T cells in the periphery, complement Tregs generated in the thymus during T cell development by broadening Treg reactivity in response to changing self and the environment. Although both TGF-β and Interleukin-2 (IL-2) synergistically promote functional pTreg development in vitro, their combined roles in inducing pTreg generation in vivo have not been thoroughly characterized and therefore are yet to be exploited for tolerizing immunotherapy. The principal limitation to in vivo use of IL-2 and TGF-β is their pleiotropy and inability to ensure co-delivery of SMAD and STAT signaling to the same cell. To overcome these challenges, we designed an IL-2-TGF-β surrogate agonist by creating a fusion protein between IL-2 and a low-affinity TGF-β mimic agonist derived from a Helminth parasite. This IL-2-TGF-β surrogate agonist was easily expressed and purified and enabled simultaneous cis-activation of both IL-2 and TGF-β signaling specifically in IL-2 receptor-expressing T cells. This IL-2-TGF-β surrogate agonist robustly induced antigen-specific, functional, and stable pTregs in vivo within peripheral lymphoid organs in OVA- or MOG35-55-immunized mice. The induced pTregs phenotypically resemble effector and colonic Rorγt⁺ pTregs, with the potential to efficiently migrate into the gut and suppress intestinal inflammation. Treatment with this agonist effectively quelled immune activation in mouse models of allergen-induced allergic inflammation and self-antigen-driven autoimmune neuroinflammation, portending a strategy for the targeted induction of antigen-specific pTregs in vivo to induce immune tolerance in inflammatory and autoimmune diseases.

overall_design: One million sorted Thy1.1⁺ naïve OT-II cells were adoptively transferred into Thy1.2⁺ Foxp3-GFP recipient mice via retro-orbital intravenous injection. This was followed by six intraperitoneal injections of 100 μg OVA protein combined with 50 pmol of the indicated proteins, administered every other day. On day 11, donor OT-II cells and host Foxp3-GFP⁺ CD4⁺ T cells were sorted from the mLNs of the respective treatment groups and submitted to MedGenome, Inc. for library preparation and RNA sequencing.

first: Masato

last: Ogishi

first: K Christopher

last: Garcia


title: Suppressive Genetic Interactions Between Haploinsufficient Mitochondrial Genes Encoded in the 22q11.2 Microdeletion Locus Define Brain and Cardiac Phenotypes

city: Middlebury College

first: Amanda

last: Crocker

state: Vermont

address: 276 Bicentennial Way

department: Neuroscience

institute: Middlebury College

summary: Genomic copy number variations, such as the 22q11.2 microdeletion syndrome, cause pleiotropic disorders that affect diverse organ systems and disrupt neurodevelopment. Deletions of the 22q11.2 locus reduce the dosage of up to 46 protein coding genes, raising questions about the identity of haploinsufficient genes and their genetic interactions contributing to 22q11.2 phenotypes. Here, we dissect functional and molecular relationships between two genes encoded within the 22q11.2 locus: the mitochondrial ribosomal protein gene MRPL40 and the mitochondrial citrate transporter SLC25A1. We show that a MRPL40 null mutation disrupts mitochondrial translation, impairs respiration, and affects multiple components of the SLC25A1 interactome including factors required for lipid metabolism, mitochondrial ribosome subunits, and the mitochondrial RNA processing machinery. In silico coessentiality network analysis revealed correlated and anticorrelated fitness interactions linking MRPL40 and SLC25A1 to mitochondrial translation, intermediate carbon metabolism, and interferon signaling. We determined that Mrpl40-null mutations are embryonic lethal in mice, but Mrpl40-/+ mice are viable and displayed embryonic cardiac development and adult behavioral phenotypes. Similarly, Slc25a1-/+ animals showed embryonic cardiac developmental defects but lacked the adult behavioral phenotypes observed in Mrpl40-/+ mice. Surprisingly, transheterozygotic Slc25a1-/+;Mrpl40-/+ mice suppressed or mitigated cardiac development, behavioral, and brain transcriptome phenotypes observed in single heterozygotic animals. These results reveal that MRPL40 and SLC25A1 are haploinsufficient genes within the 22q11.2 locus that genetically and biochemically interact to define tissue development and physiology. Our findings provide a framework for understanding the complexity and type of gene dosage interactions within the 22q11.2 deletion syndrome locus.

overall_design: RNA-seq profiling of cortex and hippocampus of WT, Mrpl -/+ heterozygous, Slc25a1 -/+ heterozygous, and Mrpl -/+, Slca1 -/+ double heterozygous animals.

first: Victor

last: Faundez

first: Stephanie

last: Zlatic


title: circular RNAs in RNASEK depleted cells

city: Daejeon

first: Seung-Jae

middle: V

address: 291 Daehak-ro

department: Biological Sciences

country: South Korea

institute: KAIST

summary: Circular RNA microarray under RNASEK depletion.

overall_design: Relative enrichment of each circular RNAs. Relative ratio was caculated as siRNASEK/siControl.

first: Sieun

last: Kim

first: Seokjin

last: Ham

first: Sung Ho

last: Boo

first: Rosa

last: Haque

first: Gee-Yoon

first: Yoon Ki


title: Early fibrotic niches establish tumour-permissive microenvironments [Experiment 2]

first: Joo-Hyeon

last: Lee

overall_design: At 2 weeks post three doses of tamoxifen induction (0.2 mg/gbw) every other day, lung tissues were collected and stromal cells (1:1 mixture of immune cells (CD45+) and both endothelial and mesenchyme cells (CD45–EpCAM–)) were isolated from Sftpc-CreERT2; Confetti and Sftpc-CreERT2; Red2Kras mice. Cells from three mice of the same genotype were pooled into a single cell suspension to generate two separate libraries (1 x Confetti and 1 x Red2Kras). Libraries were generated as described above and sequenced using the Illumina NovaSeq 6000.

first: Frances

last: England


title: FOXO1 integrates endothelial hemodynamic, inflammatory and metabolic pathways in atherosclerosis [OSS]

city: New Haven

first: Hanqiang

last: Deng

state: CT

address: 116 Court Street

department: Yale Cardiovascular Research Center

institute: Yale University

summary: Atherosclerosis occurs preferentially in regions of disturbed or low fluid shear stress (FSS), whereas physiological laminar FSS protects against disease by suppressing endothelial inflammation. Pro- vs anti-inflammatory programs are associated with glycolysis vs mitochondrial metabolism, respectively. Endothelial cells (ECs) sensing FSS from blood flow regulate these responses, but the underlying mechanisms are poorly understood. The transcription factor Forkhead box protein O1 (FOXO1) is known to regulate endothelial metabolism, yet its role in FSS-regulated endothelial inflammation remains largely unclear. We found that Oscillatory FSS and inflammatory cytokines induce, whereas physiological FSS inhibits FOXO1 nuclear translocation. RNA sequencing revealed that FOXO1 depletion in ECs upregulates the protective flow-responsive transcription factors KLF2/4 and reduces oscillatory FSS-induced inflammatory genes. Inhibition of FOXO1 nuclear translocation by physiological FSS is mediated via a KLF2-CDK2 pathway, with the latter phosphorylating FOXO1 at S249. Artery ECs-specific deletion of FOXO1 significantly reduces atherosclerotic plaque formation in hyperlipidemic mice. Inhibition of glycolysis attenuates OSS-induced FOXO1 nuclear translocation, suggesting a role for metabolic regulation. Notably, treatment with lactate promotes FOXO1 nuclear localization and lactylation, which is mediated by a lactyltransferase AARS1 (Alanyl-tRNA synthetase). These findings identify FOXO1 as a key mediator linking atheroprone flow and endothelial inflammation, suggesting potential new therapeutic targets for treating atherosclerotic cardiovascular disease.

overall_design: Human aortic endothelial cells (HAECs) were transfected with Ctrl (siCtrl) or FOXO1 (siFOXO1) siRNA for 3 days, then subjected to oscillatory shear stress (OSS; 0.5±4 dynes/cm2) for 24 hours. Total RNA was extracted and subjected to RNAseq, n=3 samples for each group.


title: circular RNAs in RNasek deficient MEFs

summary: Circular RNA microarray under Rnasek depletion.

overall_design: Relative enrichment of each circular RNAs. Relative ratio was caculated as UBC-CreERT2; Rnasek fl/fl MEFs/Rnasek fl/fl (control) MEFs .


title: Ribonuclease κ promotes longevity by preventing age-associated accumulation of circular RNA in stress granules [rnk-1]

summary: Circular RNAs (circRNAs) accumulate with age, but their functional impact on aging remains elusive. In this study, we reveal a mechanism by which ribonuclease κ (RNASEK) prevents age-dependent circRNA accumulation by promoting its degradation. Through a genetic screen targeting ribonucleases, we identified RNASEK as a specific circRNA-cleaving ribonuclease. RNASEK is downregulated during aging, causing the age-dependent increase in circRNA levels. RNASEK is necessary and sufficient for lifespan extension and healthspan maintenance in Caenorhabditis elegans. Mammalian RNASEK also directly degrades circRNAs, and is required for preventing premature aging in cultured human cells and mice, indicating its evolutionarily conserved role. Notably, we demonstrate that circRNAs localize within stress granules, where RNASEK, in collaboration with HSP90, prevents toxic aggregation of circRNAs in aged organisms. Our study establishes RNASEK as a conserved regulator of aging and offers a framework for targeting circRNAs to mitigate age-associated diseases and to extend organismal healthspan.

