title: N6-methyladenosine and the NEXT complex direct Xist RNA turnover and X inactivation dynamics [ChrRNA-seq_XistDelm6A]

city: Oxford

first: Guifeng

last: Wei

address: South Parks Road

department: Department of Biochemistry

institute: University of Oxford

summary: X chromosome inactivation (XCI) in mammals is orchestrated by the non-coding RNA Xist which together with specific interacting proteins, functions in cis to silence an entire X chromosome. Defined sites on Xist RNA carry the N6-methyladenosine (m6A) modification, and perturbation of the m6A writer complex has been found to abrogate Xist-mediated gene-silencing. However, the relative contribution of m6A and its mechanism of action remain unclear. Here we re-investigate the role of m6A in XCI by applying rapid degron-mediated depletion of METTL3, the catalytic subunit of the m6A writer complex, an approach that minimises indirect effects due to transcriptome-wide depletion of m6A. In contrast to prior studies, we find that acute loss of METTL3/m6A accelerates Xist-mediated gene silencing, and that this correlates with increased levels and stability of Xist transcripts. We show that Xist RNA turnover is mediated by the nuclear exosome targeting (NEXT) complex but is independent of the major nuclear m6A reader protein YTHDC1. Our findings demonstrate that the primary function of m6A on Xist RNA is to promote Xist RNA turnover which in turn regulates XCI dynamics.

overall_design: To investigate the role of m⁶A on Xist RNA, we generated deletions in the m⁶A-modified regions of Xist: either a 355 bp deletion in the exon 1 m⁶A region, a 153 bp deletion in the exon 7 m⁶A region, or a combination of both. Cells were either left untreated or induced with doxycycline (dox) for 24 hours to express Xist. Gene silencing was assessed by measuring the allelic ratio, defined as Xi / (Xi + Xa).

first: Neil

last: Brockdorff


title: C/EBP⍺ activates Irf8 expression in myeloid progenitors at the +56 kb

status: Public on Jun 22 2025

city: Saint Louis

first: Jing

last: Chen

state: MO

address: 600 S Euclid Ave

country: USA

institute: Washington University in St. Louis

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Development of type 1 conventional dendritic cells (cDC1) underlies the capacity to generate antiviral and antitumor immune responses. Here, we identify the basis for cDC1 development from its earliest progenitors, determining the hierarchy of several required transcription factors and uncovering a novel paradigm for superenhancer function. We produced in vivo mutations of two C/EBP binding sites that comprise the Irf8 +56 kb enhancer that abrogate IRF8 expression in all myeloid progenitors and impair cDC1 development. These sites do not bind RUNX1 or RUNX3, and C/EBPα expression instead is regulated by their action at the Cebpa +37 kb enhancer, placing RUNX factors upstream of Cepba in regulating Irf8.  Finally, we demonstrate that cis interactions between the +56 kb Irf8 enhancer and the previously reported +41 kb and +32 kb Irf8 enhancers are mandatory in the sequential progression of these stage specific constituent elements.

overall_design: Examination of H3K27ac histone modification in the primary BM CDP and MDP cells by CUT&RUN in wildtype, d1pm, d1, d12, and IRF8KO mice.

first: Theresa

middle: L

last: Murphy

first: Sunkyung

last: Kim

first: Kenneth

middle: M


title: Effect of cytokines IL1β and TNFα on  human MDA-MB-453 breast cancer cells

status: Public on Jun 23 2025

city: Aurora

first: Andrew

last: Goodspeed

state: CO

address: 12801 E 17th Ave, Mail Stop 8303

institute: University of Colorado Anschutz Medical Campus

type: Expression profiling by high throughput sequencing

summary: To investigate single cell transcriptomics of human MDA-MB-453 breast cancer cells in response to the cytokine treatment in compared with the vehicle control

overall_design: Breast cancer cell line MDA-MB-453 was treated with IL1β and TNFα or vehicle control (water) for 24 hours, the cells were then collected for single-cell RNA sequencing

first: Li-Wei

last: Kuo

first: Lyndsey

middle: S

last: Crump

first: Kathleen

middle: I

last: O'Neill

first: Michelle

last: Williams

first: Jessica

last: Christenson

first: Nicole

last: Spoelstra

first: Jennifer

middle: K

last: Richer


title: Agouti overexpression in a transgenic model regulates integrity, permeability and electrogenic amino acid transport in zebrafish intestine

city: Barcelona

first: Alfonso

last: Saera-Vila

address: Dr. Trueta 179

country: Spain

institute: Sequientia Biotech

summary: Overexpression of asip1 in transgenic zebrafish disrupts dorsoventral pigment pattern in addition to increasing food intake levels and linear growth. A higher feed intake is unnecessary in transgenic fish to enable larger and heavier growth. A plausible explanation may rely on the enhanced feeding efficiency mediated by improved nutrient absorption in transgenic animals. To test this hypothesis, wide scope transcriptomic techniques were used to pipe the potential pathways involved in the enhanced nutrient absorption and intestinal epithelium permeability/integrity, in addition, the electrogenic capacity for amino acid transport was analysed. Transcriptomic results suggested that amino acid, monocarboxylates, ionic and vitamin transmembrane transport was substantially modified. Enrichment analysis also revealed an inhibition of intestinal lipid metabolism and down-regulation of KEGG pathways related to membrane integrity suggesting an augmented intestinal laxity that may result in enhanced paracellular transport. Electrophysiological experiments carried out in Ussing chambers show that asip1 overexpression decrease membrane tissue resistance (Rt), indicating a modification of the intestinal barrier function in ASIP1 transgenic animals. Similarly, paracellular permeability was higher in transgenic zebrafish. Both the decrease in Rt and the increase in permeability point to an ASIP1-dependent decrease in the tissue barrier function. Electrogenic amino acid transport was also enhanced in transgenic animals providing a strong indication that ASIP1 fish can extract more amino acids from their diet at similar feeding levels. Both transcriptomic and electrophysiological results suggest that asip1-overexpressing zebrafish display improved nutrient absorption and by extension a higher feed efficiency which explains enhanced growth in the absence of augmented food intake. The enhanced growth of ASIP1 zebrafish potentially mediated by improved nutrient uptake and feed efficiency suggests that the melanocortin system, specifically asip1 overexpression, is a potential target for the development of genetically engineered fish displaying improved performance and no differential lipid accumulation.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for transgenic (Tg(Xla.Eef1a1:Cau.Asip1)iim04) and wild type (Tubingen) zebrafish.

first: Esther

last: Leal

first: José

last: Cerdá-Reverter


title: Analysis of individual patient pathway coordination in a cross-species single-cell kidney atlas

city: Philadelphia

first: Katalin

last: Susztak

state: Pennsylvania

address: 12-123 Smilow Translational Research Center, 3400 Civic Center Blvd

department: Renal, Electrolyte, and Hypertension Division, Department of Medicine

institute: University of Pennsylvania, Perelman School of Medicine

summary: The use of single-cell RNA sequencing in clinical and translational research is limited by cross-species differences and the challenge of identifying cell-type-specific, targetable molecular changes in individual patients. Here, we created an Integrated Single-cell Kidney Atlas including over one million cells from 140 samples, defining more than 70 conserved cell states in human and rodent models. We developed CellSpectra, a computational tool that quantifies changes in gene expression coordination across cellular functions, which we applied to kidney and lung cancer data. This tool powers our Patient-Level Single-Cell Functional Profiling Report, which highlights cell-type-specific changes in pathway gene expression coordination in individuals. Our cross-species atlas facilitates the selection of a rodent model that closely reflects the cellular and pathway-level signatures observed in a patient sample, advancing the application of single-cell methodologies in clinical precision medicine. Finally, by using experimental models, we demonstrate how our informatics approach can be applied for the potential selection of suitable therapeutics.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design:
Mouse: To obtain a more heterogenous collection of mouse models we generated snRNA-seq data from mouse disease models and controls incuding a podocyte specific G2NA APOL1 risk allele mouse and samples prepared following folic acid injection (250 mg/kg body weight in 300 mM NaHCO3) sacrificed 7 days after disease induction.
Human: New generated human data was based on biopsy samples in a biobank from an observational cohort. No interventions were part of this investigation. A total of 5 kidney biopsy specimen from patients with kidney disease were processed (more clinical metadata in manuscript supplements).

***Human raw data were not provided due to privacy concerns***

first: Konstantin

middle: A

last: Klötzer

first: Amin

last: Abedini

first: Shen

last: Li

first: Michael

last: Balzer

first: Xiujie

last: Liang

first: Jonathan

last: Levinsohn

first: Bernhard

last: Dumoulin

middle: J

last: Hogan

first: Ghazal

last: Quinn

first: Roy

middle: D

last: Bloom

first: Max

last: Schuller

first: Kathrin

last: Eller

first: Balazs

last: Halmos

first: Nancy

middle: R

last: Zhang


title: Differential microRNA profiles associated with the hydroxyurea-inducible SAR1A gene

city: Bethesda

first: Griffin

middle: P

last: Rodgers

state: MD

address: 10 Center Drive

department: NHLBI

institute: NIH

type: Non-coding RNA profiling by high throughput sequencing

summary: We used small-RNA sequencing to carry out the first comprehensive survey of miRNAs expressed in SAR1A-knockout K562 cells without or with HU treatment and assessed their potential functions related to HbF induction. our findings suggest a profile of dysregulated miRNAs associated with SAR1A downregulation which may influence HbF expression directly or indirectly targeting the transcription factors linked to gamma-globin gene regulation.

overall_design: small RNA-Seq profiling of wildtype K562 cells and SAR1A-knockout K562 cells without or with HU treatment

first: Chutima

last: Kumkhaek

first: Jianqiong

last: Zhu

first: Wulin

last: Aerbajinai

first: Wenli

last: Liu

first: Wannaporn

last: Ittiprasert

first: Griffin P


title: Maternal Stress Triggers Early-Life Eczema via Fetal Mast Cell Reprogramming [FetalMC-bulkRNAseq]

status: Public on Jun 21 2025

city: Toulouse

first: Rüçhan

last: Ekren

address: CHU Purpan – BP 3028 –  Toulouse Cedex, 3

department: INFINITY

country: France

institute: Inserm UMR 1291

summary: Prenatal stress (PS) is a repeated exposure to aversive situations during pregnancy, including high emotional strain, and suspected to impact physiological systems in infants. Pediatric eczema develops quickly after birth at flexural sites subjected to continuous mechanical constraints. While epidemiological studies have suggested an association between PS and higher risk of eczema in children, no causative biological link has been identified to date. Here we show that early-onset eczema at birth originates from molecular dysregulations of neuroimmune circuits in utero triggered by fluctuations in the maternal hypothalamic-pituitary-adrenal axis. We found that offspring from stressed pregnant dams have dysregulated Mcpt5+ mast cells and skin-projecting neurons, and quickly develop eczema in response to harmless mechanical frictions. We demonstrate that PS transiently modulates maternal corticosterone levels, which profoundly alters the developmental program of skin mast cells expressing Nr3c1 and adjacent sensory neurons conveying mechanosensation. Therapeutic normalization of corticosterone levels or genetic depletion of Mcpt5+ mast cells during stressed gestation prevents dysregulation of neuroimmune pathways and fully protects from eczema development at birth. Our findings support a new model in which early-onset pediatric eczema originates from dysregulations in fetal immune and somatosensory systems caused by fluctuations in maternal glucocorticoids induced by stress.

overall_design: Pregnant 8- to 10-week-old dams were subject to chronic prenatal stress (PS) from embryonic day (E)13.5 to E18.5. Skin samples were collected from E18.5 control (CT) and PS offspring of 12 mice per group [CT, PS]; in total n = 24), and bulk RNA sequencing of isolated fetal mast cells was conducted.

