title: GSX2 Regulates Progenitor Maturation and Regional Identity Through Transcriptional Repression in the Developing Basal Ganglia [ATAC-seq]

city: Cincinnati

first: Brian

last: Gebelein

address: 3333 Burnet Avenue, MLC 7007

institute: Cincinnati Children's Hospital Medical Center

summary: Transgenic mouse models have demonstrated the critical role of the homeodomain transcription factor Genetic-Screened Homeobox 2 (Gsx2)   in the developing basal ganglia. However, the technical limitations of the existing in vivo approaches, such as progenitor heterogeneity and lack of temporal resolution, have impeded the investigation of direct regulatory targets . This study engineered a Dox-inducible human embryonic stem cell (hESC) line to investigate the function of GSX2 protein in directed differentiation cultures that model developing lateral ganglionic eminence-like (LGE-like) progenitors. Transcriptomic, chromatin accessibility, and genomic binding studies revealed that GSX2: (1) binds to both high- and low-accessibility chromatin using varying binding site preferences; (2) alters chromatin accessibility largely through indirect mechanisms; (3) functions primarily as a transcriptional repressor; and (4) regulates key conserved target genes of both progenitor maturation and regional specification. These results provide insight into the key regulatory roles and targets of GSX2, thereby establishing a new tractable experimental system to investigate basal ganglia development.

overall_design: Triplicate, longitudinal ATAC-seq of chromatin state in transgenic H9 (WA-09) hESC line (TRE3G 2xFLAG-GSX2 WT) differentiated to LGE-like progenitors at Days 10-12 with variable transgene induction

first: Edward

last: Farrow

first: Kenneth

last: Campbell

first: Hee-Woong

last: Lim


title: Spatially Resolved Insights Into Fistulating Crohn’s Disease Pathogenesis - bulk RNA-Seq

status: Public on Sep 30 2025

city: Oxford

first: Alison

last: Simmons

address: John Radcliffe Hospital/Headley Way

department: The MRC Weatherall Institute of Molecular Medicine

country: United Kingdom

institute: University of Oxford

type: Expression profiling by high throughput sequencing

summary: The aim of this study was to investigate how over expression of  transcription factors alters the gene expression program of primary human colonic fibroblasts. Fibroblasts isolated from healthy donor biopsies were transduced with lentiviral vectors to over-express TWIST1 or OSR2, or with empty vector as a control, followed by bulk RNA sequencing. The goal was to identify transcriptional pathways and effector genes regulated by these factors, with a focus on extracellular matrix organization, morphogen signaling, and fibroblast functional states.

overall_design: Primary human colonic fibroblasts were isolated from endoscopic biopsies of healthy donors and expanded in vitro. Cells were transduced with lentiviral vectors to over-express TWIST1 or OSR2, or with empty vector as a control, followed by puromycin selection. Total RNA was extracted from each condition using the RNeasy Plus Micro kit (Qiagen). Bulk RNA sequencing libraries were prepared by Novogene and sequenced on an Illumina platform to a depth of approximately 50 million paired-end reads per sample.

first: Colleen

last: McGregor

first: Xiao

last: Qin

first: Marta

last: Jagielowicz

first: Tarun

last: Gupta

first: Zinan

last: Yin

first: Verena

last: Lentsch

first: David

last: Fawkner-Corbett

first: Vy

last: Lai

first: Paula

last: Gomez Castro

first: Esther

last: Bridges

first: Chloe

last: Hyun-Jung Lee

first: Huei-Wen

last: Chuang

first: Lei

last: Deng

first: Anna

last: Aulicino

first: Renuka

last: Teague

first: Sorayya

last: Moradi

first: Jun

last: Park

first: Jeongmin

last: Woo

first: Kexin

last: Xu

first: Ruchi

last: Tandon

first: Nicole

last: Cianci

first: Jan

last: Bornschein

first: Ling-Pei

last: Ho

first: Paulina

last: Siejka-Zielinska

first: Zoe

last: Christoforidou

first: Sarah

last: Hill

first: Johannes

last: Lehmann

first: Rhea

last: Kujawa

first: Paola

last: Vargas Gutierrez

first: Carol

last: Cheng

first: Maria

last: Greco

first: Katherine

last: Baker

first: Mark

last: Bignell

first: Bruce

last: George

first: Eve

last: Fryer

first: Michael

last: Vieth

first: Agne

last: Antanaviciute


title: Hypoglycemia induces metabolic reprogramming and neurodegeneration through SRF/MRTF-A activation in the brain