overall_design: RNA-seq profiling of wild-type N2 under control or rnk-1 RNAi conditions

first: Donghun

first: Hyemin

last: Min

first: Eunseok

last: Kang

first: Hanseul

first: Yoonji

last: Jung

first: Sujeoong

last: Kwon

first: Sangsoon

last: Park

first: Hae-Eun

middle: H

first: Eun Ji

first: Wooseon

last: Hwang

first: Eunah

first: Kun-Young

first: Jae Myoung

last: Suh

first: Gwangrog


title: Ribonuclease κ promotes longevity by preventing age-associated accumulation of circular RNA in stress granules [rnk-1rnk-2_PARE]

first: Seung-Jae

overall_design: PARE-seq profiling of wild-type N2 under control or rnk-1 rnk-2 RNAi conditions

first: Sieun

first: Seokjin

first: Sung Ho

first: Rosa

first: Gee-Yoon

first: Yoon Ki


title: C9orf72 Hexanucleotide Repeat RNA Drives Transcriptional Dysregulation Through Genome-wide Hybrid G-quadruplexes [RNA-Seq]

city: Baltimore

first: Mingming

last: Liu

state: MD

address: 615 N. Wolfe Street

department: BMB, BSPH

institute: Johns Hopkins University

summary: A hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. While repeat RNAs are implicated in disease pathogenesis, their mechanisms of actions remain incompletely understood. Here we show that GGGGCC repeat RNA engages chromatin genome-wide preferentially at promoter regions in patient cells. This interaction obstructs RNA polymerase II and transcription factors with GC-rich motifs, leading to broad transcriptional repression. Biochemical assays, single-molecule imaging, and native bisulfite sequencing analyses demonstrate that GGGGCC repeat RNA intrinsically forms DNA:RNA hybrid G-quadruplexes with cognate DNA, providing a structural basis for transcriptional interference. Stabilization of these G-quadruplex structures exacerbates neuronal vulnerability to metabolic stress in patient-derived motor neurons and cortical organoids, whereas restoring key gene dysregulation improves neuronal resistance to stress. These findings uncover a previously unrecognized trans-acting mechanism whereby repetitive RNAs form hybrid structures with genomic DNA, disrupt gene regulation, and contribute to neurodegeneration.

overall_design: RNA-seq profiling of RNA-seq data for C9-ALS B lymphocytes, CTRL B lymphocytes, C9-ALS iMNs and Isogenic control iMNs.

first: Honghe

first: Jiou

last: Wang


title: Altered ileum epithelial and immune cell profiles in the intestine of patients with MS.

city: Yokohama

first: Jonathan

last: Moody

address: 1-7-22 Suehiro-cho

country: Japan

institute: RIKEN Center for Integrative Medical Sciences

summary: The intestinal epithelium plays a critical role in immune–microbiota interactions, yet its contribution to systemic autoimmunity remains unclear. Here, we identify intestinal epithelial cells (IECs) as key initiators of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). In MS patients, IECs upregulate antigen presentation–related genes and associate with increased intestinal Th17 cell accumulation.

overall_design: Terminal ileum tissue samples were obtained by endoscopic tissue biopsy from healthy control (n=3) and patients with multiple sclerosis (MS) (n=4). Fresh-frozen human ileal biopsy sample were fixed and dissociated. Cells were analysed by 10x Genomics Chronium Single Cell Gene Expression Flex platform.

first: Shohei

last: Suzuki

first: Kentaro

last: Miyamoto

first: Tomohisa

last: Sujino


title: C9orf72 Hexanucleotide Repeat RNA Drives Transcriptional Dysregulation Through Genome-wide Hybrid G-quadruplexes [DRIP-seq]

overall_design: DRIP-seq profiling of DNA-RNA hybrids in C9-ALS iMNs and Isogenic control iMNs.


title: The human Flower isoform hFWE4 facilitates cornification in cutaneous squamous cell carcinoma

city: Omaha

first: Justin

last: Rudd

address: 2500 California Plaza

institute: Creighton University

summary: Cutaneous squamous cell carcinoma (cSCC) is the second most common human tumor and arises from keratinocytes that comprise the epidermis and its associated hair follicles. Remarkably, transformed keratinocytes within a cSCC retain some ability to execute the terminal differentiation program that typically generates a stratified epidermis. Instead of the polarized stratification towards the body surface seen in normal epidermis, cSCC keratinocytes differentiate concentrically inward to produce keratin pearls. It is well appreciated that cSCC differentiation status is correlated with degree of local invasion and potential for distant metastasis, with well differentiated tumors having a better prognosis. While molecular mechanisms that govern the differentiation process are incompletely understood, identifying candidate molecules associated with cSCC differentiation can provide new histologic markers for prognostic stratification. Here we demonstrate that Flower (FWE), a newly described regulator of lamellar body (LB) trafficking in normal epidermis, is specifically expressed within highly differentiated layers of cSCC. Genetic knockout of the hFWE gene dysregulates cornification and LB-related gene expression leading to abnormal keratinization patterns, while ectopic hFWE4 expression drives G1-arrest and exit from the proliferative basal keratinocyte population. Additionally, we find that poorly differentiated keratinocyte regions of human cSCC exhibit minimal FWE-positivity, while this population is readily detectable in well-differentiated regions. We propose that FWE represents a novel differentiation marker in cSCC that could be utilized for classifying differentiation status and prognostic stratifying these tumors.

overall_design: subcutaneous xenografts of hFWE WT or KO cells, 1*106 SCC13 cells were resuspended in 100µl of cold 1:1 sterile PBS:Matrigel (Corning 356237) and subsequently injected into the subcutaneous space on the dorsal flank of a NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl (NCG) mouse (Charles River 572). Tumors were harvested at 26d post injection for transcriptomic analysis.

first: Laura

last: Hansen


title: Compensatory interplay between clarin-1 and 1 clarin-2 deafness-associated proteins govern phenotypic variability in hearing

city: Paris

first: Sedigheh

last: Delmaghani

address: 25 rue du Dr Roux

country: France

institute: Institut Pasteur

summary: Usher syndrome type III (USH3), caused by mutations in CLRN1 encoding clarin-1, presents with progressive hearing loss, vestibular dysfunction, and retinitis pigmentosa, with striking phenotypic variability even among patients sharing identical mutations. Clarin-1's paralog, clarin-2 (encoded by CLRN2), is similarly implicated in hearing loss, suggesting potential functional interplay between these proteins. To investigate this relationship, we conducted RNA-sequencing of cochlear tissues from Clrn1-/-, Clrn2-/-, and Clrn1-/-Clrn2-/- double-knockout mice, revealing that clarin-1 and clarin-2 cooperatively regulate essential auditory processes, including mechanoelectrical transduction, ionic homeostasis, and synaptic organization. The double knockout mice exhibited synergistic disruption of these pathways, resulting in more severe hearing deficits than either single knockout, demonstrating that these proteins functionally compensate for one another. These findings suggest that CLRN2 variants may influence hearing outcomes in USH3 patients, supporting the integration of CLRN2 analysis into genetic diagnostics. By demonstrating that clarin-1 and clarin-2 share overlapping functional roles in the inner ear, this study redefines USH3 as a network-dependent disorder, offering new insights for therapeutic development. The RNA-seq dataset provides a comprehensive resource for exploring gene interactions in auditory function and advancing precision medicine approaches for hearing loss.

overall_design:
At post-natal day 21 wild-type, Clrn1-/-, Clrn2-/-, and Clrn1-/-Clrn2-/- mice were injected intraperitoneally with a lethal cocktail of Ketamine and Xylazine and inner ears collected in ice-cold 1X HBSS (ThermoFisher, 24020117). Organs of Corti were quickly dissected out of the cochlea, removing the stria vascularis and modulus, and collected in RNAs-free 2mL micro tubes to be immediately flash frozen in liquid nitrogen. Organs of Corti were collected in triplicate and kept at -80°C until RNA extraction.
RNA extraction and purification was performed using the NucleoSpin RNA, Mini kit for RNA purification (Macherey-Nagel, 740955.50), following manufacturer instructions with slight modifications in quantities of reagents used. RNA samples were measured with NanoDrop to assess RNA concentration and determine if there was any protein or ethanol contaminations. 2g RNA samples were sent for RNA-sequencing at Integragen.
RNA libraries were prepared with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina protocol according supplier recommendations. Briefly, the key stages of the protocol are successively: the purification of Poly-A containing mRNA molecules using poly-T oligo attached magnetic beads from 100ng total RNA (with the Magnetic mRNA Isolation Kit from NEB), a fragmentation using divalent cations under elevated temperature to obtain approximately 300bp pieces, double strand cDNA synthesis and finally Illumina adapters ligation and cDNA library amplification by PCR for sequencing. Sequencing is then carried out on Paired End 100b reads of Illumina NovaSeq 6000. Image analysis and base calling was performed using Illumina Real Time Analysis (3.4.4) with default parameters.
Gene expression was quantified using Integragen’s Galileo software. STAR was used to obtain the number of reads associated to each gene in the Gencode vM24 annotation (restricted to protein-coding genes, antisense and lincRNAs). Raw counts for each sample were imported into R statistical software using the Bioconductor DESeq2 package. Extracted count matrix was normalized for library size and coding length of genes to compute FPKM expression levels.
Unsupervised analysis of data used the Bioconductor edgeR package to import raw counts into R statistical software and compute normalized log2 CPM (counts per millions of mapped reads) using the TMM (weighted trimmed mean of M-values) as normalization procedure. The normalized expression matrix from the 1000 most variant genes (based on standard deviation) was used to classify the samples according to their gene expression patterns using principal component analysis (PCA). PCA was performed by FactoMineR::PCA function with “ncp = 10, scale.unit = FALSE” parameters.
The Bioconductor edgeR package was used to import raw counts into R statistical software for differential expression analysis of clarin mutant mice relative to wild-type controls. Differential expression analysis was performed using the Bioconductor limma package and the voom transformation. To improve the statistical power of the analysis, only genes expressed in at least one sample (FPKM  0.1) were considered. A q-value threshold of  0.05 and a minimum fold change of 1.5 were used to define differentially expressed genes.