first: Nadine

last: Serhan

first: Nasser

last: Abdullah

last: Gheziel

first: Alexia

last: Loste

first: Elodie

last: Labit

first: Anne-Alicia

last: Gonzalez

first: Giulia

last: Oliva

first: Pauline

last: Tarot

first: Camille

last: Petitfils

first: Gaëlle

last: Payros

first: Paolo

last: D’Avino

first: Allison

last: Voisin

first: Holly Freya Grace

last: Tinsley

first: Rebecca

last: Gentek

first: Carole

last: Brosseau

first: Marie

last: Bodinier

first: Laurent

last: Reber

first: Pierre

last: Val

first: Cezmi

last: Akdis

first: Yasutaka

last: Mitamura

first: Anand Kumar

last: Andiappan

first: Jerry Kok Yen

last: Chan

first: Florent

last: Ginhoux

first: Amaury

last: François

first: Nicolas

last: Cénac

first: Lilian

last: Basso

last: Gaudenzio


title: Maternal Stress Triggers Early-Life Eczema via Fetal Mast Cell Reprogramming [DRG-bulkRNAseq]

first: Rüçhan

last: Ekren

overall_design: Pregnant 8- to 10-week-old dams were subject to chronic prenatal stress (PS) from embryonic day (E)13.5 to E18.5. Dorsal root ganglia (DRG) were collected from 8-week old (W8) control (CT) and PS offspring of 9 mice per group [CT, PS]; in total n = 18), and bulk RNA sequencing of isolated DRG was conducted.

first: Nadine

last: Serhan

first: Nasser

last: Abdullah

last: Gheziel

first: Alexia

last: Loste

first: Elodie

last: Labit

first: Anne-Alicia

last: Gonzalez

first: Giulia

last: Oliva

first: Pauline

last: Tarot

first: Camille

last: Petitfils

first: Gaëlle

last: Payros

last: D’Avino


title: Maternal Stress Triggers Early-Life Eczema via Fetal Mast Cell Reprogramming [10x Genomics Multiome ATAC + Gene Expression]

overall_design: Pregnant 8- to 10-week-old dams were subject to chronic prenatal stress (PS) from embryonic day (E)13.5 to E18.5. Skin samples were collected from 8-week old (W8) and 24-week old (W24) control (CT) and PS offspring (pool of 4 mice per group), and single cell Multiome ATAC + Gene Expression sequencing of enriched CD45+ cells was conducted.

first: Paolo

first: Allison

last: Voisin

first: Holly Freya Grace

last: Tinsley

first: Rebecca

last: Gentek

first: Carole

last: Brosseau

first: Marie

last: Bodinier

first: Laurent

last: Reber

first: Pierre

last: Val

first: Cezmi

last: Akdis

first: Yasutaka

last: Mitamura

last: Andiappan

last: Chan

last: Ginhoux


title: CDK12/13 inactivation triggers STING-mediated antitumor immunity in preclinical models

city: Ann Arbor

first: Arul

last: Chinnaiyan

state: Michigan

address: 1500 E. Medical Center Dr

institute: University of Michigan

summary: Inactivation of cyclin-dependent kinase 12 (CDK12) characterizes an immunogenic molecular subtype of prostate cancer, marked by genomic instability and increased intratumoral T cell infiltration. However, the mechanisms underlying this phenotype and its therapeutic implications remain unclear. Here, we demonstrate that CDK12 inactivation or suppression of its paralog CDK13 activates stimulator of interferon genes (STING) signaling across multiple cancer types. Analysis of clinical cohorts receiving immune checkpoint blockade (ICB) revealed that reduced CDK12/13 expression correlates with improved survival and treatment response. Mechanistically, CDK12/13 depletion or targeted degradation induces transcription-replication conflicts and R-loop accumulation, triggering cytosolic DNA release and subsequent STING activation. Functionally, CDK12/13 degradation significantly delays tumor growth and exhibits synergistic effects with anti-PD1 therapy in various syngeneic tumor models. This combination enhances STING activity and promotes the infiltration and activation of CD8+ T cells within tumors. Notably, the anti-tumor effects of this combination require STING signaling and functional CD8+ T cells. These findings identify CDK12/13 as promising therapeutic targets for enhancing ICB efficacy, highlighting their potential to amplify STING-mediated anti-tumor immunity. This work provides a mechanistic framework for leveraging CDK12/13 antagonists in combination with ICB to improve clinical outcomes in cancer therapy.

overall_design: Two CDK12-mutant and two CDK12 wildtype metastatic prostate cancer samples were subjected to spatial transcriptomics analysis to evaluate genes that were differently expressed in CDK12 mutant samples compared to the wildtype.

first: Yi

last: Bao

first: Yu

last: Chang

first: Jean

last: Ching-Yi Tien

first: Gabriel

last: Cruz

first: Fan

last: Yang

first: Rahul

last: Mannan

first: Somnath

last: Mahapatra

first: Radha

last: Paturu

first: Xuhong

last: Cao

first: Fengyun

last: Su

first: Rui

last: Wang

first: Yuping

last: Zhang

first: Mahnoor

last: Gondal

first: Jae

last: Eun Choi

first: Jonathan

last: Gurkan

first: Stephanie

last: Miner

first: Dan

last: Robinson

first: Yi-Mi

last: Wu

first: Licheng

last: Zhou

first: Zhen

first: Ilona

last: Kryczek

first: Xiaoju

first: Marcin

last: Cieslik

first: Yuanyuan

last: Qiao

first: Weiping

last: Zou

first: Ke

last: Ding


title: The RNA-binding protein TRIM71 is essential for hearing in humans and mice and times auditory sensory organ development

city: Baltimore

first: Angelika

last: Doetzlhofer

address: 855 North Wolfe St

department: Neuroscience

institute: Johns Hopkins University School of Medicine

summary: Utilizing conditional Trim71 knockout mice, we find that Trim71 deletion during early otic development causes hearing loss and induces premature cell cycle exit and differentiation of hair cell progenitors. Transcriptomic and genetic experiments reveal that TRIM71 maintains hair cell-progenitors proliferative and undifferentiated through repressing TGF-β-type signaling.

overall_design: Cochlear sensory epithelia were harvested from E13.5 Trim71 knockout mice and their littermate controls. To induce Trim71 deletion, timed pregnant females received dox containing feed starting at E8.5. Total RNA from control and Trim71 KO cochlear epithelia were isolated after 5 days (E13.5) using enzymatic treatment.

first: Xiaojun

last: Li

first: Charles

last: Morgan


title: Promoter- and Enhancer-Dependent Cohesin Loading Initiates Chromosome Looping  to Fold Tcrb Loci for Long-Range Recombination [ChIP-Seq]

first: Craig

last: Bassing

state: PA

address: 3501 Civic Center Blvd

institute: Children's Hospital of Philadelphia

summary: Cohesin-mediated chromosome looping regulates diverse processes, including antigen receptor (AgR) gene assembly by V(D)J recombination. To understand mechanisms that coordinate genome topologies, we focused on a genetically tractable AgR locus, Tcrb. Cohesin loading and initiating loop extrusion (LE) from a nearby CTCF-binding element (CBE) required the promoter of the most 5’Vb segment, creating long-range contacts with target downstream DJb segments within the recombination center (RC). CBEs flanking the RC have multiple functions: terminators of LE originating in the Vb cluster, initiators of LE in the RC, and insulation of enhancer activity. Deletion of the Tcrb super-enhancer abolished loop extrusion from the neighboring RC but spared longrange contacts, indicating that unidirectional loop extrusion from upstream Vb segments was sufficient. Thus, Vb promoter- or enhancer-dependent cohesin loading initiates LE in opposite directions across the locus to assemble a broad Tcrb repertoire, a finding that has broad implications for genomic architecture and function.

overall_design: ChIP Seq was performed in DN thymocytes from ~5 Rag1-/- mice per experiment with either a WT Tcrb locus or with the indicated mutations with the indicated antibodies.

first: Brittney

last: Allyn


title: Maternal Stress Triggers Early-Life Eczema via Fetal Mast Cell Reprogramming [scRNAseq]

first: Rüçhan

last: Ekren

overall_design: Pregnant 8- to 10-week-old dams were subject to chronic prenatal stress (PS) from embryonic day (E)13.5 to E18.5. Skin samples were collected from 8-week old (W8) and 24-week old (W24) control (CT) and PS offspring (pool of 3 mice per group), and single-cell RNA sequencing of isolated CD45+ cells was conducted.

first: Nadine

last: Serhan

first: Nasser

last: Abdullah

last: Gheziel

first: Alexia

last: Loste

first: Elodie

last: Labit

first: Anne-Alicia

last: Gonzalez

first: Giulia

last: Oliva

first: Pauline

last: Tarot

first: Camille

last: Petitfils

first: Gaëlle

last: Payros

first: Paolo

last: D'Avino

first: Allison

last: Voisin

first: Holly Freya Grace

last: Tinsley

first: Rebecca

last: Gentek

first: Carole

last: Brosseau

first: Marie

last: Bodinier

first: Laurent

last: Reber

first: Pierre

last: Val

first: Cezmi

last: Akdis

first: Yasutaka

last: Mitamura

first: Anand Kumar

last: Andiappan

first: Jerry Kok Yen

last: Chan

first: Florent

last: Ginhoux

first: Amaury

last: François

first: Nicolas

last: Cénac

first: Lilian

last: Basso

last: Gaudenzio


title: TCF19 drives a broad transcriptional program that potentiates optimal innate and adaptive functions of antiviral NK cells

city: New York

first: Celeste

last: Dang

state: NY

address: 417 E 68th St

department: Immunology

institute: Memorial Sloan Kettering Cancer Center

summary: Natural killer (NK) cells are innate lymphocytes that play a critical role in host defense against viral infection. In addition to rapid effector cytokine production and direct cytotoxicity, NK cells exhibit features of adaptive immunity, including the capacity to undergo robust antigen-specific clonal proliferation and to generate immunological memory. However, the transcriptional programs and regulators governing dynamic NK cell responses to viral infection have not been fully uncovered. In this study, we identified Transcription factor 19 (TCF19) as a key driver of NK cell proliferation and calcium signaling in the context of mouse cytomegalovirus infection. Ablation of TCF19 was detrimental to NK cell clonal expansion and host protection against viral infection. Tcf19-/- NK cells were also unable to properly mobilize calcium downstream of antigen signaling to mediate cytotoxicity. Altogether, we find that TCF19 drives a transcriptional program that coordinates the innate and adaptive NK cell responses against viral infection.

overall_design: To investigate the role of TCF19 in NK cells during MCMV infection, we performed TCF19 scRNAseq using WT and TCF19-/- NK cells. scRNAseq was performed on FACS sorted mouse Ly49H+ CD49b+ NK cells.

first: Dang

last: Celeste

first: Kim

last: Hyunu

first: Sun

middle: C

last: Joseph


title: Loss of RBM45 inhibits breast cancer progression by reducing the SUMOylation of IRF7 to promote IFNB1 transcription

city: Dalian

first: Wang

last: Yang

address: 9 Lvshun South Rd West Section, Lüshunkou District, Dalian, Liaoning, China

department: Institute of Cancer Stem Cell

country: China

institute: Dalian Medical University

summary: Type I interferons exhibit anti-proliferative and anti-cancer activities, but their regulatory mechanisms in cancer have not been fully elucidated. RNA binding proteins are master orchestrators of gene expression regulation and are closely related to tumor progression. Here we show that the upregulated RNA binding protein RBM45 correlates with poor prognosis in breast cancer. Depletion of RBM45 suppresses breast cancer progression both in cultured cells and xenograft mouse models. Further analysis reveals that depletion of RBM45 inhibits breast cancer progression through regulating type I interferon signaling, in particular by elevating IFN-β production. Mechanistically, RBM45 coordinates with TRIM28 to regulate IFNB1 transcription. We identify that RRM2 and RRM3 domains of RBM45 are required for TRIM28 binding. Moreover, RBM45 could directly interact with IRF7, whereas RBM45 without any RRM domain attenuates its ability to interact with IRF7. Importantly, RBM45 recruits TRIM28 to IRF7 and stimulates its SUMOylation, thereby repressing IFNB1 transcription. Loss of RBM45 reduced the SUMOylation of IRF7 by reducing the interaction between TRIM28 and IRF7 to promote IFNB1 transcription, leading to the inhibition of breast cancer progression. Taken together, our finding uncovers a key role of RBM45 in modulating type I interferon signaling and cancer aggressive progression, implicating RBM45 as a potential therapeutic target in breast cancer.