city: Seoul

first: Minjeong

last: Jang

state: Asia/Oceania

address: 75, Nowon-ro, Nowon-gu, Seoul, Republic of Korea

country: South Korea

institute: Korea Institute of Radiological and Medical Sciences (KIRAMS)

summary: Hypoglycemia, a common complication in diabetic patients receiving insulin or hypoglycemic agents, as well as in individuals undergoing prolonged fasting, can cause brain damage; however, the molecular mechanisms underlying this effect remain poorly understood. To shed light on this issue, we investigated the molecular basis of hypoglycemia-induced neurodegeneration using both in vitro and in vivo hypoglycemic models. Starvation-induced hypoglycemia triggers neurodegenerative features in human neurons and glial cells, mirroring those observed in the brains of hypoglycemic mice. Notably, neurons activated a survival mechanism via serum response factor (SRF) and myocardin-related transcription factor A (MRTF-A)-mediated metabolic reprogramming, utilizing extracellular matrix (ECM) components as alternative energy sources. This adaptation led to excessive production of urea cycle by-products, which in turn induced neurodegeneration, astrocyte reactivity, and microglial activation. Importantly, glucose refeeding reversed these neurodegenerative features by deactivating SRF/MRTF-A signaling. Together, our results uncover a neuron-intrinsic mechanism linking glucose deprivation to reversible neurodegeneration via SRF/MRTF-A, offering potential targets for preventing hypoglycemia-associated brain damage.

overall_design: Total RNA from 3D-cultured ReN cells under hypoglycemic and steady-fed conditions for 24 and 48 hours was isolated using the QIAzol lysis reagent (79306, Qiagen, Germany). RNA quality was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was performed using an ND-2000 spectrophotometer (Thermo Inc., DE, USA).

first: Hong Nam

last: Kim


title: GlyT1 Inhibition by ALX-5407 Attenuates Allograft Rejection Through Suppression of Th1 Cell Differentiation

city: tianjin

first: hu

last: lu

state: State

address: fu kang lu

country: China

institute: TMU

summary: Objective: Transplant rejection driven by Th1 cell-mediated immune responses remains a critical challenge. This study aimed to investigate the role of glycine transporter 1 (GlyT1/SLC6A9) in Th1 differentiation and evaluate the therapeutic potential of its inhibitor, ALX-5407, in attenuating allograft rejection. Methods: RNA sequencing, flow cytometry, and qRT-PCR were employed to analyze GlyT1 expression in Th1-polarized CD4⁺T cells. ALX-5407 (0.5–500 nM) was tested in vitro under Th1-polarizing conditions. A murine skin allograft model (BALB/c to C57BL/6) was established to assess graft survival and immune responses. Combination therapy with rapamycin and ALX-5407 was evaluated through histopathology, immunofluorescence, and splenocyte profiling. Mechanistic insights were derived from RNA-seq, KEGG/GO enrichment, and Western blotting. Results: GlyT1 expression was significantly upregulated in Th1 cells and rejection cohorts. ALX-5407 suppressed Th1 differentiation, reducing IFN-γ⁺CD4⁺T cells proportions (p < 0.05) and activation markers (CD25, CD69), while inducing apoptosis via caspase-3 activation and BCL-2 downregulation. Although ALX-5407 monotherapy failed to prolong graft survival, combination with rapamycin synergistically enhanced efficacy (p = 0.018), reduced inflammatory infiltration, and attenuated splenic Th1 polarization. Mechanistically, ALX-5407 inhibited MAPK signaling but activated the PI3K-AKT-mTOR pathway, which rapamycin counteracted to amplify suppression. Conclusions: GlyT1 serves as a metabolic checkpoint in Th1 differentiation, and its inhibition by ALX-5407 attenuates allograft rejection through dual suppression of Th1 function and apoptosis induction. Synergy with rapamycin highlights a novel combinatorial strategy to mitigate rejection with reduced toxicity. These findings position GlyT1 targeting as a promising approach for clinical translation in transplantation immunotherapy.