first: Maureen

last: Wentling


title: Mapping 3’ untranslated regions reveal compromised alternative polyadenylation in human right ventricle failure

city: Houston,

first: Hari Krishna

last: Yalamanchili

state: TX

address: 1250 Moursund St #1125,

institute: Baylor College of Medicine

summary: Right ventricular failure (RVF) impacts the prognosis of pulmonary arterial hypertension (PAH). Alternative polyadenylation (APA) generates transcript diversity and is implicated in diseases, including left heart failure. However, understanding genome-wide polyadenylation maps in RVF patients with PAH is limited, with no studies investigating RVF-specific APA events. This study mapped 3' untranslated regions (3'UTRs) to explore APA dynamics in RVF development, comparing healthy donors and RVF from patients with PAH.

overall_design: Right ventricular control and failure samples, as well as human cardiac fibroblasts treated with control or CPSF6 siRNA.

first: Rajarajan

last: Thandavarayan

last: Yamalanchili


title: C9orf72 Hexanucleotide Repeat RNA Drives Transcriptional Dysregulation Through Genome-wide Hybrid G-quadruplexes [ChIRP-seq]

overall_design: ChIRP-seq profiling of GGGGCC repeat RNA-associated DNA or RNA targets in C9-ALS B lymphocytes, CTRL B lymphocytes, C9-ALS iMNs and Isogenic control iMNs.


title: C9orf72 Hexanucleotide Repeat RNA Drives Transcriptional Dysregulation Through Genome-wide Hybrid G-quadruplexes [ATAC-Seq]

overall_design: ATAC-seq profiling of ATAC-seq data for footprinting analysis in C9-ALS iMNs and Isogenic control iMNs.


title: Ribonuclease κ promotes longevity by preventing age-associated accumulation of circular RNA in stress granules [rnk-1rnk-2]

first: Seung-Jae

last: Lee

overall_design: RNA-seq profiling of wild-type N2 under control or rnk-1 rnk-2 RNAi conditions

first: Sieun

last: Kim

first: Seokjin

last: Ham

first: Sung Ho

last: Boo

first: Donghun

first: Hyemin

last: Min

first: Eunseok

last: Kang

last: Haque

first: Hanseul

first: Yoonji

last: Jung

first: Sujeoong

last: Kwon

first: Sangsoon

first: Hae-Eun

first: Eun Ji

first: Wooseon

first: Eunah

first: Kun-Young

first: Jae Myoung

first: Gwangrog


title: C9orf72 Hexanucleotide Repeat RNA Drives Transcriptional Dysregulation Through Genome-wide Hybrid G-quadruplexes [ChIP-Seq]

first: Mingming

last: Liu

overall_design: ChIP-seq profiling of PolII and MAZ binding analysis in C9-ALS iMNs and Isogenic control iMNs.

first: Honghe

first: Jiou

last: Wang


title: FOXO1 integrates endothelial hemodynamic, inflammatory and metabolic pathways in atherosclerosis [LSS]

first: Hanqiang

last: Deng

overall_design: Human umbilical vein endothelial cells (HUVECs) were transfected with Ctrl (siCtrl) or FOXO1 (siFOXO1) siRNA for 3 days, then subjected to low shear stress (LSS; 3 dynes/cm2) for 24 hours. Total RNA was extracted and subjected to RNAseq, n=3 samples for each group.


title: Repeated Disuse Atrophy in Aged Rat Skeletal Muscle (Reduced Representation Enzymatic Seq / Methylome)

city: Oslo

first: Adam

last: Sharples

address: 220 Sognsveien

country: Norway

institute: Norwegian School of Sport Sciences

type: Methylation profiling by high throughput sequencing

summary: Disuse-induced muscle atrophy commonly occurs following illness, injury, or falls and becomes increasingly frequent with ageing. Whether skeletal muscle retains a “memory” of repeated disuse remains unknown. We investigated repeated lower- limb immobilization in young adults and a refined aged rat model, integrating physiological, multi-omic, immunohistochemical, biochemical, and primary human muscle stem cell (MuSC) analyses. To enable robust age comparisons, we integrated previously published young rat data with newly generated aged rat data. In young human muscle, repeated disuse elicited attenuated transcriptional perturbations in oxidative and mitochondrial pathways, suggestive of a protective molecular memory, despite similar atrophy to initial disuse. In contrast, aged muscle exhibited a detrimental memory, characterized by greater atrophy, exaggerated suppression of aerobic metabolism genes despite recovery after initial disuse, NAD+ and mitochondrial DNA depletion, and activation of proteasomal, extracellular-matrix, and DNA-damage pathways. Whereas young rats recovered muscle mass after initial disuse, aged rats failed to do so. Across species, repeated disuse induced DNA hypermethylation and downregulation of aerobic metabolism and mitochondrial gene networks. NR4A1 and NR4A3 were among the strongest disuse- suppressed genes; NR4A1 acquired recovery-phase hypermethylation that maintained its transcriptional repression, while NR4A3 was the most downregulated gene after initial atrophy and remained persistently suppressed into recovery. Acetylcholine receptor subunit genes (CHRNA1, CHRND) were epigenetically primed, demonstrating hypomethylation and strong upregulation after disuse, and further amplification after repeated atrophy, while CHRNG was selectively induced after repeated atrophy only. NMRK2, an NAD+ biosynthesis gene, was the most downregulated gene across both atrophy periods, and supplementation with its substrate, nicotinamide riboside (NR), improved myotube size in MuSCs derived post-atrophy. Overall, repeated disuse atrophy imprints a molecular memory in skeletal muscle shaping transcriptional resilience in young adults and exaggerated susceptibility in aged muscle.

overall_design: Ethical approval was obtained, and experimental procedures were conducted with the permissions within a project license granted (PA693D221, Jonathan Jarvis, Liverpool John Moores University, UK) under the British Home Office Animals (Scientific Procedures) Act 1986. Aged (23 ± 1 months) male Fischer 344 rats (Charles River) weighing 354 ± 54 g were housed at 20°C and 45% relative humidity on an alternating 12 hr light and 12 hr dark cycle, with food and water available ad libitum. Animals were assigned to 1 of 4 groups (n = 3 per group) including a control group (sham control) and 3 experimental groups (atrophy, recovery and repeated atrophy). All experimental groups received tetrodotoxin (TTX) to the common peroneal nerve (CPN), preventing contraction of the ankle dorsiflexor muscles. TTX binds to the voltage-gated sodium channel in the nerve cell membranes and so prevents the conduction of action potentials. Therefore muscles innervated by the blocked CPN cannot be activated in order to contract. This induces disuse atrophy of the hind limb dorsiflexor muscles i.e., the tibialis anterior (TA) and extensor digitorum longus (EDL), and is advantageous vs. denervation or hindlimb unloading as it allows normal conduction in the tibial nerve to achieve plantarflexion, and therefore animals can undertake locomotion for daily living. The atrophy and recovery groups received a single dose of TTX (6-7 d) followed by 9 d of TTX abstinence to recover (e.g., TTX and recovery), whereas the repeated atrophy group received a second TTX dose (5-6 d) following recovery (e.g., TTX - recovery - repeated TTX). The sham control group consisted of n = 1 for each timepoint (TTX/recovery/repeated TTX) that received the same surgical procedures as the experimental groups but in the absence of TTX administration. Note: Samples 364_L and 364_R were identified as PCA outliers after initial sequencing, despite DNA quantity and quality being comparable to other samples. These were re-sequenced (364_L_rpt, 364_R_rpt) along with two PCA-clustered samples from the original run (370_R, 369_L; re-sequenced as 370_R_rpt, 369_L_rpt). Resequencing yielded consistent performance, so analyses were pooled, effectively increasing technical replicates. Differential methylation analyses, as reported in https://doi.org/10.1101/2025.10.16.681134, were performed for each pairwise comparison on (i) the original sequencing data and (ii) combined original and resequenced replicates. Both approaches identified largely overlapping significant differentially methylated regions (DMRs; FDR ≤ 0.05), with a small number of unique yet significant DMRs in each. Pooling DMRs from (i) and (ii) was therefore justified to maximize sensitivity and capture both shared and unique relevant regions, while maintaining rigor since all DMRs met stringent significance thresholds. This approach reduces false negatives, improves confidence in true signals, and ensures that potentially meaningful loci are not excluded from downstream discovery analyses.