overall_design: To investigate the effects of the knockdown  of RBM45 gene on breast cancer MCF7 cells, we established two RBM45 knockdown MCF7 cell lines and then performed RNA-seq sequencing analysis.

first: Chaoqun

last: Chen

first: Yuesheng

last: Lv


title: RBM25 depletion suppresses colon cancer progression through regulating alternative splicing of MNK2

summary: To explore  the molecular mechanism of RBM25 depletion in inhibiting colon cancer progression,we performed high-throughput mRNA sequencing (mRNA-seq) in HCT116 cells with RBM25 knockdown.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for HCT116 cells with RBM25 knockdown or not

first: Lili

last: Zhi

first: Wenjing

last: Zhang

first: Yang

last: Wang


title: Identification of splicing factors signature predicting prognosis risk and the mechanistic roles of novel oncogenes in HNSCC

summary: Head and neck squamous cell carcinoma (HNSCC) is the most frequent subtype of head and neck cancer generally with a poor prognosis and limited treatment options due to its highly heterogeneous malignancy. In this study, we screened functional splicing regulatory RNA binding proteins (RBPs) that are closely related with prognosis of patients with HNSCC and showed significant expression difference between HNSCC tumors and normal tissues. Based on this finding, we have selected six candidate genes (HNRNPC, ZCRB1, RBM12B, SF3A2, SF3B3, and SRSF11) to construct a prognostic prediction model and validated the prognostic model for predicting patient survival. We found that the risk score predicted by our model can serve as an independent prognostic predictor, notably, HNSCC tumors showed enhanced expression of SF3B3, HNRNPC, and ZCRB1, which increases a patient's risk score in the discovered prediction model. The investigation of underlying mechanism validated that knockdown of SF3B3, HNRNPC, and ZCRB1 separately induced a substantial impairment of HNSCC cell survival. Conversely, single overexpression of one of the three genes promoted tumor cellular proliferation. High throughput RNA sequencing analysis and identification revealed that changes in the expression of SF3B3 and HNRNPC remarkably affected the splicing isoforms of genes related to cell cycle regulation, whereas the depletion of ZCRB1 contributed to notable DNA damage. Additionally, the prognostic prediction model's risk score was suggested to be related with immune infiltration score, particularly, SF3B3 has a negative correlation with CD8A expression. Therefore, our findings provide promising prognosis predictors and potential therapeutic targets for better treatment efficacy of HNSCC.

overall_design: To investigate the effects of the knockdown or overexpression of SF3B3, HNRNPC, and ZCRB1 genes on hypopharyngeal carcinoma FaDu cells, we established both overexpression and knockdown cell lines based on the FaDu cell line and then performed RNA-seq sequencing analysis.

first: Fang

last: Huang


first: Hyunu

last: Kim

overall_design: To investigate the binding sites of TCF19 in NK cells during MCMV infection, we performed TCF19 CUT&RUN using WT and TCF19-/- mice. CUT&RUN was performed on FACS sorted mouse Ly49H+ CD49b+ NK cells.

first: Joseph

last: Sun


title: RNA N6-methyladenosine modulates endothelial atherogenic responses to disturbed flow in mice

status: Public on Jun 20 2025

city: Tianjin

first: Bochuan

last: Li

address: Qixiangtai

department: Department of Physiology and Pathophysiology

institute: Tianjin Medical University

summary: Atherosclerosis preferentially occurs in atheroprone vasculature where human umbilical vein endothelial cells (HUVECs) are exposed to disturbed flow. Disturbed flow is associated with vascular inflammation and focal distribution. Recent studies have revealed the involvement of epigenetic regulation in atherosclerosis progression. N6-methyladenosine (m6A) is the most prevalent internal modiﬁcation of eukaryotic mRNA, but its function in endothelial atherogenic progression remains unclear. Here, we show that m6A mediates the EGFR signaling pathway during EC activation to regulate the atherosclerotic process. Oscillatory stress (OS) reduced the expression of METTL3, the primary m6A methyltransferase. Through m6A sequencing and functional studies, we determined that m6A mediates the mRNA decay of the vascular pathophysiology gene EGFR which leads to EC dysfunction. m6A modification of the EGFR 3’UTR accelerated its mRNA degradation. Double mutation of the EGFR 3’UTR abolished METTL3-induced luciferase activity. Adenovirus-mediated METTL3 overexpression significantly reduced EGFR activation and endothelial dysfunction in the presence of OS. Furthermore, TSP-1, an EGFR ligand, was specifically expressed in atheroprone regions without being affected by METTL3. Inhibition of the TSP-1/EGFR axis by using shRNA and AG1478 significantly ameliorated atherogenesis. Overall, our study revealed that METTL3 alleviates endothelial atherogenic progression through m6A-dependent stabilization of EGFR mRNA, highlighting the important role of RNA transcriptomics in atherosclerosis regulation.

overall_design: Examination of gene expressive levels, m6A enrichment levels in HUVECs treat with ST and OS for 6 hours.


title: Enhancer Poising Enables Pathogenic Gene Activation by Noncoding Variants [RNA-seq]

city: Irvine

first: Evgeny

last: Kvon

state: CA

address: 836 Health Sciences Road

institute: University of California, Irvine

summary: Single nucleotide variants within enhancers—non-coding DNA elements that regulate transcription—often lead to aberrant gene activation and contribute to a wide range of genetic disorders (Claussnitzer et al. 2015; Doan et al. 2016; Turner et al. 2017; Yanchus et al. 2022; Lettice et al. 2008). The mechanism by which ectopic gene activation occurs through these gain-of-function enhancer mutations remains poorly understood. Using the ZRS, a benchmark disease-associated enhancer of Sonic hedgehog (Shh), as a model, we demonstrate that poised (i.e., accessible but inactive) chromatin sensitizes Shh to aberrant activation in anterior limb bud, leading to polydactyly. In the anterior limb cells of wild-type mice, Shh is inactive, but the ZRS is accessible and marked by enhancer-associated histone modifications. We demonstrate that this poising signature explains how over 20 independent rare variants within the ZRS cause Shh misexpression in the same anterior limb bud cell population, resulting in similar limb malformations, despite affecting binding sites for different activators and repressors. Disabling pioneer transcription factor binding to the ZRS suppresses its poised state in anterior cells, prevents aberrant activation of the ZRS by rare variants, and fully rescues limb malformations in variant knock-in mice. A thorough examination of other disease-associated enhancers with pathogenic gain-of-function variants revealed that they are all poised in tissues with ectopic activity. We use this poising signature to predict and validate in vivo ectopic forebrain activity of previously uncharacterized autism-associated non-coding variants. Our findings suggest that spatial enhancer poising, likely a byproduct of development, leads to susceptibility to non-coding mutations offering a potential mechanistic explanation for the burden of disease-associated non-coding variants.

overall_design: Purified populations of anterior (mCherry+) and central (mCherry-) hindlimb cells were obtained from H11^hZRS404G>A-mCherry transgenic reporter mice at embryonic day 11.5. Each tissue domain was dissected, dissociated into single cells, and then fluorescently sorted. Sorted cells were snap-frozen before being processed for total RNA extraction. cDNA libraries were generated and sequencing was performed by Novogene at a median depth of 25M reads.

first: Ethan

middle: W

last: Hollingsworth

middle: Z


title: Enhancer Poising Enables Pathogenic Gene Activation by Noncoding Variants [CUT&RUN]

overall_design: CUT&RUN for ZIC3 was performed on bulk dissected anterior and posterior regions of E11.5 wild type mouse hindlimb buds


title: Enhancer poising enables pathogenic gene activation by noncoding variants [CUT&Tag]

overall_design: CUT&Tag followed by sequencing was performed on 30-60k fluorescently-sorted cells from anterior, central, and posterior domains of E11.5 mouse hindlimb buds. H3K4me1, H3K27ac, and ZIC2 antibodies were used. CUT&Tag direct, protocol version 4 published by the Henikoff lab was used.


title: SNU449 and SNU449 sorafenib-resistant cell line gene expression profiling

city: Seoul

first: Song

last: Jae Jin

address: Room 330, Ludlow Faculty Building, 50-1 Yonsei-ro, Seodaemun-gu

department: Biomedical science

country: South Korea

institute: Yonsei University College of medicine

summary:
We used microarrays to investigate transcriptomic changes between parental SNU449 and sorafenib-resistant SNU449 cells under various shRNA knockdown conditions.
Samples included control, sorafenib-resistant, Ad-shHSP27, Ad-shTGFβ1, Ad-shHSP27-shTGFβ1 and Ad-shTGFβ1-shGRP78 knockdown conditions.

overall_design: Total RNA was extracted from five experimental conditions and hybridized to GeneChip™ Human Gene 2.0 ST Arrays to compare gene expression changes associated with sorafenib resistance and molecular perturbation via shRNA.

first: Jaejin

last: Song

first: Sujin

last: Choi


title: A Ψ–Ψ codon–anticodon pairing in nonsense suppression and translational recoding

city: Rochester

first: Yi

last: Pan

address: 601 Elmwood Ave

department: Department of Biochemistry & Biophysics

institute: University of Rochester Medical Center

summary: Pseudouridine (Ψ) is known for decades, but its flexibility in base-pairing remains unclear. This study engineers artificial box H/ACA guide RNAs to direct pseudouridylation at the uridine of a premature termination codon (PTC; UAA, UAG, or UGA) within an intron-less mRNA and U36 of the anticodon of a matching tRNA in yeast and human cells. Targeted pseudouridylation leads to the formation of a Ψ-Ψ codon-anticodon pair, which, together with the other two Watson-Crick base pairs in the codon-anticodon duplex, significantly improves codon-anticodon recognition, robustly promoting PTC read-through. The intron-less mRNA level remains unchanged with or without guide RNAs. Additionally, pseudouridylation does not impact tRNA stability or charging. Our results show that nonsense suppression is promoted by the high affinity of the Ψ-Ψ pair, which is verified by melting curve analysis. This work has identified an unusual Ψ-Ψ base pair, which contributes significantly to codon-anticodon recognition and translational recoding.

overall_design: BY4741 strain was transformed with mCherry(K14UAG) reporter gene and guide RNA (gRNA) expression cassette for targeted pseudouridylation. The cassette contains targeted gRNAs, including PTC(K14ΨAG)-gRNA and tRNA_Lys(CUΨ36)-gRNA, for treated samples, or control gRNAs for control samples. Cells were cultured to mid-log phase before harvesting. Samples were processed by TB-SEQ, Inc. for RNA-seq and ribosome profiling (RIBO-seq).

first: Yi-Tao

last: Yu


title: Enhancer poising enables pathogenic gene activation by noncoding variants [ATAC-seq]

overall_design: ATAC-seq was performed on 50-100k fluorescently-sorted cells from anterior, central, and posterior domains of E11.5 mouse hindlimb buds. Sorted cells were lysed in nuclear lysis buffer for 5 min on ice before being spun down for transposition with Tn5 and then library preparation. Omni-ATAC-seq was performed following Corces et al, 2017 protocol.