overall_design: The CD4⁺T cells sorted from the spleen were polarized into Th0 and Th1 subsets under standard conditions for 3 days. For ALX-5407 treatment, cells were exposed to 500 nM ALX-5407 or vehicle (0.1% DMSO) for 72 h.

first: Xiaohan

last: Zhang

first: Weiqi

first: Jianghao

last: Wei

first: Shuai

last: Jin

first: Zhen

last: Wang

first: Hui

first: Gang

last: Feng

first: Jie

last: Zhao


title: GSX2 Regulates Progenitor Maturation and Regional Identity Through Transcriptional Repression in the Developing Basal Ganglia [ChIP-Seq]

first: Brian

last: Gebelein

state: OH

country: USA

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: ChIP-seq of FLAG-epitope-tagged GSX2 WT transgenic protein after 12 hours of doxycycline induction in LGE-like progenitors

first: Edward

last: Farrow

first: Kenneth

last: Campbell

first: Hee-Woong

last: Lim


title: Recurrent Splicing Programs Define a Conserved Axis of Tumor Heterogeneity and Prognosis

first: Nathan

last: Salomonis

state: Ohio

address: 3401 Burnet Ave, Cincinnati, OH 45229

department: Biomedical Informatics

institute: Cincinnati Children's Medical Hospital

summary: Alternative splicing is a widespread but largely undercharacterized contributor to tumor heterogeneity, lineage plasticity, and clinical outcomes. To systematically define the pan-cancer landscape of alternative splicing, we leveraged a sensitive unsupervised strategy to iteratively discover and functionally describe the universe of coordinated and uncoordinated splicing events across 25 malignancies and >10,000 patients. This analysis uncovered more than 1,000 distinct splicing-based tumor subtypes, including ~200 with prognostic significance and captured three times the number of poor survival splicing events than conventional analyses. Among these tumor subtypes, we describe a recurrent, poor-prognosis lung adenocarcinoma subtype enriched for but not genetically dependent on RBM10 mutations, and a neuroendocrine-like breast cancer subtype exhibiting brain-specific microexon splicing. These subtypes recur across multiple tumor types, revealing conserved splicing programs that transcend tissue of origin. Comparison of splicing across malignancies revealed nine core pan-cancer modules linked to regulation of distinct RNA binding proteins and reciprocal regulation of lineage differentiation programs. These findings establish an orthogonal classification system based on splicing dysregulation, independent of canonical splicing factor mutations, with implications for prognostic biomarker discovery and therapeutic targeting.

overall_design: RNA-seq of A549 RBM10 knockdown cells using shRNA.

first: Audrey

last: Crowther


title: Bacterial infections shape cardiac macrophages’ response to ischemia

city: Cologne

first: Simon

last: Geissen

address: Kerpener Strasse 62

country: Germany

institute: University Hospital Cologne

summary: Background: Patients with bacterial infections are at increased risk for subsequent cardiovascular events. Whether infections’ effects on innate immune cells within the cardiovascular system influence subsequent pathologies remains unclear. Here, we explore cardiac myeloid cells’ chronic adaptations to a preceding bacterial insult and implications for subsequent myocardial ischemia. Methods: We used various flow cytometry protocols to assess cardiac immune cells, peripheral leukocytes and hematopoietic stem and progenitor cells in bone marrow and spleen. A genetic fate mapping model was used to determine cardiac macrophages’ origin after bacteremia. Cardiac leukocytes were analyzed using scRNAseq. Nanoparticle-mediated RNAi was used to target macrophages in-vivo. Results: Cardiac macrophage numbers increased sharply, and numerical changes alongside subset alterations persisted over time. Fate mapping pointed towards local origin as the primary macrophage source after infection. Profiling macrophage heterogeneity using scRNAseq, we identified two previously unknown subpopulations remaining after resolution of infection. Whereas heightened metabolic activity was one subset’s primary feature, the other displayed excessive chemotactic properties, which amplified cardiac leukocyte recruitment and inflammation after a subsequent ischemic injury. Targeting cardiac macrophages’ surplus inflammatory activity after infection using nanoparticle-enabled, macrophage-directed RNAi kept disproportionate subsequent ischemic inflammation at bay. Conclusions: Bacteremia induces long-lasting changes in the cardiovascular system’s innate immune cells’ composition. This may amplify myocardial inflammation after a subsequent ischemic injury.