first: Daniel

middle: C

last: Turner


title: FGFR Inhibition Overcomes Therapeutic Resistance and Immune Evasion in Oncogenic PIK3CA-Driven Serous Endometrial Cancers

city: Boston

first: Jean

last: Zhao

state: MA

address: 450 Brookline Ave

institute: Dana Farber Cancer Institute

summary: We developed the first clinically relevant, immunocompetent SEC mouse model incorporating PIK3CA mutation, TP53 loss, and MYC overexpression. Through comprehensive analysis integrating mouse models, human cell lines, xenografts, and clinical samples, we investigated mechanisms of PIK3CA-targeted therapy resistance. Single-cell transcriptional profiling identified FGFR1/2 association with intrinsic resistance and FGFR3 with acquired resistance. Dual inhibition of FGFR and PI3Ka achieved enhanced tumor suppression. Additionally, we discovered that FGFR signaling promotes immune evasion by downregulating MHC-I/HLA-mediated antigen processing and presentation. Importantly, FGFR inhibition not only reversed immune suppression by restoring antigen presentation but also demonstrated a synergistic effect with anti-PD-1 therapy, enhancing immunotherapy response and anti-tumor immune memory. Our findings reveal FGFR's dual role in therapy resistance and immune evasion, supporting FGFR inhibition as a promising strategy to enhance treatment outcome in SEC patients.

overall_design: In this study, we developed the first clinically relevant, immunocompetent transgenic mouse model of SEC driven by PIK3CA mutation, P53 loss, and C-MYC overexpression. To model tumor recurrence, we withdrew doxycycline from a cohort of tumor-bearing mice and monitored them for several months. We then collected primary and recurrent tumors for both snRNA-seq and RNA-seq analysis.

first: Xin

last: Cheng


title: C9orf72 Hexanucleotide Repeat RNA Drives Transcriptional Dysregulation Through Genome-wide Hybrid G-quadruplexes [RAP]

overall_design: RAP-DNA profiling of GGGGCC repeat RNA-associated DNA regions in C9-ALS iMNs and Isogenic control iMNs.


title: C9orf72 Hexanucleotide Repeat RNA Drives Transcriptional Dysregulation Through Genome-wide Hybrid G-quadruplexes [MeDIP-Seq]

overall_design: Profiling of 5-methyl cytosine and 5-hydroxymethyl cytosine in C9-ALS iMNs and Isogenic control iMNs.


title: Impact of vitamin D deficiency on defective endometrial decidualization and the repressive role of VDR in the epigenomic network [CUT&Run]

city: Durham

first: MyeongJin

last: Yi

state: NC

address: 111 TW Alexander Dr

institute: NIEHS

summary: The identification of the factors regulating female reproduction is critical to understanding how the environment and nutrition impact female fertility and reproductive health. Vitamin D and its cognate receptor, VDR, are recognized for their role in calcium homeostasis; however, their broader impact on female reproduction remains underexplored. Here, we demonstrate that vitamin D and VDR are involved in the hormonal induction of uterine stromal cell differentiation, known as decidualization. Mice fed a vitamin D-deficient diet displayed an impaired hormonally induced decidual response. In a human endometrial stromal cell line, T-HESC, VDR decreases during decidualization. siRNA knockdown of VDR in T-HESC enhanced decidualization, while overexpression of VDR inhibited decidualization. Chromatin accessibility and histone modification analyses revealed that VDR functions as a chromatin regulator, restricting accessibility and maintaining transcriptional repression in specific genomic regions. Transcriptomic analyses confirmed that VDR broadly modulates gene expression, with most ligand-mediated effects dependent on its presence. These findings identify VDR as a key regulator of transcriptional and chromatin landscapes in endometrial stromal cells, offering novel insights into vitamin D signaling in reproduction. This study highlights the potential of targeting vitamin D pathways to treat uterine disorders associated with impaired decidualization.

overall_design:
Experimental Design: 2 Different Experiments
Experiment 1. Identification of cistromic changes in human endometrial stromal cells by the receptor (VDR) and the ligand (1,25(OH)2D3).
There are 8 groups, and 24 samples in total. All libraries were prepared from the human endometrial stromal cell line (T-HESC).
1) Control lentiviral GFP vector transduced T-HESCs treated with vehicle and precipitated with VDR antibody, n=3
2) Control lentiviral GFP vector transduced T-HESCs treated with vehicle and precipitated with IgG, n=3
3) Control lentiviral GFP vector transduced T-HESCs treated with 1,25(OH)2D3 and precipitated with VDR antibody, n=3
4) Lentiviral VDR plasmid transduced T-HESCs treated with 1,25(OH)2D3 and precipitated with IgG, n=3
5) Lentiviral VDR plasmid transduced T-HESCs treated with vehicle and precipitated with VDR antibody, n=3
6) Lentiviral VDR plasmid transduced T-HESCs treated with vehicle and precipitated with IgG, n=3
7) Lentiviral VDR plasmid transduced T-HESCs treated with 1,25(OH)2D3 and precipitated with VDR antibody, n=3
8) Lentiviral VDR plasmid transduced T-HESCs treated with 1,25(OH)2D3 and precipitated with IgG, n=3
Experiment 2.  Impact of VDR and 1,25(OH)2D3 on histone modifications
There are 8 groups, and 24 samples in total. All libraries were prepared from the human endometrial stromal cell line (T-HESC).
1) Non-targeting pooled siRNA-transfected T-HESCs and precipitated with H3K4me3, n=3
2) Non-targeting pooled siRNA-transfected T-HESCs and precipitated with H3K27me3, n=3
3) Non-targeting pooled siRNA-transfected T-HESCs and precipitated with H3K27ac, n=3
4) Non-targeting pooled siRNA-transfected T-HESCs and precipitated with IgG, n=3
5) VDR siRNA-transfected T-HESCs and precipitated with H3K4me3, n=3
6) VDR siRNA-transfected T-HESCs and precipitated with H3K27me3, n=3
7) VDR siRNA-transfected T-HESCs and precipitated with H3K27ac, n=3
8) VDR siRNA-transfected T-HESCs and precipitated with IgG, n=3
Data Analysis Strategy: Dr. Austin Bell-Hensley, Dr. Tianyuan Wang, and Dr. Abdull Massri from the informatics group supported Dr. MyeongJin Yi with the analysis.
1) Remove adaptor 2) QC with trim-galore 3) Map the pair-end reads 4) Count reads mapped to chrM 5) Remove duplicated reads 6) Remove unmapped reads 7) Remove reads mapped to chrM 8) Prepare SAM file based on trimmed 9bp reads 9) Random sampling to the same depth 10) Compare between different groups

first: Skylar

last: Montague Redecke

first: Tianyuan

first: Austin

last: Bell-Hensley

first: Abdull

last: Massri

first: Francesco

last: DeMayo


title: Ciliated cells prevent tumorigenesis through an immune-independent mechanism of STING-mediated tumor suppression

city: Ann Arbor

first: Brian

last: Magnuson

state: MI

address: 1500 East Medical Center Drive

institute: University of Michigan

summary: Mitigating DNA damage in the fallopian tube epithelium (FTE) is essential for preventing tubo-ovarian high-grade serous carcinoma (HGSC). Here we demonstrate that Stimulator of Interferon Genes (STING) is abundantly expressed in the ciliated cells of the FTE and functions as a critical immune-independent tumor suppressor. Using patient samples, mouse models, and organoid systems, we demonstrate that ciliated cells mount a dual protective response to ovulation-associated genotoxic stress: intrinsic STING-driven apoptosis and extrinsic clearance of neighboring damaged secretory cells via TNFα secretion. This surveillance mechanism markedly limits DNA damage accumulation within the epithelial microenvironment. Crucially, while these mechanisms are vital for maintaining homeostasis and reducing genomic instability, they fail to impact p53-deficient precursor lesions as both intrinsic and extrinsic pro-apoptotic processes rely on functional p53 signaling. These findings redefine ciliated cells as key guardians of genome integrity rather than passive bystanders and implicate early loss of STING-high ciliated cells as a pivotal event in HGSC initiation with potential relevance for prevention and therapeutic intervention.

overall_design: Organoids derived from wild-type mouse (B6) oviductal epithelium were cultured in the presence of PBS (control) or human follicular fluid (treatment) for 24 h (4 replicates per condition for a total of 8 samples).

first: Jose

last: Colina

first: Rong

last: Wu

first: Kathleen

last: Cho

first: Analisa

last: DiFeo


title: JAK2V617F Myeloproliferative Neoplasms Support Parallel Evolution of Independent Leukemic Clones

city: St Louis

first: Grant

last: Challen

state: MO

address: 660 Euclid Avenue

department: Medicine

institute: Washington University School of Medicine

summary: Myeloproliferative neoplasms (MPNs) are hematological diseases predominantly driven by the JAK2V617F mutation. Progression from chronic-phase MPN to secondary acute myeloid leukemia (sAML) is a severe complication as it dramatically worsens disease prognosis. While sAML transformation is classically linked to MPN clones acquiring additional mutations, the absence of JAK2V617F in sAML cases originating from JAK2-mutant MPNs suggests alternative mechanisms. Utilizing patient samples and in vivo modeling, we establish that sAML clones can emerge independently of JAK2-mutant cells. These leukemic clones undergo positive selection in the pro-inflammatory MPN environment leading to their predominance in the hematopoietic system. Genetic and pharmacological inhibition of IL-12 and TNFa mitigates this competitive advantage. Our data establish a new paradigm and show that disease progression in MPN can arise from parallel acute myeloid leukemia (pAML) clones.