title: Combined Flt3L and CD40 agonism restores dendritic cell driven T cell immunity in mouse models and patients with pancreatic cancer [mouse]

city: St. Louis

first: David

middle: G

last: DeNardo

state: Missouri

address: 425 S Euclid Ave

institute: Washington University

summary: T cell directed immunotherapies have largely failed to demonstrate clinical efficacy in patients with pancreatic ductal adenocarcinoma (PDAC). This broad resistance may result from poor tumor antigenicity and an immunosuppressive tumor microenvironment (TME). We hypothesize that tumor immunity in pancreatic cancer patients is further limited by systemic and tumor-intrinsic suppression of conventional dendritic cells (cDC). Here, we find that low tissue expression of Flt3L may in part underly cDC deficits in PDAC tissues. We show in both autochthonous murine models and samples from a clinical trial, that treatment with systemic Flt3L and CD40 agonists can restore cDC number and function in the PDAC TME. Alone, CD40 agonism failed to fully engage cDC activity; yet when combined with Flt3L, the dual therapy triggered a cDC driven type-I immune response characterized by CD8+ T cell infiltration, interleukin-12 production, and a reciprocal interferon (IFN) response. In mice, type-1 cDCs (cDC1) were directly responsible for the influx of CD8+ T cells, which in-turn supported cDC1 survival via IFN-γ, resulting in a synergistic feed-forward loop. Lastly, while we find that combination Flt3L and CD40 agonism improve tumor immunity, they also increase the number and effector phenotype of FOXP3+ regulatory T cells (Tregs) in a process dependent on type-2 cDCs. Tregs were found to limit therapeutic efficacy by inhibiting mature cDC subsets, and concurrent Treg depletion led to improved tumor control. These data support the continued investigation of combined Flt3L and CD40. agonism, which represent a promising strategy to engage anti-tumor immunity in advanced human malignancies.

overall_design: KPPC mice were treated with FLT3L, a CD40 agonist, or combination therapy for 12 days. Cells were isolated from the pancreas and FACs sorted to isolate CD45+ cells and CAF populations.

first: Graham

last: Hogg


title: The Notch ligand Jagged1 plays a dual role in cochlear hair cell regeneration

first: Angelika

last: Doetzlhofer

summary: Hair cells (HCs) within the inner ear cochlea are highly specialized mechano-sensory cells that enable us to detect sound. In humans and other mammals, HC loss is permanent and a leading cause of deafness. Recent studies in newborn mice revealed that supporting cells (SCs) have the capacity to form cochlear HCs, and that inhibition of Notch signaling dramatically increases the otherwise low rate of SC-to-HC conversion. It has been proposed that in the absence of HCs, the SC-specific Notch ligand Jagged1 (JAG1) mediates the HC-repressive role of Notch signaling. Here we show that conditional deletion of Jag1 at postnatal day2 (P2) increases the rate of HC formation/regeneration in cochlear tissue and organoids. However, Jag1 deficiency also reduces the expression of key progenitor and metabolic genes and attenuates PI3K-Akt-mTOR signaling in cochlear SCs and Kölliker’s cells and we show that Notch1 and Notch2 are critical for mediating these pro-growth functions of Jag1. Conversely, we show that increasing JAG1/Notch signaling enhances the mitotic capacity of cochlear SCs/ Kölliker’s cells. Finally, we show that JAG1 expression declines in SCs as they undergo maturation, and that stimulation of JAG1 signaling boosts the ability of maturing cochlear SCs to form HCs in an mTOR-dependent manner.

overall_design: We established organoid culture with postnatal day 2 cochlear epithelilal cells. Then we added doxycycline in the culture medium at the beginning of plating the cells. At 7 days of expansion, organoid culture was harvested with cell recovery solution to dissolve the matrigel in the 3D culture. Finally the cells were washed with PBS for three times, and total RNA from organoids was extracted using the miRNeasy Micro Kit as described previously. Then the total RNA samples were used for RNA-seq analysis.

first: Xiaojun

first: Charles

last: Morgan

first: Lin


title: Acyl CoA binding protein as a potential driver of pathological aging.

city: PARIS

first: Guido

last: Kroemer

address: 15 rue de L'école de médecine

institute: Centre de Recherche des Cordeliers

summary: The tissue hormone acyl coenzyme A binding protein (ACBP, encoded by the gene diazepam binding inhibitor, DBI) has been involved in various facets of pathological aging. Here, we show that ACBP plasma concentrations are elevated in (close-to-)centenarians commensurate with their health deterioration, correlating with a reduced glomerular filtration rate and a surge in senescence-associated cytokines. In a mouse model of chronic kidney injury by cisplatin, we observed that ACBP neutralization by means of a monoclonal antibody (mAb) prevented histopathological and functional signs of organ failure. ACBP inhibition also prevented the senescence of tubular epithelial cells and glomerular podocytes induced by cisplatin or doxorubicin, respectively, as measurable by the immunohistochemical detection of cyclin dependent kinase inhibitor 1A (CDKN1A, best known as p21). Senescence was also blocked by anti-ACBP mAb in additional mouse models of accelerated aging. This applies to liver damage induced by a combination of high-fat diet and carbon tetrachloride, where hepatocytes become senescent. In addition, administration of anti-ACBP mAb prevented natural and doxorubicin-accelerated cardiomyocyte senescence. We performed single nucleus RNA sequencing to study the transcriptome of hearts that had been exposed to doxorubicin and/or anti-ACBP in vivo. In cardiomyocytes, doxorubicin caused the anti-ACBP-reversible dyregulation of mRNAs coding for cardioprotective proteins involved in autophagy, fatty acid oxidation, mitochondrial homeostasis and oxidative phosphorylation. Altogether, these findings plead in favor of a broad antiaging effect of ACBP neutralization across different organ systems.

overall_design:
Nuclei extracted from left ventricles from C57Bl/6J female mice aged 12 weeks, after four weekly cycles of:
- Control (n = 2): PBS and mouse IgG2a isotype control
- Anti-ACBP/DBI (n = 2): PBS and Anti-ACBP/DBI mAb
- DOXO (n = 2): Doxorubicin and mouse IgG2a isotype control
- Anti-ACBP/DBI + DOXO (n = 2): Doxorubicin and anti-ACBP/DBI mAb

first: Léa

last: Montégut

first: Vincent

last: Carbonnier

first: Delphine

last: Le Corre

first: Sophie

last: Mouillet

first: Isabelle

last: Martins


title: Nanoplastics Enhance Transmembrane Transport of Carcinogens and Induce Cellular Stress Responses as Revealed by mRNA Sequencing Analysis

city: Boston

first: Bjoern

last: Reinhard

address: 590 Commonwealth Ave

institute: Boston University

summary: Nanoplastics were generated from common consumer plastics (PET, HDPE, PS, PVC) and exposed to simulated marine weathering for up to 10 weeks. Fourier-Transform Infrared (FT-IR) spectroscopy and ζ-potentials measurements revealed continuous changes in the composition of the nanoplastics consistent with oxidation. Although the chemical composition and oxidation of the nanoplastics influenced their ability to sorb Polycyclic Aromatic Hydrocarbons (PAHs), for all investigated conditions, sorption of PAH to nanoplastics achieved effective PAH concentrations that were orders of magnitude higher than the solubility limit in water. In an intestinal membrane model, PAH-loaded nanoplastics enhanced the overall PAH transport into and across the membrane, with HDPE achieving the highest intracellular PAH concentration. RNA sequencing of cell membranes exposed to nanoplastics revealed significant transcriptional changes, including upregulation of oxidative stress and detoxification pathways (NQO1, CYP1A1, CYP1B), especially in response to PAH-loaded nanoplastics, while genes associated with basic cell functions, such as cellular migration (NCKAP5, MACROD2) and division (KIF20A) were downregulated. These findings suggest that nanoplastics can increase bioaccessibility and bioavailability of hydrophobic carcinogens and enhance cellular stress, raising concerns about the potential environmental and health impacts associated with nanoplastics as carriers of hydrophobic environmental toxins.

overall_design: Cells were exposed to PET, PVC, HDPE, or PS with no weathering (native), 4 weeks or 10 weeks of weathering. All nanoplastics conditions were considered with and without PAH loading and for an exposure duration of 24 h. We also included cells exposed to free PAHs at their respective solubility limit in water (WaterPAH, 3 biological replicates) and untreated membranes as no treatment controls (NoTreat, 4 biological replicates)


title: PPP6R3-mediated dephosphorylation regulates mRNA translation during spermatogonial differentiation

city: Jinan

first: Qian

last: Fang

state: Shandong

address: 44 Wenhua West RD. Lixia District

institute: Shandong University

summary: Protein dephosphorylation mediated by phosphatases regulates spermatogenesis. However, which proteins are dephosphorylated and how they regulate spermatogenesis are largely unknown. Here, we show that germline-specific deletion of protein phosphatase 6 regulatory subunit 3 (PPP6R3), which determines substrate specificity of protein phosphatase 6 catalytic subunit, causes abnormal spermatogonial differentiation and male infertility, accompanied by translation inhibition. PPP6R3 directly interacts with EIF3C and EIF4G1 in KIT+ spermatogonia. Decreased levels of non-phosphorylated EIF3C and EIF4G1 after PPP6R3 deletion attenuate their enrichment for mRNAs associated with spermatogonial differentiation, and increased phosphorylation levels promote their degradation. Specifically, the phosphorylation status both of EIF3CS39 and EIF4G1S1217 are significantly upregulated in mutant mice. Overexpression of EIF3CS39A and EIF4G1S1217A mutants in Ppp6r3-cKO spermatogonial progenitor cells compensates for their differentiation efficiency by upregulating the translation rates of differentiation-associated mRNAs. Our findings demonstrate that dephosphorylation of EIF3C and EIF4G1 mediated by PPP6R3 is critical for translation activation during spermatogonial differentiation.

overall_design: Testis samples from P9 wild-type and Ppp6r3-cKO mice were collected to conduct transcriptomics profiling. Briefly, total RNA was extracted using TRIzol reagent, and then mRNA enrichment was performed by Oligo(dT) magnetic beads. Following the generation of cDNA libraries, Illumina sequencing was performed. The paired-end clean reads were aligned to mouse mm10 genome using Hisat2 (v2.0.5).

first: Hongbin

last: Liu


title: Granzyme K+ CD8 T cells slow tauopathy progression by targeting microglia

first: Kory

last: Johnson

address: 10/3B01, 9000 Rockville Pike

department: DIR IT & Bioinformatics

institute: NINDS/NIH

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Regulation of somatic hypermutation by higher-order chromatin structure [MutPE-Seq]

city: Freiburg im Breisgau

first: Maximilian

middle: Christian

last: von der Linde

address: Stefan-Meier-Straße 17

department: Institute for Medical Bioinformatics and Systems Medicine (IBSM)

country: Germany

institute: University Medical Center Freiburg

summary: The generation of protective antibodies by somatic hypermutation (SHM) is essential for antibody maturation and adaptive immunity. SHM involves co-transcriptional mutagenesis of immunoglobulin variable (V) regions regulated by enhancers located hundreds of kilobases away. How 3D chromatin structure affects SHM is poorly understood. Here, we measure higher-order interactions on single alleles of the human immunoglobulin heavy chain locus (IGH) using Tri-C. We find that SHM is underpinned by a multiway hub wherein the V region is proximal to all enhancers. Cohesin-mediated loop extrusion is dispensable for IGH transcription and hub architecture. Transcription and mutagenesis of IGH switch regions, which are necessary for antibody class-switch recombination, creates new chromatin loops that can form without cohesin. However, these additional loops do not compromise hub integrity, V region transcription or SHM. Thus, antibody maturation occurs within a multiway hub accommodating several gene-enhancer loops wherein transcription and mutagenesis of different segments can occur non-competitively.