overall_design: C57Bl6/J mice were subject to cecal ligation and puncture (CLP); 6 weeks later, animals were sacrificed and hearts were flushed and digested. From Single cell dispension, CD45 positive cells were isolated by fluorescence activated cell sorting and immediately prepped for single cell sequencing by 10x genomics workflow. Animals of the same age without treatment were used as a control.

first: Alexander

last: Hof

first: Elvina Santhamma

last: Philip

first: Friedrich Felix

last: Hoyer


title: Determinants of successful AAV delivery of HIV-1 bNAbs in early life

city: Pittsburgh

first: Greg

last: Delgoffe

state: PA

address: 5051 Centre Avenue, 4051

department: Microbiology and Immunology

institute: University of Pittsburgh

summary: A single AAV vector dose delivering broadly neutralizing antibody 3BNC117 at birth provided over 3 years of protection against HIV-like infection in infant rhesus macaques. Neonatal administration was most effective due to immune tolerance, which reduced anti-drug antibodies and enabled sustained bNAb expression. This approach offers a promising one-time gene therapy strategy to prevent perinatal and adolescent HIV-1 infections. A total of 65 rhesus macaques (Macaca mulatta) of Indian origin were utilized in this study

overall_design: Newborn infant and juvenile rhesus macaques were divided into nine groups in total and treated with different AAV vectors delivering HIV-1 broadly neutralizing agents at different timepoints and blood plasma was collected to perform RNA-sequencing.

first: Martins

last: Mauricio

first: Daniel

last: Hagen

first: Isha

last: Mehta

first: Amir

last: Ardeshir


title: Sequential Platinum and PARP Inhibition Enhances PD1 Immunotherapy Efficacy in Murine Brca2 Mutated Pancreatic Cancer [RNAseq_2]

city: Philadelphia

first: John

middle: C

last: McVey

state: Pennsylvania

address: 3400 Spruce st

institute: University of Pennsylvania

summary: Pancreatic ductal adenocarcinoma (PDAC) is a challenging malignancy to treat, but emerging evidence suggests that specific subtypes may respond more favorably to certain therapies. BRCA-mutated PDAC represents a distinct subtype that is particularly sensitive to DNA-damaging therapies. The current standard of care for advanced BRCA-mutated PDAC involves induction platinum-based chemotherapy followed by maintenance therapy with a poly (ADP-ribose) polymerase inhibitor (PARPi). However, the randomized phase III POLO trial, upon which this standard is based, did not demonstrate an improved overall survival in patients who received olaparib compared to those who received placebo, highlighting the need for new therapeutic approaches. Additionally, there is a lack of robust models that recapitulate the tumor microenvironment of BRCA mutated PDAC, limiting the development of next-generation maintenance treatment options. In this study, we developed a syngeneic and immunocompetent mouse model of Brca2-mutated PDAC.  The model demonstrated high sensitivity to cisplatin plus gemcitabine, but limited efficacy of PARPi monotherapy. Induction with platinum-based chemotherapy sensitized tumors to PARPi maintenance therapy and promoted an exhausted, T cell-inflamed tumor microenvironment. However, resistance emerged which was associated with CDX2 expression and tumor differentiation. The addition of anti-PD1 treatment to PARPi maintenance enhanced tumor regression and prolonged overall survival. These findings provide preclinical support for ongoing clinical trials investigating immunotherapy with PARPi as a maintenance strategy in homologous recombination-deficient PDAC.

overall_design: Bulk RNA sequencing assessing impact of Gemcitabine/Cisplatin followed by maintenance Olaparib or placebo in a BRCA2 mutated mouse model

first: Max

last: Wattenberg

first: Gregory

middle: L

last: Beatty


title: Tumor suppressor genes p53 and p21 influence metformin’s antitumor efficacy in KRAS-mutated HCT116 colorectal cancer cells