overall_design: To investigate the differential gene expression patterns of Tet2-mutant hematopoietic stem and progenitor cells in either a Jak2-mutant or wild-type environment, we established Cre-mediated deletion and knock-in models. Competitive BM transplantation was performed. Post-transplant cells were isolated 10 and 18-weeks post transplant for analysis of gene expression patterns by RNA sequencing.

first: Tyler

middle: M

last: Parsons

first: Aishwarya

last: Krishnan

first: Infencia

last: Xavier Raj

first: Andrew

middle: L

last: Young

first: David

last: O'Leary

first: Jason

last: Arand

first: Maggie

last: Cox

first: Stephen

last: Oh


title: The LMO2-LDB1-TAL1 complex regulates transcription networks in AML

city: Charlottesville

first: Francine

middle: E.

last: Garrett-Bakelman

state: VA

address: 1340 Jefferson park Ave.

department: Medicine and BMG

institute: University of Virginia

summary: Relapsed acute myeloid leukemia (relAML) remains a clinical challenge. We have shown that epigenetic heterogeneity may contribute to transcriptional dysregulation and disease progression in AML, but the specific transcriptional programs have not been identified. To characterize relAML transcriptional landscapes, we analyzed molecular profiles from patient-matched diagnostic and relapse AML specimens. A subset of differentially expressed genes (DEGs) that were disparate in direction of expression change identified two patient subtypes, but were not associated with specific molecular events. We predicted transcriptional regulators (TRs) that might regulate the expression patterns observed. The expression patterns of the top TRs predicted for the disparate genes associated with clinical outcomes in independent AML patient cohorts. The top TRs predicted included members of the LMO2 multi-subunit complex (TAL1 and LDB1).  LMO2/LDB1/TAL1 complex members were also identified as potential TRs of DEGs in a patient derived xenograft mouse model of relapse.  Analysis of DepMap data identified LMO2-dependent cells with a subset highly expressing TAL1, suggesting coordinated regulation. TAL1 co-purified in immunoprecipitation for LMO2 and LDB1 followed by tandem mass spectrometry analysis in HEL and K562 cells. Loss of function experiments targeting LMO2/LDB1/TAL1 in AML cell lines associated with reduced cell growth, downregulation of cell cycle genes, and a negative association with gene expression patterns observed in relapsed patients with increased TAL1 expression. Data from primary AML specimens and functional analyses of AML cell lines supports an essential role for the LMO2/LDB1/TAL1 complex in relAML. Targeting the complex or downstream effectors could provide novel therapeutic considerations for relAML patients.

overall_design: RNA-seq of LMO2 and LDB1 knockdown in K562 cells and TAL1 knockdown in HEL cells.


title: Repeated Disuse Atrophy in Aged Rat Skeletal Muscle (RNA-seq / Transcriptome)

first: Adam

last: Sharples

overall_design: Ethical approval was obtained, and experimental procedures were conducted with the permissions within a project license granted (PA693D221, Jonathan Jarvis, Liverpool John Moores University, UK) under the British Home Office Animals (Scientific Procedures) Act 1986. Aged (23 ± 1 months) male Fischer 344 rats (Charles River) weighing 354 ± 54 g were housed at 20°C and 45% relative humidity on an alternating 12 hr light and 12 hr dark cycle, with food and water available ad libitum. Animals were assigned to 1 of 4 groups (n = 3 per group) including a control group (sham control) and 3 experimental groups (atrophy, recovery and repeated atrophy). All experimental groups received tetrodotoxin (TTX) to the common peroneal nerve (CPN), preventing contraction of the ankle dorsiflexor muscles. TTX binds to the voltage-gated sodium channel in the nerve cell membranes and so prevents the conduction of action potentials. Therefore muscles innervated by the blocked CPN cannot be activated in order to contract. This induces disuse atrophy of the hind limb dorsiflexor muscles i.e., the tibialis anterior (TA) and extensor digitorum longus (EDL), and is advantageous vs. denervation or hindlimb unloading as it allows normal conduction in the tibial nerve to achieve plantarflexion, and therefore animals can undertake locomotion for daily living. The atrophy and recovery groups received a single dose of TTX (6-7 d) followed by 9 d of TTX abstinence to recover (e.g., TTX  recovery), whereas the repeated atrophy group received a second TTX dose (5-6 d) following recovery (e.g., TTX  recovery  repeated TTX). The sham control group consisted of n = 1 for each timepoint (TTX/recovery/repeated TTX) that received the same surgical procedures as the experimental groups but in the absence of TTX administration.

first: Daniel

last: Turner


title: Siglec-F Protects Against Elastase-induced Lung Inflammation and Emphysema in Mice

city: Aurora

first: Qihua

last: Ye

state: CO

address: Research Complex II  12700 East 19th Avenue

department: Pulmonary

institute: University of Colorado Anschutz Medical Campus

summary: Airspace macrophages (AMs) are essential for maintaining lung homeostasis. Their activity must be tightly controlled to prevent excessive immune responses and tissue damage, such as emphysematous alveolar destruction. Siglec-F is a canonical marker of AMs, but its functional role in lung disease remains unclear. Here, we investigated the role of Siglec-F in a murine model of elastase-induced emphysema. We found that Siglec-F-deficient mice exhibited worsened emphysematous phenotypes, along with accumulation and transcriptional reprogramming of resident AMs (RAMs). Transcriptomic profiling of Siglec-F-deficient RAMs revealed dysregulation of key pathways involved in tissue repair, including extracellular matrix degradation, TGF-β signaling, and phagocytosis. These findings demonstrate that Siglec-F is critical for restraining RAM activity and preserving alveolar integrity during injury. Our study provides new insight into the immunoregulatory function of Siglec-F in a tissue-destructive context and highlights the potential protective roles of Siglec signaling in chronic lung disease.

overall_design: Bulk RNA-seq of FACS-sorted CD11c+ resident airspace macrophages (RAMs) from the lung lavage of Siglecf+/+ (WT) and Siglecf-/- (KO) mice on days 0, 3, 7, 21 post porcine pancreatic elastase (PPE) administration.

first: Naoko

last: Hara

first: Yan

last: Hu

first: Marika

last: Orlov

first: Bradford

middle: J

last: Smith

first: Tonya

last: Brunetti

first: Rachel

last: Blumhagen

first: Zhenghui

last: Liu

first: Bifeng

last: Gao

first: Melanie

last: Königshoff

first: William

last: Janssen

first: Christopher

last: Evans


title: Repeated Disuse Atrophy in Adult Human Skeletal Muscle (Reduced Representation Bisulfite-Seq / Methylome)

overall_design: Ethical approval was granted by the Regional Committees for Medical and Health Research Ethics (REK, Application ID 227924). Informed consent was obtained. The study was registered with the Norwegian Agency for Shared Services in Education and Research (Sikt) under reference number 627824. In a repeated measures design, young healthy adult human (male & female) participants (age 26.5 ± 4.1, height 177.5 ± 7.9 cm, weight 84 ± 20.9 kg) underwent 2 weeks/wks (14 days/d) disuse atrophy (via unilateral limb immobilization) followed by ~7 wks (49 ± 5 d) recovery followed by 2 wks repeated disuse atrophy (unilateral limb immobilization). Muscle biopsies were obtained from the vastus lateralis muscle from the immobilized leg. Briefly, local anaesthesia (Xylocaine with 10 mg/ml lidocaine + 5 µg/ml adrenaline, AstraZeneca, Södertälje, Sweden) was administered to the biopsy area and muscle tissue was obtained from the mid-belly (10% proximal of the mid-thigh) of the m. vastus lateralis (VL) muscle using a modified Bergström technique (6 mm Pelomi-needle, Albertslund, Denmark) with manual suction (i.e., 50 ml syringe connected to the Bergström needle). Some outliers samples, included in this GEO sample record, were identified and removed before downstream differential methylation analysis included in the published paper here: https://advanced.onlinelibrary.wiley.com/doi/10.1002/advs.202522726, this included samples: 7B, 7A1, 4R, 5A2, 15A2, 12R.


title: Gene expression profile at single cell level of iPSC-derived enteric neural lineages from Parkinson's Disease patients

city: Erlangen

first: Bruno

last: Ghirotto

address: Kussmaulalle 4

country: Germany

summary: iPSC-derived enteric neural lineages are a mixed culture of enteric neurons and glial cells. We used scRNAseq to dissect differences at single cell level between patients and control lines.