overall_design: Examination of the multiway-interactome at the IGH locus through the chromatin conformation caputure technique Tri-C.

first: Ursula

last: E. Schoeberl

first: Johanna

last: Fitz

first: Renan

last: Valieris

first: Bernd

last: Bauer

first: Daniel

last: Malzl

first: Kimon

last: Froussios

first: Julia

last: Costea

first: Franziska

last: Schmidt

first: Jeremy

last: Pflaum

first: Bianca

last: Bartl

last: Albrecht

first: Eva-Maria

last: Wiedemann

first: Adriana

last: Cantoran Garcia

first: Mihaela

last: Peycheva

first: Marta

last: Rizzi

first: Anton

last: Goloborodko

first: Israel

last: Tojal da Silva

first: Rushad

last: Pavri


title: Regulation of somatic hypermutation by higher-order chromatin structure [Tri-C]

first: Maximilian

last: von der Linde

first: Ursula

last: E. Schoeberl

last: Fitz


title: m6A-seq profiling in Mettl3 Ctrl and dTAG-13 treated mESCs

status: Public on Jun 19 2025

city: Oxford

first: Guifeng

last: Wei

address: South Parks Road

country: United Kingdom

institute: University of Oxford

type: Methylation profiling by high throughput sequencing

summary: dTAG-13 treated METTL3_FKBP12(F36V) is sufficient to functionally deplete m6A methylation catalysed by METTL3/14 complex.

overall_design: We would like to confirm the functional m6A loss by dTAG-13 treatment in the Mettl3_FKBP12(F36V) cells. The Female mouse embryonic stem cells expressing dox-inducible Xist were employed to generate this dTAG line because Xist is heavily methylated by m6A so that it can be used as a marker to test the presence or absence of m6A modification. In this study, cells are either treated with dTAG-13 or not for 24 hours, and then added the doxycycline to induce Xist expression for another 24 hours. Total RNAs from these treated cells were isolated [Optional: and fragmented with independent Drosophila total RNAs in parallel].  All the fragmented RNAs were quantified well by Nanodrop and Qubit, then the equal ms of fragmented mESC RNAs (300ug) were mixed with 30ug of Drosophila total RNAs and subjected to standard m6A-seq. Fragmented mESC total RNA were used input-RNA-seq and used for the conventional m6A-seq analysis.

first: Neil

last: Brockdorff


title: N6-methyladenosine and the NEXT complex direct Xist RNA turnover and X inactivation dynamics [ChrRNA-seq; METTL3i]

summary: X chromosome inactivation (XCI) in mammals is orchestrated by the non-coding RNA Xist which together with specific interacting proteins, functions in cis to silence an entire X chromosome. Defined sites on Xist RNA carry the N6-methyladenosine (m6A) modification, and perturbation of the m6A writer complex has been found to abrogate Xist-mediated gene-silencing. However, the relative contribution of m6A and its mechanism of action remain unclear. Here we re-investigate the role of m6A in XCI by applying rapid degron-mediated depletion of METTL3, the catalytic subunit of the m6A writer complex, an approach that minimises indirect effects due to transcriptome-wide depletion of m6A. In contrast to prior studies, we find that acute loss of METTL3/m6A accelerates Xist-mediated gene silencing, and that this correlates with increased levels and stability of Xist transcripts. We show that Xist RNA turnover is mediated by the nuclear exosome targeting (NEXT) complex but is independent of the major nuclear m6A reader protein YTHDC1. Our findings demonstrate that the primary function of m6A on Xist RNA is to promote Xist RNA turnover which in turn regulates XCI dynamics.

overall_design: To investigate the role of m6A on Xist RNA, we treated the cells (Dox inducible Xist) either with DMSO or METTL3 inhibitor (STM2457) for 6 hours followed by dox-induced Xist expression of 24 hours with continued DMSO/METTL3i treatment. The concentration of METTL3 inhibitor used in this study is 10uM, 20uM, and 30uM. Gene silencing is measured as allelic ratio, i.e. Xi/(Xi+Xa).


title: N6-methyladenosine and the NEXT complex direct Xist RNA turnover and X inactivation dynamics [ChrRNA-seq]

overall_design: To investigate Xist-mediated chromosomal silencing following the depletion of various RNA m6A modification-related proteins, chromatin-associated RNAs (ChrRNA) were isolated to assess allelic gene expression. The dTAG degron strategy was used to rapidly deplete the proteins of interest, minimizing secondary or indirect effects caused by chronic protein inactivation. ChrRNAs were isolated from cells with an endogenous inducible Xist model (iXist-ChrX), derived from mouse embryonic stem cells. The allelic ratios of X-linked genes and Xist RNA expression levels were systematically and comprehensively compared.

first: Mafalda

last: Almeida


title: N6-methyladenosine and the NEXT complex direct Xist RNA turnover and X inactivation dynamics [RNA-Seq]

overall_design: To identify genes whose expression changes are directly driven by METTL3/m6A, we treated METTL3-FKBP12F36V cells with or without dTAG-13 for 26 hours to acutely remove METTL3, minimizing secondary or indirect effects associated with long-term knockout or knockdown of METTL3.


title: Single RNA-Seq on GBM cells from human GBM sample

city: Wuhan

first: Xuan

last: Liu

address: Donghu Road, No. 115, Wuchang District, Wuhan, Hubei Province, P.R. China

institute: Wuhan university

summary: Glioblastoma multiforme (GBM), a highly malignant and heterogeneous brain tumor, contains various types of tumor and non-tumor cells. Whether GBM cells can trans-differentiate into non-neural cell types, including mural cells or endothelial cells, to support tumor growth and invasion remains controversial. Here we generated two genetic GBM models de novo in immunocompetent mouse brains, mimicking essential pathological and molecular features of human GBMs. Single-cell RNA sequencing showed that patterns of copy-number variations (CNVs) of mural cells and endothelial cells were distinct from those of GBM cells, indicating discrete origins of GBM cells and vascular components. Furthermore, lineage tracing and transplantation studies demonstrated that, although blood vessels in GBM brains underwent drastic remodeling, GBM cells did not give rise to non-neural cell types in the brain. Intriguingly, GBM cells could randomly express mesenchymal markers, including those for mural cells, during gliomagenesis. Most importantly, single-cell CNV analysis of human GBM specimens also strongly suggested that GBM cells and vascular cells are separate lineages. Instead, non-neural cell types expanded by proliferation during tumorigenesis. Therefore, cross-lineage trans-differentiation of GBM cells is very unlikely to occur during gliomagenesis. Our findings advance understanding of cell lineage dynamics during gliomagenesis, and have implications for targeted treatment of GBMs.

overall_design: RNA profiles of Individual cells from tumor sites in  human GBM sample

first: Fangyu

last: Wang

first: Shaowen

last: Li

first: Yan

last: Zhou


title: N6-methyladenosine and the NEXT complex direct Xist RNA turnover and X inactivation dynamics [SLAM-Seq]

overall_design: To investigate the stability of Xist RNA following the depletion of various m6A RNA modification-related proteins (METTL3, YTHDC1, ZCCHC8), the dTAG degron system was employed. This method allows for rapid protein depletion, thereby minimizing secondary or indirect effects that may arise from prolonged protein inactivation. The SLAMseq strategy was used to analyze total RNA. Total RNA was isolated from cells of an inducible Xist model (iXist-ChrX) with a Bgl stem loop inserted in exon 7, derived from mouse embryonic stem cells.


title: Microglia regulate GABAergic neurogenesis in prenatal human brain through IGF1

status: Public on Jun 18 2025

city: San Francisco

first: Xianhua

last: Piao

institute: University of California, San Francisco

summary: We conducted snRNAseq to investigate the transcriptomics of postmortem prenatal and peirnatal human brains.

overall_design: Single nuclei suspension were prepared from whole (GW22-23), periventricle (GW30-PW3), or cortex(GW30-PW3) regions of postmortem huamn tissues. DAPI+, PU.1+ and OLIG2+ nuclei have been sorted with Fluorescence-activated cell sorting (FACS) and sequenced.

first: Andi

last: Wangzhou

first: Diankun

last: Yu


title: Peroxisome Proliferator-activated Receptor Agonist IVA337 Alleviates Inflammation

city: Shanghai

state: 279 Zhouzhu RD, Pudong New District

address: Zhou

institute: Shanghai University of Medicine and Health Sciences

summary: Metabolic dysfunction-associated steatohepatitis (MASH), an advanced stage of metabolic dysfunction-associated steatotic liver disease (MASLD), is characterized by significant hepatic fibrosis and inflammation. The pan-peroxisome proliferator-activated receptor (pan-PPAR) agonist IVA337 (lanifibranor) has demonstrated potential as an anti-MASH therapeutic, although its mechanisms of action remain incompletely understood. This study explores the effects and mechanisms of IVA337 using two distinct MASH models: 2D primary human hepatic stellate cells (HSCs) stimulated with TGFß1, and 3D liver spheroids comprising primary hepatocytes, HSCs, and nonparenchymal cells. In TGFß1-stimulated HSCs, IVA337 effectively suppressed the expression of fibrosis-related genes, including PAI1, COL1A1, and SMA, as well as the inflammatory gene IL-6. We successfully established 3D mouse and human liver spheroid models of MASH, characterized by reduced lipid content and elevated fibrotic gene expression. IVA337 treatment not only attenuated fibrotic gene expression but also restored lipid content in the MASH spheroids, as evidenced by BODIPY staining. Immunostaining further confirmed a reduction in SMA and collagen levels following IVA337 treatment. Bulk RNA sequencing and Gene Ontology (GO) analysis revealed lipid metabolism-related genes as key effectors downstream of IVA337. Additionally, IVA337 modulated multiple signaling pathways, including IL-17, TNF, NF-kappa B, PI3K-AKT, and MAPK. Collectively, these findings demonstrate that IVA337 effectively mitigates fibrosis development in both 2D and 3D MASH models by restoring lipid homeostasis and regulating crucial fibrotic and inflammatory pathways.

overall_design: Primary human HSCs and spheroids were treated with or without IVA337, then RNA-sequencing were performed with these samples.

first: Shuai

last: Wu

first: Xiaodan

first: Meng

last: Yan

first: Yifu

last: Ding

first: Lijuan

last: Zhang

first: David

last: Brenner

first: Xiao

first: Tatiana

last: Kisseleva


title: T-bet regulates the maintenance and ASC differentiation potential of lymph node and lung effector memory B cell subsets

city: Birmingham

first: Frances

last: Lund

state: AL

address: 1720 2nd Ave. South

department: Microbiology

institute: University of Alabama at Birmingham

summary: While human and mouse memory B cells (Bmem) can express T-bet, its role in regulating Bmem function is largely unknown. We characterized multiple transcriptionally distinct clusters of mature, somatically-mutated nucleoprotein (NP)-specific Bmem in LNs and lungs of influenza-infected mice. Although none of the Bmem expressed the plasma cell (PC) lineage commitment factors Blimp1, one cluster was enriched for Tbx21+ cells. Like the previously described human T-bet+ effector Bmem (eBmem) population, Tbx21+ mouse Bmem upregulated gene networks associated with effector metabolism, protein synthesis and the unfolded protein response. Using constitutive and inducible mouse models to ablate T-bet in B cells, we showed that T-bet expression by Bmem was required for persistence of LN and lung eBmem with rapid in vitro and in vivo PC differentiation potential. Thus, T-bet marked NP+ eBmem that were poised to differentiate and was required for maintenance of eBmem that respond in the lung following viral re-exposure.

overall_design: On two separate days, on D30 following PR8 (H1N1) influenza infection mdLN from Tbx21 reporter were isolated and pooled. Influenza specifc (NP+) and non-specific (NPneg) memory B cells were isolated by fluorescence activated cell sorting (FACS) and stained with CITE-seq antibodies (ADT and HTO). GEX, VDJ, and ADT libraries were prepared using the 10X platform.