city: Education City

first: Georges

last: Nemer

state: Doha

address: Penrose Road

department: College of Health and Life Sciences

country: Qatar

institute: Hamad Bin Khalifa University

summary: Colorectal cancer (CRC) continues to be the third most common cancer globally, often diagnosed at advanced stages due to a lack of early biomarkers. Current therapeutic methods frequently exhibit variable efficacy, highlighting the need for new treatment strategies. Metformin, a widely used antihyperglycemic agent, has recently garnered attention for its potential anti-tumor properties. In this study, we evaluated the effects of tumor suppressor genes TP53 and CDKN1A (p21) on the efficacy of metformin in a KRAS-mutant CRC model, HCT116 cells (harboring a G13D mutation in KRAS). Using the parental (p53+/+, p21+/+) cells and isogenic knockout (KO) versions for p53 and p21, we assessed cell viability, cell cycle distribution, and their transcriptomic responses to metformin treatment. Metformin significantly reduced cell proliferation in a dose- and time-dependent manner, with parental cells exhibiting the greatest sensitivity (approximately 20% viability reduction at 2 mM and 40% reduction at 10 mM after 24 h, p<0.05; and a 40% viability reduction at 5 mM and 60-70% reduction at 10 mM after 48 h, p<0.01). The loss of p53 or p21 notably decreased this sensitivity, with p53 KO cells responding only after 48 h of treatment, and p21 KO cells responding only at the higher dose of 10 mM after an incubation time of 72 h. The cell cycle analysis indicated a substantial G0/G1 arrest in parental cells (from approximately 55% of untreated cells to around 65% with 8 mM metformin at 24 h, p<0.05), while p53-/- and p21-/- cells did not show significant arrest (p>0.05). Our transcriptomic profiling revealed extensive differential gene expression in parental cells (1399 DEGs; 902 downregulated, 497 upregulated), in contrast to the limited responses in p53-/- (270 DEGs) and p21-/- cells (32 DEGs). Importantly, key genes involved in DNA replication stress (FAM111A), MAPK signaling (DUSP5), and inflammation modulation (TNFAIP3) were commonly regulated across the genotypes. Our findings emphasize that metformin’s antiproliferative effects in KRAS-mutant CRC are significantly influenced by the p53–p21 tumor suppressor axis, offering a more defined rationale for genotype-based precision oncology strategies.

overall_design: To investigate global transcriptomic changes associated with Metformin treatment of colorectal cancer cells, we performed bulk RNA sequencing on HCT116 cell lines and their isogenic p53 -/- and p21-/- derivatives, all sampled simultaneously. Each sample in this study comprises two biological replicates.

first: Asma

last: Saeed

first: Jamila

last: Hijazi

first: Zainab

last: Bachir

first: Pierre

last: Khoueiry

first: Assaad

last: Eid

first: Nadine

last: Darwiche

first: Maria Teresa

last: Bengoechea-Alonso

first: Johan

last: Ericsson

first: Borbala

last: Mifsud


title: Sequential Platinum and PARP Inhibition Enhances PD1 Immunotherapy Efficacy in Murine Brca2 Mutated Pancreatic Cancer [RNAseq_1]

overall_design: Bulk RNA sequencing assessing impact of Gemcitabine/Cisplatin or Olaparib in a BRCA2 mutated mouse model


title: A genome-wide CRISPR screen identifies the TNRC18 gene locus as a novel regulator of inflammatory signaling [CRISPR Screen]

city: North Chicago

first: Fedik

last: Rahimov

state: IL

address: 1 N. Waukegan Road

department: Genomics Research Center

institute: AbbVie

summary: Interleukin-1β (IL-1β) is dysregulated in chronic inflammatory diseases, yet the genetic factors influencing IL-1β production remain largely unknown. Myeloid-derived cells are the primary producers of IL-1β, which prompted a genome-wide CRISPR knockout screen in the human myeloid-derived U937 cell model treated with lipopolysaccharide (LPS) to mimic inflammatory conditions and sorted for high and low intracellular IL-1β levels. A total of 295 genes were identified as regulators of IL-1β production, including known mediators such as TLR4, JAK-STAT and IL-10 receptor. Notably, 57 out of the 295 genes overlapped with loci associated with human inflammatory diseases, including the TNRC18 gene locus associated with multiple diseases in the Finnish population. U937 cells engineered with the Finnish-enriched rs748670681 risk allele demonstrated decreased levels of mRNA for TNRC18 and an adjacent gene WIPI2, reduction in LPS-dependent gene activation and cytokine production, but elevation of interferon-responsive gene programs. Transcriptomic profiles for individual knockouts of TNRC18 and WIPI2 attributed the loss of LPS-dependent signaling primarily to TNRC18, which occurs through the modulation of H3K27 acetylation around inflammatory regulatory regions via TNRC18 and its protein interaction network. In contrast, the loss of WIPI2 is characterized by an exacerbation of interferon signaling. Collectively, these findings delineate the global regulatory mechanisms of IL-1β production and provide molecular insights to the role of the rs748670681 variant as a pleiotropic risk factor for inflammatory diseases.