overall_design: On day 70 of differentiation, we obtained single cells from iPSC-ENLs using a papain-DNAse digestion protocol and cells were submitted to scRNAseq analysis with 6000 cells targeted per sample and 50,000 reads targeted per cell

first: Ghirotto

last: Bruno

first: Gupta

last: Pooja

first: Winner

last: Beate


title: Histone methyltransferase DOT1L affects the development of dendritic cell subsets [ChIP-Seq]

city: Amsterdam

first: Fred

last: van Leeuwen

address: Plesmanlaan 121

department: Gene Regulation

country: Netherlands

institute: Netherlands Cancer Institute

summary: Dendritic cells (DCs) are important orchestrators of immune responses and can be divided into two conventional DC subsets, cDC1s and cDC2s, and a plasmacytoid DC (pDC) subset, which are involved in the activation of T cells and production of type I interferons (IFNs), respectively. The development of the DC subsets occurs in the bone marrow (BM) under control of different transcription factors and epigenetic modifiers. The histone methyltransferase DOT1L, known as a druggable target in cancer, is emerging as a key epigenetic regulator of differentiation in lymphocytes. DOT1L writes methylation of lysine 79 of histone H3 in gene bodies of actively transcribed genes. Deletion or inhibition of DOT1L affects the differentiation trajectory of innate and adaptive immune cells, leading to altered cell states. Recent studies suggest that DOT1L also plays a role in DC function. Here, we investigated the role of DOT1L in the development of DCs in in vitro BM cultures and in vivo using a tamoxifen-inducible mouse model. H3K79me2 ChIPseq data of sorted DC subsets from the BM revealed distinct H3K79me2 peaks for all three subsets, which suggested differential regulation of the subsets by DOT1L. A comparison of the H3K79me2 ChIPseq data to bulk RNAseq of sorted DC subsets from the BM revealed a stronger correlation between H3K79me2 and global transcription in pDCs and cDC2s compared to cDC1s. In line with this, we observed that Dot1l deletion led to a decrease in pDCs and CpG A-stimulated IFNα production and an increase in cDC2s in in vitro BM cultures, while cDC1s were unchanged. In vivo deletion of Dot1l led to a decrease in common myeloid progenitors (CMPs), monocyte DC progenitors (MDPs), and common DC progenitors (CDPs) in the BM, while common lymphoid progenitor (CLP) and cDC2 numbers were increased. Inhibition of DOT1L resulted in a similar phenotype, which linked the observed effects to the methyltransferase activity of DOT1L. Interestingly, all Dot1l-KO DC subsets exhibited an enrichment of pathways related to antigen presentation and the immune response and MHCII expression was upregulated in Dot1l-KO pDCs and cDC2s in vivo. To conclude, our data indicate that DOT1L function differentially affects the development of DCs with predominant effects in pDCs and cDC2s.

overall_design: Chromatin immunoprecipitation DNA sequencing (ChIP-seq) for H3K79me2 on isolated and FACS sorted pDC, DC1 and DC2 populations (N=2 biological replicates). Chromatin from S. cerevisiae containing HA tagged H3 and lacking H3K79 methylation was spiked into all samples and co-immunoprecipitated to normalize and quantify the data.

first: Willem-Jan

last: de Leeuw

first: Anne

last: Mensink

first: Rianne

last: Bouma

first: Joke

last: den Haan


title: A viral transcriptional regulator from a dsRNA virus antagonizes the innate immune response by manipulating the host co-transcription factor DHX9

city: Tianjin

first: Xueyang

last: Pang

state: Tianjin

address: 22 Meteorological Observatory Road, Heping District, Tianjin

institute: TMU

summary: Viral transcriptional regulators (vTRs) have emerged as potent factors that shape the host anti-viral gene expression programs. Delineating the molecular mechanisms by which vTRs inhibit or promote transcription would provide fundamental insights for developing anti-viral strategies. Using Mammalian orthoreovirus (REOV) as a model system, we identified a new viral mechanism by which viruses antagonize the host innate immune response. We found that the REOV outer capsid protein σ3 functions as a vTR to suppress NF-κB gene expression via its direct interaction with the host helicase DHX9. Mechanistically, σ3 impairs the initial recruitment of Pol II by disrupting the interaction between DHX9 and Pol II. More interestingly, σ3 suppresses DHX9 helicase activity, resulting in the aberrant accumulation of R-loops at promoter-proximal regions, thereby affecting Pol II pause-release and ultimately suppressing NF-κB gene expression. Together, our findings reveal an unprecedented strategy employed by a viral protein that regulates anti-viral gene expression by directly modulating the host transcription factor DHX9.

overall_design: R-Loop occupancy on chromatin upon TNFa, IFNβ or REOV treatment in 293T cells were assessed by Cut&Tag assay.

first: Shiyu

first: Yixiao

last: Zhu

first: Meleana

last: Hinchman

last: Qiao

first: Xuejun

last: Li

first: Weiqian

last: Dai

first: Yingying

last: Tong

first: Man

last: Xing

first: Jiling

last: Ren

first: John

last: Parker

last: Guo

first: Dongming

last: Zhou


title: Repeated Disuse Atrophy in Adult Human Skeletal Muscle (RNA-seq / Transcriptome)

first: Adam

last: Sharples

overall_design: Ethical approval was granted by the Regional Committees for Medical and Health Research Ethics (REK, Application ID 227924). Informed consent was obtained. The study was registered with the Norwegian Agency for Shared Services in Education and Research (Sikt) under reference number 627824. In a repeated measures design, young healthy adult human (male & female) participants (age 26.5 ± 4.1, height 177.5 ± 7.9 cm, weight 84 ± 20.9 kg) underwent 2 weeks/wks (14 days/d) disuse atrophy (via unilateral limb immobilization) followed by ~7 wks (49 ± 5 d) recovery followed by 2 wks repeated disuse atrophy (unilateral limb immobilization). Muscle biopsies were obtained from the vastus lateralis muscle from the immobilized leg. Briefly, local anaesthesia (Xylocaine with 10 mg/ml lidocaine + 5 µg/ml adrenaline, AstraZeneca, Södertälje, Sweden) was administered to the biopsy area and muscle tissue was obtained from the mid-belly (10% proximal of the mid-thigh) of the m. vastus lateralis (VL) muscle using a modified Bergström technique (6 mm Pelomi-needle, Albertslund, Denmark) with manual suction (i.e., 50 ml syringe connected to the Bergström needle).

first: Daniel

last: Turner


title: Inflammation-enhanced synapse-specific phagocytosis by adult APP microglia in a microfluidic neuron–microglia co-culture model

city: LILLE

first: Florence

last: MAURIER

address: 1 rue du Professeur Calmette BP 245

department: Plateformes Lilloises en Biologie & Santé

institute: Institut Pasteur de Lille

summary: Microglia play a critical role in synapse remodeling and neuroinflammation, both of which are dysregulated in Alzheimer’s disease (AD). However, most in vitro models rely on neonatal or immortalized microglia, limiting their relevance to adult pathophysiological context. Here, we present a compartmentalized microfluidic co-culture platform that enables spatially controlled interactions between primary cortical neurons and adult microglia from wild-type (WT) and APP-transgenic mice. This system allows precise functional analysis of microglia–synapse interactions under defined inflammatory conditions. Upon lipopolysaccharide (LPS) stimulation, APP microglia exhibited exaggerated morphological activation, elevated IL-1β secretion, and selectively increased engulfment of synaptic material. In contrast, phagocytosis of non-specific substrates such as pHrodo™ Zymosan remained unchanged, suggesting a substrate-specific enhancement of microglial phagocytic activity. Blocking the complement receptor CD11b abolished the LPS-induced increase in synaptic uptake, confirming the role of complement-dependent pathways. Transcriptomic profiling revealed robust inflammatory responses in both genotypes, with selectively heightened expression of proinflammatory genes in APP microglia, consistent with a primed immune phenotype. Importantly, increased synaptic uptake occurred without measurable loss of global synaptic connectivity, highlighting the specificity and sensitivity of the system to detect microglial functional changes. This model captures genotype-dependent microglial reactivity (revealing phenotypes not fully captured by transcriptomic rofiling) and provides a physiologically relevant, tractable in vitro platform for dissecting microglial contributions to synaptic pathology in neurodegenerative disease.

overall_design: Adult cortical microglia were isolated from 10–11-month-old wild-type and APP-transgenic mice, maintained in vitro for four weeks, and treated with vehicle or lipopolysaccharide (100 ng/mL, 6 h). After a 16 h recovery, total RNA was extracted, libraries prepared using Lexogen QuantSeq 3′ mRNA-Seq FWD (with UMIs and ERCC spike-ins), and sequenced on an Illumina NovaSeq 6000 (single-end 100 bp). Nineteen samples were collected across four conditions: WT+Vehicle (n=4), WT+LPS (n=5), APP+Vehicle (n=5), APP+LPS (n=5).

first: Dolores

last: Siedlecki-Wullich

first: Devrim

last: Kilinc

first: Jean-Charles

last: Lambert

first: Lukas

last: Iohan

first: Delphine

last: Beury

first: Myriam

last: Delacre

first: David

last: Hot


title: m6A epitranscriptome regulates axonogenesis and reprogramming of retinal ganglion cells [RNA-seq]

city: Guangzhou

first: SHUYI

last: CHEN

state: Guangdong

address: 54 S. Xianlie Rd

department: State Key Laboratory of Ophthalmology

institute: Zhongshan Ophthalmic Center, SYSU

summary: induced retinal ganglion cells (iRGCs) is an excellent system to generate RGC-like neurons for translational and theoretical studies. N6-methyladenosine (m6A) is the most prevalent internal modification in mRNAs. Using this iRGC system, we demonstrated that Mettl3, the core component of the m6A-methyltransferase complex, promotes iRGC axon development and the final iRGC reprogramming outcome.

overall_design: mouse embryonic fibroblasts (MEFs) infected with Ascl1/Brn3b/Islet1 overexpressing lentiviruses of different treatment groups were collected at day 5 after induction, and processed for RNA-seq.