first: Christopher

last: Risley

first: Mchael

last: Schultz

first: Rameeza

last: Allie

first: Shanrun

first: Jessica

last: Peel

first: Anoma

last: Nellore

last: Fucile

last: Scharer

first: Jeremy

last: Boss

first: Troy

last: Randall

first: Alex

last: Rosenberg


title: N6-methyladenosine and the NEXT complex direct Xist RNA turnover and X inactivation dynamics

first: Guifeng

last: Wei


title: Bcl11a is essential for B-1a cell maintenance during aging

city: shanghai

first: Yong

last: Yu

address: 1239 Siping Road

department: School of Life Sciences and Technology

institute: Tongji University


title: Cytoskeletal control in adult microglia is essential to restore neurodevelopmental synaptic and cognitive deficits

city: Diepenbeek

first: Ibrahim

last: HAMAD

state: Yes

address: Agoralaan Building C

department: Hasselt University

country: Belgium

summary: We have employed a single-cell sequencing approach using 10x Genomics scRNAseq to study the impact of Disc1 locus impairment (LI) on adult microglia. This study revealed that actin dysregulation and enhanced phagocytosis characterize the transcriptomic profile of Disc1 LI microglia.

overall_design: CD45lowCD11b+ immune cells were sorted out of the brains of 23-week-old homozygous mutated (Disc1 LI/LI) and wild-type (Disc1 WT/WT) littermate controls (n = 6 males per group) using a BD FACS ARIA II. Cells were analyzed using scRNAseq.

first: Sofie

last: Kessels

first: Yeranddy

last: Alpizar

last: Hamad

first: Ilse

last: Dewachter

first: Akira

last: Sawa

first: Bert

last: Brône


title: Microglia regulate GABAergic neurogenesis in prenatal human brain through IGF1 [MGEorganoid_scRNAseq]

first: Xianhua

last: Piao

summary: We conducted scRNAseq to investigate the transcriptomics of induced microglia (iMG) and iMG containing MGE-oriented organoids.

overall_design: Human embryonic stem cells-induced microglia (iMG) were transplanted to 4-week-old MGE organoids. We conducted scRNAseq to investigate the transcriptomics of 6-week-old MGE organoids with and without iMG. We also used Fluorescence-activated cell sorting (FACS) to enrich GFP-labelled iMG and condcuted scRNAseq.

first: Diankun

first: Andi

last: Wangzhou

first: Samhita

last: Jain


title: Rapid and long-lasting remodelling of the blood transcriptome following bariatric surgery

city: Basel

first: DBM Bioinformatics

last: Core Facility

state: BS

address: Hebelstrasse 20

department: Departement of Biomedicine

country: Switzerland

institute: University of Basel

summary:
Bariatric (or metabolic) surgery is the most effective treatment for severe obesity, resulting in sustained weight loss and rapid improvement in metabolic outcomes. However, it is currently unknown what molecular changes are induced by the surgery, how they relate to the health improvements, how early they occur, and whether they are maintained over time.
In this study, we characterized early gene expression changes following surgery in blood samples from the “Personalised Medicine for Morbid Obesity” cohort. We observed widespread changes in gene expression only a few days after surgery. Pathways related to immune response were affected, in particular with a decreased expression of the NF-κB pathway and neutrophil cytotoxic activity genes, suggesting a reduction of the inflammatory state typically associated with obesity. Metabolic signaling was also affected, with decreased expression of genes from pathways related to glucagon and insulin secretion, and key regulators such as the appetite-controlling hormone ghrelin. Comparisons to publicly available transcriptomics datasets showed that beyond these specific changes, bariatric surgery induces a transcriptome-wide reversal of expression changes associated with obesity and with forms of type 2 diabetes related to obesity. Comparisons to transcriptomics studies with longer-term follow-ups after bariatric surgery showed that a large fraction of early-induced expression changes likely persist for several months.
Taken together, we show that previously unreported early changes in blood gene expression after bariatric surgery provide in-depth insights into the resolution of the chronic inflammation associated with severe obesity and its connection with metabolic improvement.

overall_design:
Whole blood samples were obtained for standard laboratory assessments and transcriptome characterization with RNA-sequencing for 51 individuals from the “Personalised Medicine for Morbid Obesity” cohort who underwent bariatric surgery. Matched RNA-sequencing samples collected before, and after surgery (median of 11 days) were available for 24 study participants.

>>>Submitter states that raw data are not available due to patient privacy concerns.<<<

first: Julien

last: Roux

last: Blakemore

first: Claudia

last: Cavelti-Weder


title: Synthetic IL-21 cytokine mimetic promotes T cell function

first: Stephanie

last: Dougan

address: 450 Brookline Ave

institute: Dana Farber Cancer Institute

summary: Synthetic cytokine mimetics of IL-2 have been developed, including neoleukin-2 (Neo2), which has been shown to have enhanced therapeutic efficacy over IL-2, with reduced toxicity. IL-21 has also been used in TIL therapy to promote T cell expansion. Here, we tested the efficacy of Neo2, native IL-21, or synthetic IL-21 (21h10) in treating mice bearing B16 tumors that received ACT with melanoma-specific T cells (TRP1High and TRP1Low ; TCRs recognizing antigen with high or low affinity). We performed single-cell RNA-sequencing on these tumors to analyze cytokine therapy-induced changes in the microenvironment.

overall_design: Two days prior to tumor inoculation, naïve melanoma-specific T cells (TRP1High and TRP1Low) were adoptively transferred to wild-type mice. B16 tumors were inoculated and 5 days post tumor inoculation I.P. cytokine treatments commenced every day until day 15 when tumors from each cohort were labelled with hashtag antibodies and subjected to single-cell RNA-sequencing.

first: Jungho

last: Chun

first: Birkley

last: Lim

first: Kevin

last: Zhangxu

first: Michael

last: Walsh

first: Tavus

last: Atajanova


title: Master transcription factor binding sites constitute the core of early replication control elements [Repli-seq]

status: Public on Jun 17 2025

city: San Diego

first: David

last: Gilbert

state: California

address: 3525 John Hopkins Ct Ste 200

institute: San Diego Biomedical Research Institute

summary: Eukaryotic genomes replicate in a defined temporal order called the replication timing (RT) program. RT is developmentally regulated with potential to drive cell fate transi-tions, but mechanisms controlling RT remain elusive. We previously identified “Early Replication Control Elements” (ERCEs) necessary for early RT, domain-wide transcrip-tion, 3D chromatin architecture and compartmentalization in mouse embryonic stem cells (mESCs) but, deletions identifying ERCEs were large and encompassed many putative regulatory elements. Here, we show that ERCEs are compound elements whose RT activity can largely be accounted for by multiple sites of diverse master tran-scription factor binding (subERCEs), distinguished from other such sites by their long-range interactions. While deletion of subERCEs had large effects on both transcription and RT, deleting transcription start sites eliminated nearly all transcription with moder-ate effects on RT. Our results suggest a model in which subERCEs respond to diverse master transcription factors by functioning both as transcription enhancers and as el-ements that organize chromatin domains structurally and support early RT, potentially providing a feed-forward loop to drive robust epigenomic change during cell fate transi-tions.

overall_design: Replication timing profiling of hybrid mESCs containing various deletions of suspected cis-elements regulating replication timing

first: Jesse

last: Turner

first: Laura

last: Hinojosa-Gonzalez

first: Takayo

last: Sasaki

first: Satoshi

last: Uchino

first: Athanasios

last: Vouzas

first: Mariella

last: Soto

first: Abhijit

last: Chakraborty

first: Karen

middle: E

last: Alexander

first: Cheryl

last: Fitch

first: Amber

middle: N

last: Brown

first: Ferhat

last: Ay


title: ERAP1 deficiency exacerbates DSS-induced colitis and alters sulfasalazine efficacy via immune dysregulation

city: Swon

first: Hye-Myung

last: Ryu

address: 206, World cup-ro

department: Microbiolgy

institute: Ajou Hospital

summary:
UIntroduction: Ulcerative colitis (UC) is a life-threatening heterogeneous condition characterized by inflammation of the colon. Endoplasmic reticulum aminopeptidase 1 (ERAP1) is essential for antigen processing and immune regulation, however, its specific role in UC pathogenesis and therapeutic response remains unclear. This study aimed to investigate the role of ERAP1 in the response to sulfasalazine, a standard treatment for UC, using an ERAP1-heterozygous (ERAP1+/-) mouse model susceptible to colitis.

Methods: Wild-type (WT) and ERAP1+/- mice were treated with 2.5% dextran sulfate sodium to induce colitis, followed by sulfasalazine administration. Colitis severity was assessed through histopathology. Immune cell populations, including neutrophils, dendritic cells, T cells, and NK1.1+ cells, were analyzed using flow cytometry. RNA sequencing of colonic tissues was performed to assess gene expression changes associated with reduced ERAP1 expression.

Results: ERAP1+/- mice exhibited mildly increased susceptibility to DSS-induced colitis, with greater weight loss and distinct alterations in immune cell infiltration compared to WT mice. These differences were further pronounced after sulfasalazine treatment. RNA sequencing identified 428 differentially expressed genes between ERAP1⁺/⁻ and WT mice. Among these, 28 genes were previously associated with colitis or colorectal cancer, of which 11 were upregulated and 17 downregulated in ERAP1+/- mice. RT-qPCR confirmed significantly elevated expression of Anxa9, Atp2a1, and Hepacam2 in ERAP1+/- mice after sulfasalazine treatment, indicating a differential therapeutic response.

Conclusion: Collectively, our findings show that partial ERAP1 deficiency promotes immune dysregulation, alters the expression of inflammation-associated genes, and impairs sulfasalazine efficacy. Therefore, ERAP1 may serve as a key regulator in the pathogenesis of UC and a potential target for therapy.

overall_design: RNA-seq profiling of wild type and ERAP1 hetero type( ERAP1⁺/⁻ ) intestine.

first: HyeMyung

first: Bushra

last: Riaz

first: S. M. Shamsul

last: Islam

first: Rahar

last: Babita

first: Je Kyung

last: Seong

first: Ho

last: Lee

first: Eunjoo H

first: Seonghyang

last: Sohn


title: RNA-programmable cell type monitoring and manipulation in the human cortex with CellREADR

city: Durham

first: Jeffrey

last: Russ

state: NC

address: 311 Research Drive

institute: Duke University

summary: Reliable and systematic access to diverse cell types is necessary for understanding the organization, function, and pathophysiology of human neural circuits. Methods for targeting human neural populations are scarce and currently center on identifying transcriptional enhancers and engineering viral capsids. Here we demonstrate the utility of CellREADR, a programmable RNA sensor-effector technology that couples cellular RNA sensing to effector protein translation, for accessing, monitoring, and manipulating specific neuron types in human cortex, ex vivo. We designed CellREADRs to target two subpopulations, CALB2 GABAergic interneurons and FOXP2 glutamatergic projection neurons, then validated targeting specificity using histological, electrophysiological, and transcriptomic methods. CellREADR expression of channelrhodopsin and GCamp enabled the manipulation and monitoring of these populations in live cortical microcircuits. By demonstrating specific, reliable, and programmable experimental access to human neuronal subpopulations, our results highlight CellREADR’s potential for studying neural circuits and treating brain disorders.

overall_design: Patchseq data from CellREADR-labeled human cortical neurons. After electrophysiological recording, individual nuclei were extracted from neurons, RNA was purified and converted to cDNA for each nucleus, and RNA sequencing was performed.