overall_design: Human monocytic U937 cells that stably express Cas9 and LC3 GFP reporter were infected with the Brunello sgRNA library in three biological replicates. The Brunello CRISPR-Cas9 knockout library contains 76,441 sgRNAs targeting 19,114 protein coding genes with 1,000 non-targeting controls. Cell populations expressing the top and bottom 10% intracellular IL-1β levels were enriched using fluorescence activated cell sorting (FACS). Genomic DNA was isolated, the guide casette was amplifed by PCR, and then sequenced by next generation sequencing.

first: Charles

last: Lu

first: Jing

last: Wang

first: Joshua

last: Stender


title: A machine learning framework to predict cancer metabolomics from gene-expression data

city: London

first: George

last: Poulogiannis

address: 237 Fulham Road

department: Cell and Molecular Biology

institute: The Institute of Cancer Research

summary: Metabolomics provides a direct functional readout of a tumor’s physiology. Yet, it is lagging behind other omics technologies in facilitating disease monitoring and prognostication. This stems partly from the scarcity of large-scale metabolomic studies, but also the analytical complexities of detecting diverse metabolites with varying physicochemical properties and concentrations. To address this, we developed a machine learning framework using both tumor tissue and cell line samples across multiple cancer types that allows prediction of metabolomics from gene expression data. Two different model types were selected and trained for tissues and cell lines with their generalization capacity validated on independent cohorts, accurately predicting as high as 70-80% of tested metabolites. This work offers a scalable and efficient machine learning pipeline to determine metabolic from transcriptomic signatures, opening avenues to reconstruct and study the metabolic landscape of samples across novel and existing datasets lacking direct metabolomics measurements.

overall_design: RNA-sequencing profile of MCF7 (PIK3CA wild-type (WT) and E545K mutant (MUT)) and MCF10A (PIK3CA wild-type (WT), E545K and H1047R mutant (MUT)) isogenic cell lines.

first: Theodoros

last: Karalis

first: Aurelien

last: Tripp

first: Manas

last: Kohli


title: Stable lentiviral-mediated expression of Cytochrome P450 2D6 in HepaRG cells: New means for in vitro assessment of xenobiotic biotransformation and cytotoxicity.

city: Rennes

first: Pascal

last: Loyer

address: CHU Pontchaillou

country: France

institute: Inserm

summary: The human hepatic HepaRG cell line is often considered as the closest surrogate to primary culture of normal human hepatocytes (PHH) for toxicity studies. However, differentiated HepaRGcells express very low levels of the cytochrome P450 2D6 (CYP2D6) protein, which is essential for the biotransformation of nearly 25% of drugs on the market. To overcome this limitation, infection of progenitor HepaRG cells were performed using lentiviral particles containing a transgene encoding a single mRNA translated into a polypeptide undergoing proteolytic cleavage via the T2A peptide to produce both human CYP2D6 and GFP proteins. Differentiated HepaRG cells transduced with this lentivirus stably expressed GFP and catalytically active CYP2D6 enzyme at levels close to those found in PHH with high CYP2D6 activities. Using the CYP2D6 transgenic HepaRG cells, we showed that tramadol was metabolized into both, N- and O-desmethyl tramadol as observed in human serum in contrast with the production of N-desmethyl tramadol only in parental HepaRG cells via the CYP3A4 activity. Similarly, after perhexiline (PHX) treatments, higher IC50 were found in CYP2D6 expressing HepaRG cells associated to lower mitochondrial damages compared to those found in parental cells for the same PHX concentrations. Gene profiling between parental and transgenic cells demonstrated that the CYP2D6 expressing HepaRG cells had kept their ability to proliferate and differentiate with low impact on the expression of the hepatocyte specific functions, however, we identified a limited set of genes including NXF3 and TRIM63 that were up-regulated in CYP2D6 expressing HepaRG cells. By knocking-down GFP and CYP2D6 expression using CRISP/Cas9 technology in the transgenic cell line, we demonstrated that NXF3 and TRIM63 induction was triggered by the mRNA encoding GFP and CYP2D6 but not by the lentiviral transgene inserted into the HepaRG cell genome. Together, these data confirmed that the CYP2D6 transgenic HepaRG cells represent a suitable optimized transgenic model of HepaRG cells to evaluate biotransformation and toxicity of specific compounds metabolized by CYP2D6.