first: Shuyi

last: Chen


title: Transcriptome profiling of secondary hair follicle stem cells during cashmere growth [RNA-Seq]

city: Shenyang

first: Huiling

last: Xue

address: 120 Dongling Road,110866 Shenyang,China

institute: Shenyang Agricultural University

summary: To investigate how various histone modifications influence gene expression, transcriptome sequencing was performed on secondary hair follicle stem cells (SHFSCs) collected in parallel with the ChIP-seq samples, and the resulting data could be integrated with ChIP-seq for joint analysis.

overall_design: Secondary hair follicle stem cells (SHFSCs) were isolated and cultured from the dorsal skin of cashmere goat during the early anagen (April) and mid anagen (October) phases. RNA sequencing was conducted on cells from both phases, with two biological replicates per phase (male or female samples).

first: Xinyue

last: Liang

first: Bohan

last: Liu

first: Yanlei

first: Shengda

last: Sheng


title: Single-cell RNA-seq analysis was performed on CD45-positive cells isolated from mouse mammary tumors.

city: Beijing

first: Yabing Nan

last: Yabing Nan

address: 17 Panjiayuan Nanli, Chaoyang Diistrict

institute: Cancer Hospital Chinese Academy of Medical Sciences

summary: Tumors derived from BP cells of mouse mammary cancer were collected after treatment with vehicle control, olaparib alone, or the combination of olaparib and an IRG1 inhibitor. CD45-positive cells were isolated from these tumors for single-cell RNA-seq analysis to investigate the impact of the combination therapy on the tumor immune microenvironment.

overall_design: Tumor tissues collected from mice treated with vehicle, olaparib alone, or the combination of olaparib and IRG1-IN-1 were enzymatically dissociated into single-cell suspensions using a collagenase/hyaluronidase-based digestion buffer. CD45 positive immune cells were then enriched using a CD45 positive selection kit (18945, StemCell Technologies). For each treatment group, single-cell suspensions from four individual tumors were pooled by centrifugation and resuspended in CryoStor CS10 (100-1061, StemCell Technologies) for preservation. Single-cell RNA sequencing was performed by Novogene using the 10x Genomics platform.

first: Yabing

first: Qiwei

last: Wang

first: Jean

last: Zhao


title: Histone methyltransferase DOT1L affects the development of dendritic cell subsets [RNA-Seq]

first: Fred

last: van Leeuwen

overall_design: RNAseq of sorted murine dendritic cell populations (pDC, DC1 and DC2) 12 days after the genetic ablation of Dot1l,by tamoxifen injection, as well as non-injected WT controls, to investigate the effect of loss of Dot1l on the transcriptome.

first: Willem-Jan

last: de Leeuw

first: Anne

last: Mensink

first: Rianne

last: Bouma

first: Joke

last: den Haan


title: Triploidy disrupts hormonal and paracrine signaling and enforces male development in zebrafish

city: Liběchov

first: Tomáš

last: Tichopád

address: Rumburská 89

country: Czech Republic

institute: ÚŽFG

summary: Sex differentiation in zebrafish is governed by a complex interplay of genetic and endocrine cues. Triploid zebrafish, which are largely sterile, consistently develop as males, but the mechanisms remain elusive. Here, we combined histological and transcriptomic analyses to examine how triploidy and exposure to 17α-ethinyl estradiol (EE2) influence sex differentiation in zebrafish. Triploidy disrupted hormonal and paracrine signaling, with downregulation of fshr and amh, upregulation of igf3, suppression of ptger2a and dio1, collectively driving complete masculinization. In diploids, EE2 produced diverse gonadal outcomes, ranging from testes and ovotestes to fully differentiated ovaries. By contrast, EE2 promoted early germ cell proliferation in triploids but failed to induce ovarian differentiation, reinforcing a fixed male trajectory. Triplody and EE2 administration altered meiosis and spermatogenesis, consistent with downregulation of klhl10 and constrained retinoic acid signaling through dhrs3a and/or cyp26b1. At the molecular level, both triploidy and EE2 converged on suppression of early steroidogenic genes, including star and cyp11a1, limiting androgen and estrogen biosynthesis. Together, these findings reveal how ploidy reshapes endocrine regulation and responsiveness and reveal shared and unique molecular pathways by which EE2 influences zebrafish gonadal fate.

overall_design: exposure to 17α-ethinyl estradiol (EE2) in 2n  and 3n zebrafish

first: Tomas

last: Tichopad

first: Aaron

last: Torres

first: Martin

last: Psenicka

first: Roman

last: Franěk


title: Characterization of Oral-to-Blood Microbial DNA Translocation in Chronic Cocaine Use Disorder

city: Charleston

first: Stefano

last: Berto

state: SC

address: 68 President Street, BE208

institute: MUSC

summary: Chronic cocaine use exhibits a compromised gut barrier, systemic microbial translocation, and systemic and neural inflammation, ultimately contributing to long-term health issues. However, there is a debate about whether cocaine itself is capable of inducing inflammation. In the current study, we investigate whether the oral cavity can be the source of blood microbial DNA translocation in chronic cocaine users.

overall_design: We recruited individuals with chronic cocaine use disorder and non-drug-using controls and collected paired saliva and blood samples. Microbial DNA was isolated, and the microbial 16S rRNA V4 region was sequenced. Single-cell RNA sequencing (scRNAseq) was performed on peripheral blood mononuclear cells (PBMC) to assess immune perturbations

first: Suganya

last: Subramanian

first: Douglas

last: Johnson

first: Wei

last: Jiang


title: Genomic characterization of mouse hindlimb skeletal muscles with divergent slow myofiber content at an embryonic and adult time point [ATAC-Seq]

city: Cambridge

first: Terence

last: Capellini

address: 11 Divinity Avenue

department: Human Evolutionary Biology

institute: Harvard University

summary: Here, we characterize the genomic landscape of four mouse hindlimb muscles that differ in slow myofiber content at two time points (E18.5 and adult) to identify novel enhancers that may underlie the expression of genes involved in myofiber development and physiology. RNA-seq analysis revealed that fast- and slow-biased muscles show divergent patterns of gene regulation beginning at a late embryonic time point and persisting through adulthood. Genes differentially expressed between adult fast- and slow-biased muscles were enriched with biological pathways unique to each myofiber type, with glycolytic pathways characterizing adult fast-biased muscles and mitochondrial biogenesis and lipid metabolism characterizing adult slow-biased muscles. By integrating differential expression analysis (RNA-seq) with differential accessibility analysis (ATAC-seq) we identified twelve conserved, muscle-specific candidate enhancers nearby differentially expressed genes that regulate cell metabolism and the genetic markers of myofiber type. Nine candidate enhancers significantly increased and three significantly decreased luciferase reporter activities in C2C12 cells, highlighting the dynamic role of tissue-specific enhancers and repressors on development and differentiation. However, further validation is needed to see if these enhancers regulate the expression of target genes and the effect on muscle myofiber content. Collectively, these results highlight the importance of conserved, skeletal muscle-specific enhancers on skeletal muscle development and adult myofiber phenotypes and provide additional genomic targets for further validation.

overall_design: Examination of open chromatin regions (ATAC-seq) and differentially expressed genes (RNA-seq) in mouse hindlimb muscles with divergent slow myofiber content at an embryonic (E18.5) and adult (P56) time point.

first: Samantha

last: Queeno

first: Alexander

last: Okamoto

middle: D

first: Kirstin

middle: N

last: Sterner


title: Histone modification clocks for robust cross-species biological age prediction and elucidating senescence regulation

city: Bad Nauheim

first: Lei

last: Gu

state: Hessen

address: Parkstraße 1

department: Epigenetics

institute: Max Planck Institute for Heart and Lung Research

summary: By analyzing ChIP-seq data across six tissues and six histone marks, we developed 36 tissue-specific histone modification-based epigenetic clocks that detect age acceleration in leukemia and reversal following treatment. Many age-associated loci showed nonlinear trajectories peaking at midlife, and super-enhancer fragmentation was observed with age. Functional validation of an H3K27ac peak near IGF2BP3 confirmed its role in senescence via TRA2A regulation. These clocks also generalized to Drosophila melanogaster, highlighting the evolutionary conservation and utility of histone modifications as aging biomarkers.

sample_organism:
Drosophila melanogaster
Homo sapiens

platform_organism:
Drosophila melanogaster
Homo sapiens

overall_design:
RNA-seq: Wild-type HL-60 cells were transfected with a CRISPR-dCas9-HDAC plasmid along with a piRNA plasmid targeting the region near the IGF2BP3 promoter. Two days post-transfection, both wild-type and transfected cells were harvested for total RNA isolation and poly(A)-selected mRNA sequencing.
ChIP-seq: Adult flies at 1, 3, 5, 13, 20, 33, 45, 55, 60, and 63 days post-eclosion were collected for ChIP-seq.