first: Elizabeth

last: Matthews

middle: B

first: Yongjun

last: Qian

first: Shengli

last: Zhao

first: Peyton

last: Thompson

first: Muhib

last: Methani

first: Matthew

middle: L

last: Vestal

first: Josh

last: Huang

first: Derek

last: Southwell


title: Ontogeny-specific induction of the KMT2A::AFF1-fusion drives development of a distinct CD24 positive pre-leukemic state

city: Lund

first: Charlotta

last: Böiers

state: Lund

address: BMC B12

country: Sweden

institute: Lund University,

summary:
Infant Acute Lymphoblastic Leukemia (ALL) driven by the KMT2A::AFF1 onco-fusion is an aggressive, poor prognosis
disease with few co-operative mutations. The fusion originates in utero, yet the embryonic initiating steps of disease
development remain poorly understood. Here, we present a novel murine KMT2A::AFF1 model, that provides key insights into
KMT2A::AFF1 pre-leukemia, relevant to human disease. The model enables precise oncogene induction, and upon targeting
hematopoietic stem and progenitor cells (HSPCs) a selective negative impact on proliferation of hematopoietic stem cells
(HSCs) was observed, regardless of developmental state during induction. However, a unique CD24 PreProB subset expanded
exclusively within the KMT2A::AFF1 embryonic context. This population was absent when targeting lymphoid progenitors,
highlighting the importance of the cell of origin for leukemic development. The CD24 PreProB subset displayed key features
of pre-leukemic stem cells, including lineage plasticity and aberrant engraftment ability. In line with their pre-malignant
phenotype, single-cell transcriptomics revealed a signature consistent with stemness, and notable, up-regulation of Hmga2, a
regulator of self-renewal. The signature was critically transferable to human KMT2A::AFF1 patients. Furthermore, given that
CD24 is a potential therapeutic target, our findings uncover a distinct embryonic pre-leukemic state with direct relevance to
human disease.

overall_design:
A murine KMT2A::AFF1 model was used to investigate the impact of the oncogene duringe embryonic development and compared it to adult state. A Col1a1-tetO human KMT2A::AFF1 fusion gene was placed under the control of a Tet-op promoter (KMT2A::AFF1 ). The fusion was made Cre inducible by crossing with the Rosa26-ZtTA strain in which the tetracycline-trans-activator (tTA)
contains a STOP cassette flanked by LoxP sites. The expression of KMT2A::AFF1 was induced in embryonic
hematopoietic stem and progenitor cells (HSPCs) using Vav-Cre recombinase.
Control (cre-)and KMT2A::AFF1+ (cre+) cells from embryo (embryonic day 18.5) and postnatally induced adult mice were stained with CITE seq antibodies (ADTs and HTOs) . Three populations from each developmental time-point and genotype (cre- and cre+) were purified by FACS; LSK, PreProBs and ProBs.

first: Ariana

middle: S

last: Calderón

first: Roshanak

last: Ghazanfari

first: Zahra

last: Masoumi

first: Shabnam

last: Kharazi

first: Sara

last: Palo

first: Stefan

last: Lang

first: Kristijonas

last: Žemaitis

first: Mohamed

last: Eldeeb

first: Agatheeswaran

last: Subramaniam

first: Shamit

last: Soneji

first: Ronald

last: Stam

first: David

last: Bryder


title: High-throughput Transcriptomics Screen of 137 Per- and Polyfluoroalkyl Substances (PFAS) in U-2 OS Cells

city: RTP

first: Monique

last: Hazemi

address: 109 T.W. Alexander Dr

institute: US EPA

summary: High-throughput transcriptomics (HTTr) was undertaken to quantify and characterize the biological effects of chemicals observed in human-derived in vitro test systems. A set of 137 structurally diverse PFAS was tested using the HTTr assay in the human-derived cell type U-2 OS (osteosarcoma). The PFAS studies were carried out as a part of a larger screening study of a total of 1323 chemicals. Chemicals were randomized to treatment plates (cells plus media) in triplicate. Plates also contained reference treatments for quality control purposes. The TempO-Seq human whole transcriptome assay (Yeakley et al., 2017) was used to identify biological pathway altering concentrations (BPACs) and characterize the biological activity of test chemicals. For this analysis, a catalog of 11,037 gene expression signatures collated from multiple public sources were analyzed using concentration-response modeling of signature enrichment scores determined using single-sample gene set enrichment analysis (ssGSEA) (Barbie et al. 2009) with DESeq2-moderated fold change values as input (Love et al. 2014). A sample was considered active if 25 or more signatures were concentration-responsive and the BPAC was calculated as the 5th percentile of benchmark concentrations (BMCs) of active signatures.

overall_design: In a previous study, we established experimental and computational workflows for HTTr screening in a human cell model (Harrill et al., 2021). The TempO-Seq human whole transcriptome assay (Yeakley et al., 2017) was used to identify biological pathway altering concentrations (BPACs) and characterize the biological activity of test chemicals. The present study applies those methods to a larger chemical space focused on Per- and Polyfluoroalkyl Substances (PFAS). Here, we screened 144 PFAS chemical samples corresponding to 137 unique PFAS substances in U-2 OS (osteosarcoma) cells using HTTr and experimental conditions similar to those described previously (Harrill et al., 2021).  Each chemical sample was tested in a eight-point dilution series with one technical replicate well per chemical sample and dose level combination per culture, repeated across three independent cultures.  For most chemical samples, the tested concentration range was 0.03 to 100 µM with half-log spacing. The highest tested concentration for some chemicals was as low as 16.7 µM, but half-log spacing across eight dilutions was used for all chemicals.  U-2 OS cells were treated with PFAS chemicals for 24 hours prior to sampling for TempO-Seq analysis.

first: Joshua

last: Harrill

first: Logan

last: Everett

first: Derik

last: Haggard

first: Joseph

last: Bundy

first: Sarah

last: Davidson-Fritz

first: Imran

last: Shah

first: Richard

last: Judson

first: Clinton

last: Willis

first: Jo

last: Nyffeler

first: Felix

last: Harris

first: Gabrielle

last: Byrd


title: Disruption of KLHL37-N-Myc complex restores N-Myc degradation and arrests neuroblastoma growth in mouse models

city: Hangzhou

first: Meidan

last: Ying

address: No.866 Yuhangtang Road

department: College of Pharmaceutical Sciences

institute: Zhejiang University

summary: MYCN amplification accounts for the most common genetic aberration in neuroblastoma and strongly predicts the aggressive progression and poor clinical prognosis. However, clinically effective therapies that directly target N-Myc activity are limited. N-Myc is a transcription factor, and its stability are tightly controlled by ubiquitination-dependent proteasomal degradation. Here, we discovered that Kelch-like protein 37 (KLHL37) played a crucial role in enhancing the protein stability of N-Myc in neuroblastoma. KLHL37 directly interacted with N-Myc to disrupt the N-Myc/FBXW7 interaction, thereby stabilizing N-Myc and enabling tumor progression. Suppressing KLHL37 effectively induced the degradation of N-Myc and exhibited a profound inhibitory effect on the growth of MYCN-amplified neuroblastoma. Notably, we identified RTA-408 as an inhibitor of KLHL37 to disrupt KLHL37-N-Myc complex, promoting the degradation of N-Myc and suppressing neuroblastoma in vivo and in vitro. Moreover, we elucidated the therapeutic potential of RTA-408 for neuroblastoma by utilizing the PDC and PDX tumor models. RTA408's anti-tumor effects may not be exclusively via KLHL37, and specific KLHL37 inhibitors are expected to be developed in the future. These findings not only uncover the biological function of KLHL37 in regulating N-Myc stability, but also indicate that KLHL37 inhibition is a promising therapeutic regimen for neuroblastoma, especially in MYCN-amplified patients.

overall_design: RNA-seq profiling of SK-N-BE(2) xenograft model (shCtrl, shKLHL37 #1, shKLHL37 #2). RNA-seq profiling of SK-N-DZ xenograft model (Control, RTA-408 (50mg/kg), RTA-408 (100mg/kg)

first: Senfeng

last: Xiang

first: Pengfei

last: Chen

first: Xiaoxian

last: Shi

first: Hanqi

last: Cai

first: Zihan

last: Shen

first: Luyyang

last: Liu


title: Direct RNA long read transcriptomic data of 293T cells containing different promoters

city: Worcester

first: Athma

last: Pai

state: MA

address: 55 Lake Avenue North

department: RTI

institute: UMass Chan Medical School

summary: We applied direct RNA long read sequencing for characterization of transcripts from constructs inserted into HEK293T mammalian cells with different promoters. Direct RNA sequencing was performed on an Oxford Nanopore GridION device using the Direct Sequencing Kit (SQK-RNA004, date accessed 15 May 2024), MinION RNA flow cell (FLO-MIN00RA), and data pre-processing was performed with MinKNOW (v24.06.10).

overall_design: Direct RNA- long read sequencing on polyclonal 293T cells containing synthetic contructs that differ in promoter identity.

first: Daniels

last: Rachels

first: Valeria

last: Sanabria


title: POU3F2 regulates canonical Wnt signaling via SOX13 and ADNP to expand the neural progenitor population [RNAseq_SOX13NPCs]

city: Boston

first: Courtney

last: Benoit

address: 60 Fenwood Road

department: Neurology

institute: Brigham and Women's Hospital

summary: Loss-of-function mutations in the transcription factor POU3F2 have been identified in individuals with neurodevelopmental disorders. To elucidate the mechanistic role of POU3F2 in human neurodevelopment, we induced POU3F2 disruption in human neural progenitor cells (NPCs). Mutation of POU3F2 in NPCs causes reduced baseline canonical Wnt signaling and decreased proliferation, resulting in premature specification of radial glia. Additionally, POU3F2 levels across genetically diverse NPCs significantly associate positively with baseline canonical Wnt signaling and negatively with markers of radial glia specification. Through a series of unbiased analyses, we show that SOX13 and ADNP are transcriptional targets of POU3F2 which mediate POU3F2’s effects on Wnt signaling in human NPCs. Finally, we describe five individuals with autism spectrum disorder that harbor loss-of-function mutations in POU3F2, enhancing the genetic evidence for its critical role in human neurodevelopment. Together, these studies define POU3F2 as an activator of canonical Wnt signaling and mechanistically link two high-confidence autism genes, ADNP and POU3F2, in the regulation of neurodevelopment.

overall_design: CRISPR/Cas9-edited and respective control iPSC lines were differentiated into neural progenitor cells (NPCs) using a dual-SMAD inhibition protocol adapted from Topol, et al. (2015). SOX13 WT and MUT NPCs were harvested at D21 and subjected to RNA-sequencing. This data is presented in Figure 7I-K.

last: Pearse

first: Tracy

last: Young-Pearse


title: POU3F2 regulates canonical Wnt signaling via SOX13 and ADNP to expand the neural progenitor population [RNAseq_ROSMAPNPCs]

overall_design: iPSC lines from the ROSMAP cohort (n=33) were differentiated into neural progenitor cells (NPCs) using a dual-SMAD inhibition protocol adapted from Topol, et al. (2015). NPCs were harvested at D21 and subjected to RNA-sequencing. This data is presented in Figure 5.

first: Richard


title: Single-Cell and Spatial Multi-Omics Reveal Mechanical Stress Driving Heterogeneous Xylem Development in Populus [LCM Spatial RNA-seq]

city: Taipei

first: Ying-Chung Jimmy

last: Lin

address: No. 1, Sec. 4, Roosevelt Rd.