first: Niu

last: Zhixin

first: Gu

last: Lei


title: A viral transcriptional regulator from a dsRNA virus antagonizes the innate immune response by manipulating the host co-transcription factor DHX9 [RNA-Seq]

first: Xueyang

last: Pang

summary: Viral transcriptional regulators (vTRs) have emerged as potent factors that shape the host anti-viral gene expression programs. Delineating the molecular mechanisms by which vTRs inhibit or promote transcription would provide fundamental insights for developing anti-viral strategies. Using Mammalian orthoreovirus (REOV) as a model system, we identified a new viral mechanism by which viruses antagonize the host innate immune response. We found that the REOV outer capsid protein σ3 functions as a vTR that limits RNA polymerase II (Pol II) recruitment to the promoter regions of NF-κB-dependent genes via its direct interaction with the host helicase DHX9. σ3 competes with DHX9 for its interaction with Pol II, which led to marked attenuation of Pol II loading onto chromatin. More interestingly, σ3 also suppresses DHX9 helicase activity, leading to aberrant accumulation of R-loops at promoter-proximal regions and a decrease in NF-κB-dependent gene expression. Together, our findings uncover an unprecedented strategy of a viral protein that regulates anti-viral gene expression by directly manipulating host transcription factor DHX9.

overall_design: Plasmids expressing σ3 were transfected into HEK293 cells using PEI. At 48 h post-transfection, cells were treated with 10 ng/ml TNF-α for 3 h. Total RNA was extracted with TriZOL for library construction and subsequent sequencing.

first: Shiyu

last: Liu

first: Yixiao

last: Zhu

first: Meleana

last: Hinchman

last: Qiao

first: Xuejun

last: Li

first: Weiqian

last: Dai

first: Yingying

last: Tong

first: Man

last: Xing

first: Jiling

last: Ren

first: John

last: Parker

last: Guo

first: Dongming

last: Zhou


title: Impact of vitamin D deficiency on defective endometrial decidualization and the repressive role of VDR in the epigenomic network

first: MyeongJin

last: Yi

summary: The identification of the factors regulating female reproduction is critical to understanding how the environment and nutrition impact female fertility and reproductive health. Vitamin D and its cognate receptor, VDR, are recognized for their role in calcium homeostasis; however, their broader impact on female reproduction remains underexplored. Here, we demonstrate that vitamin D and VDR are involved in the hormonal induction of uterine stromal cell differentiation, known as decidualization. Mice fed a vitamin D-deficient diet displayed an impaired hormonally induced decidual response. In a human endometrial stromal cell line, T-HESC, VDR decreases during decidualization. siRNA knockdown of VDR in T-HESC enhanced decidualization, while overexpression of VDR inhibited decidualization. Chromatin accessibility and histone modification analyses revealed that VDR functions as a chromatin regulator, restricting accessibility and maintaining transcriptional repression in specific genomic regions. Transcriptomic analyses confirmed that VDR broadly modulates gene expression, with most ligand-mediated effects dependent on its presence. These findings identify VDR as a key regulator of transcriptional and chromatin landscapes in endometrial stromal cells, offering novel insights into vitamin D signaling in reproduction. This study highlights the potential of targeting vitamin D pathways to treat uterine disorders associated with impaired decidualization.

overall_design: Experimental Design: There are 2 groups, and 6 samples in total. All ATACseq libraries were prepared from the human endometrial stromal cell line (T-HESC). 1) Non-targeting siRNA transfection (control) n=3 2) VDR siRNA transfection (VDR knockdown) n=3  Data Analysis Strategy: Dr. Tianyuan Wang and Dr. Austin Bell-Hensley from the informatics group supported Dr. MyeongJin Yi with the analysis. 1) Remove adaptor 2) QC with trim-galore 3) Map the pair-end reads 4) Count reads mapped to chrM 5) Remove duplicated reads 6) Remove unmapped reads 7) Remove reads mapped to chrM 8) Prepare SAM file based on trimmed 9bp reads 9) Random sampling to the same depth 10) Compare between different groups (siNT vs siVDR)

first: Tianyuan

last: Wang

first: Austin

last: Bell-Hensley

first: Francesco

last: DeMayo


title: m6A epitranscriptome regulates axonogenesis and reprogramming of retinal ganglion cells

first: SHUYI

last: CHEN

overall_design: mouse embryonic fibroblasts (MEFs) infected with ABI-2A-GFP lentiviruses were collected at day 5 after induction (DAI 5) and DAI 13 , and processed for MeRIP-Seq.

first: Shuyi

last: Chen


title: Phosphorylation of SF3B1 by CDK11 1 orchestrates spliceosome activation via SNIP1 dependent RES complex recruitment

city: Pamplona

first: Igor

last: Ruiz de los Mozos

state: Nyp

address: C. del Sadar s/n

department: Department of Personalized Medicine

country: Spain

institute: NASERTIC

summary: Splicing Factor 3b Subunit 1 (SF3B1), a core component of the spliceosome, undergoes dynamic phosphorylation and dephosphorylation during the splicing cycle to regulate pre-mRNA splicing. Twenty-eight threonine/proline repeats are phosphorylated by CDK11 during spliceosome activation and remain phosphorylated in the catalytically active spliceosomes. The function of phosphorylated SF3B1 (P-SF3B1), and the identity of spliceosomes stalled by CDK11 inhibition remain unclear. Using quantitative proteomics of chromatin-associated spliceosomes, we identify a previously uncharacterized intermediate complex BOTS964, arrested by CDK11 inhibitor OTS964, that incorporates the nineteen-related (NTR) but not nineteen (NTC) complex. iCLIP-seq revealed that P-SF3B1 engages with the U6 snRNA internal stem-loop (ISL), suggesting a potential role in stabilizing the RNA catalytic core. We further demonstrate that P-SF3B1 is recognized by forkhead-associated (FHA) domain of SNIP1, which promotes recruitment of retention and splicing (RES) complex during spliceosome activation. Acute SNIP1 depletion disrupts RES incorporation, causes widespread splicing defects, and promotes hyperphosphorylation of SF3B1 by CDK11. Mutations in SNIP1 FHA domain, including the neurodevelopmental disorder associated E366G variant, impair P-SF3B1 binding, pre-mRNA splicing, and cell viability. Together, these findings uncover the phosphorylation-dependent CDK11/P-SF3B1/SNIP1 signaling axis that is critical for pre-mRNA splicing and cellular proliferation and provide a mechanistic insight into its dysregulation in disease.

overall_design: Examination of RNA-seq in the presence or absence of SNIP1 protein on HCT116 dTAG SNIP1 cells.

first: Pavla

last: Gajduskova

last: Ruiz de Los Mozos

first: Milan

last: Hluchy

first: Antonina

last: Myskova

first: Katerina

last: Zabrady

first: Peter

last: Manik

first: Neda

last: Dragisic

last: Hanakova

first: David

last: Potesil

first: Sara

last: Bologna

first: Jiri

last: Novacek

first: Caroline

last: Friedel

first: Konstantinos

last: Tripsianes

first: Zbynek

last: Zdrahal

first: Dalibor

last: Blazek


title: Gene expression in Tetrahydrocurcumin-treated HUVEC cells

city: Taipei

first: Yi-Lin

last: Chiu

address: No.161, Sec. 6, Minquan E. Rd., Neihu Dist., Taipei City 11490, Taiwan

department: Department of Biochemistry, College of Biomedical Sciences

country: Taiwan

institute: National Defense Medical University

summary: Endothelial nitric oxide synthase (eNOS) plays a crucial role in maintaining endothelial cell function and preventing atherosclerosis. Tetrahydrocannabinol is a metabolite that has better bioactivity than curcumin. This study investigated the effect of the PI3K/AKTpathway on eNOS expression and NO production induced by tetrahydrocannabinol (THC) in human umbilical vein endothelial cells. The findings reveal that both PI3Kand AKT are essential for THC-induced eNOS phosphorylation and subsequent NO production.

overall_design: Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in EGM-2 medium and treated with either vehicle (DMSO) or tetrahydrocurcumin (THC) at concentrations of 2.5, and 5 µM for 24 hours.

first: Tsai-Jung

last: Lin

first: Tsung-Neng

last: Tsai


title: Genomic characterization of mouse hindlimb skeletal muscles with divergent slow myofiber content at an embryonic and adult time point [RNA-Seq]

first: Terence

last: Capellini

first: Samantha

last: Queeno

first: Alexander

last: Okamoto

first: Kirstin

last: Sterner


title: Integrating Trajectory Inference and Self-Explainable Predictive Models to Explore Cell State Transitions in Breast Cancer at Single-  Cell Resolution

city: Rende

first: Rosamaria

last: Lappano

state: Cs

address: via P. Bucci

country: Italy

institute: University of Calabria

summary:
Breast cancer is characterized by a highly heterogeneous cellular environment composed of diverse malignant clones and components of the tumor microenvironment (TME) that collectively influence the progression of the disease. Single-cell RNA sequencing (scRNA-seq) offers a powerful tool to dissect this complexity, enabling high-resolution characterization of tumor heterogeneity and the functional interactions within the TME. Moreover, it supports the discovery of clinically relevant subpopulations and potential therapeutic targets. In this study, we present a novel scRNA-seq dataset from an infiltrating ductal breast cancer, profiling over 5,000 cells and identifying six distinct clusters spanning cancer and TME populations. To
explore the molecular drivers of cell state transitions, we integrate pseudotime trajectory inference with interpretable, tree-based machine learning. This approach enables the identification of key genes and expression thresholds associated with dynamic phenotypic shifts. Unlike black-box models, our framework yields transparent, rule-based insights into transcriptional reprogramming during tumor evolution. The resulting dataset, along with an accessible and transparent analytical pipeline, offers a valuable resource for the breast cancer research community and lays the groundwork for future studies aimed at refining molecular classification and precision therapy development.

overall_design: We analyzed single cell RNA-seq data from breast cancer tissue.