department: Department of Life Science

country: Taiwan

institute: National Taiwan University

summary: Xylem, the predominant tissue for structural support, forms tension wood with G-layer-rich fibers under mechanical stress. Despite being recognized over a century ago, three key biological questions remained unclear: (1) Are fibers in normal and tension wood distinct cells due to morphological differences? (2) Do tension wood fibers arise from different lineages? (3) What are the key genes regulating tension wood formation? We conducted single-cell RNA-seq on normal, tension and opposite xylem. Fibers in normal and tension wood belong to the same cell type and lineage. Differential developmental speed and cell-type ratio in tension and opposite xylem were further validated by spatial transcriptomics and metabolomics. Phosphoproteomics uncovered mechanical sensing mechanisms conserved between stems and roots across angiosperms. We identified a group of genes involved in the cell fate transition in tension wood. The knowledge on the heterogeneity of cell development offers insights for optimizing biomass production and bioenergy yield.

overall_design: Illumina RNA sequencing of normal and tension stem differentiating xylem (SDX) in P. trichocarpa

first: Jo-Wei Allison

last: Hsieh

first: Pin-Chien

last: Liou

first: Chia-Chang

first: Xiufang

last: Dai

first: Chen-Wei

last: Hu

first: I-Fan

last: Wang

first: Jr-Fong

last: Dang

first: Yi-Chi

last: Ho

first: Kai-Wen

last: Cheng

first: Wenjing

last: Xu

first: Shang-Che

last: Kuo

first: Chung-Ting

last: Kao

first: Dian-Xuan

last: Yang

first: Ray

first: Ke

last: Xiao

first: Jeng-Shane

first: Cheng-Chih

last: Hsu

first: Chuan-Chih

first: Te-Lun

last: Mai

first: Yuxiang

first: Mei-Chun

last: Tseng

first: Wei

last: Li

first: Ying-Lan


title: POU3F2 regulates canonical Wnt signaling via SOX13 and ADNP to expand the neural progenitor population [scRNAseq_POU3F2NPCs]

first: Courtney

last: Benoit

overall_design: CRISPR/Cas9-edited and respective control iPSC lines were differentiated into neural progenitor cells (NPCs) using a dual-SMAD inhibition protocol adapted from Topol, et al. (2015). POU3F2 WT and MUT NPCs were harvested at D21, subjected to single-cell RNA-sequencing, and the data was integrated with (1) snRNA-seq of iNs, ebAstros, and iMGLs, or (2) scRNA-seq of human fetal brain at Carnegie Stage (CS) 14. This data is presented in Figure 3A-H.

first: Richard

last: Pearse

first: Tracy

last: Young-Pearse


title: Master transcription factor binding sites constitute the core of early replication control elements [BRU-Seq]

first: David

middle: M

last: Gilbert

overall_design: Nascent transcription profiling by Bru-seq of hybrid mESCs containing various deletions of suspected cis-elements regulating replication timing

first: Jesse

last: Turner

first: Laura

last: Hinojosa-Gonzalez

first: Takayo

last: Sasaki

first: Satoshi

last: Uchino

first: Athanasios

last: Vouzas

first: Mariella

last: Soto

first: Abhijit

last: Chakraborty

first: Karen

last: Alexander

first: Cheryl

last: Fitch

first: Amber

last: Brown

first: Ferhat

last: Ay


title: POU3F2 regulates canonical Wnt signaling via SOX13 and ADNP to expand the neural progenitor population [RNAseq_POU3F2NPCs]

overall_design: CRISPR/Cas9-edited and respective control iPSC lines were differentiated into neural progenitor cells (NPCs) using a dual-SMAD inhibition protocol adapted from Topol, et al. (2015). POU3F2 WT and MUT NPCs were harvested at D21 and subjected to RNA-sequencing. This data is presented in Figure 1E-G.


title: Engineering biomimetic bone marrow niche with gene modified Mesenchymal Stromal Cells for ex vivo culture of human Hematopoietic Stem and Progenitor cells

city: Bagayam, Vellore

first: Srujan Kumar

last: Marepally

state: Tamil Nadu

address: Christian Medical College Campus,

country: India

institute: Centre for Stem Cell Research

summary: Hematopoietic Stem and Progenitor Cells (HSPCs) gene therapy has shown significant progress, with commercial approval for at least four distinct haematological disorders, and poised for a rapid expansion in the upcoming years. Despite these advancements, the ex vivo culture of HSPCs continues to present significant challenges. The stress induced by ex vivo culture can negatively impact transplantation outcomes, while the need for exogenous cytokine supplementation contributes to the high costs associated with gene therapy products. We developed genetically modified human bone marrow MSCs (GM-MSCs) secreting cytokines such as stem cell factor (SCF), Thrombopoietin (TPO), FMS-like tyrosine kinase-3-ligand (FLT3L), and interleukin-3 (IL3), closely resembling bone marrow cellular niche to augment HSPCs culture. HSPCs proliferate on GM-MSCs akin to standard conditions, devoid of external cytokine supplementation and these HSPCs retain their stem cell characteristics, colony-forming potential, stemness gene signatures, and capacity for long-term multilineage reconstitution in NBSGW mice. We demonstrate that our biomimetic feeder layer supports and alleviates stress associated with Homology Directed Repair (HDR) mediated gene-editing of HSPCs for fetal haemoglobin activation for a potential application in β-hemoglobinopathies gene therapy. Our GM-MSCs offer a compelling alternative to traditional cytokine supplementation by establishing a biomimetic bone marrow niche that fosters HSPC expansion while maintaining their stemness. These findings underscore the potential of engineered MSCs to revolutionize ex vivo HSPCs culture, ultimately enhancing their therapeutic value for gene therapy applications.

overall_design: HSPCs were cultured in standard condition and feeder layer with both wide type bone marrow MSCs and GM-MScs. In standard condition and WT-MSCs condition external cytokine were added where as GM-MSCs devoid of external cytokine. After 5days, cultured cells were collected for RNA sequencing.

first: Sevanthy

last: Suresh

first: Vigneshwaran

last: Venkatesan

first: Saravanabhavan

last: Thangavel

first: Srujan

middle: K


title: Activating nuclear receptor subfamily 2 group F member 2 in adipocyte stem cells rescues beige adipocyte metabolism impaired by excess early-life omega-6 fatty acids

city: Oklahoma City

first: Michael

middle: C.

last: Rudolph

state: OK

address: 975 NE 10th St. BRC North rm 364

department: Biochemistry and Physiology

institute: OUHSC

summary:
Background and aims: Developmental exposure to an elevated ratio of omega-6 (n6) to omega-3 (n3) fatty acids (FA) is linked to increased infant body fat and risk of future childhood obesity. We demonstrated in mice that the high n6/n3 developmental exposure reduced nuclear receptor subfamily 2 group F member 2 (NR2F2) in Adipocyte Stem Cells (ASCs), coincident with an altered ASC mitochondrial expression profile and increased white adipose accumulation in pups. This suggested that NR2F2- low ASCs might adopt a nutrient-storage phenotype. Here, we tested the hypothesis that NR2F2 is required in ASCs to undergo beige adipogenesis and metabolism needed during postnatal life for energy and thermogenesis.

Methods: C57BL/6J dams were randomized to either n6-rich or balanced n6/n3 control diets at the time of mating and underwent normal gestation and parturition. On postnatal day 12 (PND12), whole-body offspring metabolism was quantified by indirect calorimetry in conjunction with 13C-palmitate and 13C-glucose tracing. Inguinal fat pad ASCs were isolated by flow cytometry to assess adipocyte differentiation potential, global gene expression and proteomics, and mitochondrial oxidation. NR2F2 was transiently re-activated in vitro in ASCs with its ligand, 1-deoxysphingosine (1-DSO), and NR2F2 was ablated in ASCs ex vivo using homozygous floxed Nr2f2 pups to determine loss of function, ensure specificity of 1-DSO treatment during gain of function.

Results: Excess developmental n6-FA exposure reduced whole-body 13C-palmitate and 13C-glucose oxidation, diminished PND12 pup energy expenditure, and increased triacylglyceride accumulation in inguinal adipose. In ASCs isolated from n6-FA exposed pups, NR2F2 was decreased. These NR2F2-low ASCs formed downstream adipocytes with decreased beige metabolic regulators peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), peroxisome proliferator-activated receptor gamma (PPARγ), PR domain containing 16 (PRDM16), and uncoupling protein 1 (UCP1), had lower glycolysis and lipid metabolism enzymes, oxidized lipid and glucose at lower rates, and had increased lipogenic enzymes. Ex vivo deletion of Nr2f2 from ASCs recapitulated the metabolic deficits observed in the adipocytes derived from NR2F2-low ASCs isolated from n6-FA exposed pups. NR2F2 loss disrupted beige regulator induction, reduced adipocyte FAO, and promoted lipogenesis pathways, mirroring the n6-FA phenotype. Transient NR2F2 activation of ASCs from n6-FA pups using 1-DSO restored induction of beige regulators, increased mitochondrial oxidative phosphorylation enzymes, reduced lipogenic/storage pathways, ultimately enhancing nutrient oxidation.

Conclusions: These findings demonstrate that excess n6-FA developmental exposure disrupts NR2F2- mediated ASC fate determination, leading to formation of nutrient-storing, lipogenic adipocytes. This work highlights NR2F2 as an important upstream or parallel regulator necessary for beige adipogenesis and underscores its activation as a potential therapeutic approach to mitigate early-life obesity risk.

overall_design: Biological triplicates from control (Adeno-GFP) versus NR2F2-deleted (Adeno-Cre-GFP) were used. Adipocyte Stem-like cells were isolated by FACS from postnatal day 12 pups, NR2F2 was deleted using adenovirus-Cre in vitro, and cells were differentiated into adipocytes. Dams were provided the control n6:n3 fatty acid ratio diet.

first: Snehasis

last: Das


title: Cranial radiotherapy profoundly affects glia without inducing widespread cellular senescence

city: Utrecht

first: Stefan

last: Prekovic

address: Universiteitsweg 100

department: CMM

country: Netherlands

institute: UMC Utrecht

summary:
Background
More than 70% of cancer patients who underwent cranial radiotherapy have cognitive problems, suggesting that they might undergo accelerated brain aging due to the cancer treatment. Radiotherapy is known to induce cellular senescence in tumors and in various cell types in vitro. Therefore, we hypothesized that cranial radiotherapy induces cellular senescence in the brain.

Methods
We treated male C57BL/6 mice with various dosages of (fractionated) CT-guided cranial radiotherapy. The brains were analyzed for various senescence and glial markers using immunohistochemistry, histochemistry, Cosmx SMI and RT-qPCR. To contextualize the findings regarding cranial radiotherapy in young animals and assess whether these changes parallel natural aging, we studied the brains from 77-week-old female mice, which is considered middle-aged to old representing early aging.

Results
Surprisingly, we found no increase in markers for cellular senescence in the brain after cranial radiotherapy. However, we did detect profound changes in different types of glia. In early-aged mice we again did not detect an increase in senescence markers, but we observed the same directionality in the effects on glia. These effects on glia were milder compared to those upon cranial radiotherapy.

Conclusion
Overall, cellular senescence in the healthy brain seems a rather uncommon phenomenon and not induced upon cranial radiotherapy, but profound changes in different types of glia were detected upon cranial radiotherapy.

overall_design: This study contains 1 slide of a CosMx run, capturing a piece of the cortex, thalamus and hippocampus of control and irradiated (3x 10Gy) mice.

first: Laura

last: Kuil

first: Roan

middle: H

last: van Scheppingen

first: Yoran

last: Leter

first: Berend

last: Röring

first: Mark

last: de Gooijer

first: Caroline

last: van Heijningen

first: Annette

last: Compter

first: Olaf

last: van Tellingen

first: Sanne

last: Schagen


title: Right atrial snRNAseq analysis

city: Minneapolis

first: Kurt

last: Prins

state: MN

address: 2231 6th St Se

institute: University of Minnesota

summary: We evaluated the effects of pulmonary artery banding on the cellular landscape of the right atrium using single nucleus RNAseq

overall_design: Control pigs, PAB pigs had RA free wall nuclei isolated and were analyzed by single nucleus RNAseq