overall_design: Brb-seq profiling of parental and stably transduced CYP2D6+ HepaRG cells at day2, day4, day7, day14, day32, day37 and day44.

first: Hugo

last: Coppens-Exandier

first: Franz

last: Mackanga

first: Thomas

middle: A

last: Darde

first: Corentin

last: Bonhomme

first: Catherine

last: Ribault

last: Gicquel

first: Romain

last: Pelletier

first: Flyuza

last: Roshchina

first: Estelle

last: Schaefer

first: Christophe

last: Chesné

first: Agnès

last: Jamin

first: Anne

last: Corlu


title: A20 stabilizes IRF8: De novo trogocytic cDC2s orchestrate robust anti-tumor immunity in cDC1-deficient A20cKO mice

city: SUWON-SI

first: Jinjoo

last: Lee

state: - Select -

address: SEOBU-RO, JANGAN-GU

department: Biology

institute: SUNGKYUNKWAN UNIVERSIT

summary: Our study explored the role of the ubiquitin-editing enzyme A20 (TNFAIP3) in regulating  conventional dendritic cell (cDC) differentiation and its implications for anti-tumor immunity. We found that A20 stabilizes IRF8, essential for type 1 cDC (cDC1) development, by removing K48-linked polyubiquitin chain. DC-specific A20-depleted (A20cKO) mice showed a significant reduction in cDC1 levels but exhibited robust anti-tumor immunity due to superior MHC-I-mediated antigen presentation by de novo type 2 cDCs (cDC2s). These de novo cDC2s showed distinct gene expression profiles compared to wild-type cDC2s and acquired the ability to trogocytose adjacent tumor cells. NF-κB-dependent TNFα and TBK1/IRF3-mediated type I interferon secreted by cDCs in A20cKO mice played crucial roles in cDC2’s acquisition of cross-dressing characteristics, facilitating tumor-specific cytotoxic T lymphocyte responses even when cross-dressed with allo-tumor cells. This study highlights the crucial role of A20 in the development of cDC1s through IRF8 stabilization and unveils the unique anti-tumor potential of A20cKO cDC2s through their cross-dressing capabilities.

overall_design: DC1s and DC2s of the WT-BMDC and A20 iKO-BMDC derived from 3 mice each were isolated into using FACS and analyzed using QuantSeq 3’ mRNA-Seq.

first: KeeHyun

last: Kwon

first: Yong-soo

last: Bae


title: Histon lactylation drives HAS2 expression in gastric cancer tumorigensis after H.pylori infection [ChIP-seq]

city: Nanjing

first: Han

last: Chen

state: Jiangsu

address: #300 Guzhou Road, Gulou District

institute: Nanjing Medical University

summary: Histone lactylation, an epigenetic modification, promotes tumor growth and immune evasion in various cancers. Our study found increased pan-lysine lactylation and H3K18la in gastric tissues, linked to poor prognosis. Inhibiting glycolysis and silencing lactate dehydrogenase reduced H3K18la, boosting CD8+ T-cell activity against gastric cancer. H3K18la activated HAS2 transcription, enhancing MYC nuclear transport and PD-L1 expression..Our data provide a molecular framework for Histone lactylation in the process of gastric cancer.

overall_design: The ChIP-Seq with anti-H3K18la antibodies in AGS cell

first: Xiaoying

last: Zhou


title: PGR Isoform Modulation via Enhancer Activation Regulates Progesterone Signaling in Human Endometrial Stromal Cells [RNA-Seq]

city: Research Triangle Park

first: Francesco

middle: John

last: DeMayo

state: North Carolina

address: 111 T W Alexander Dr

department: DIR

institute: NIEHS

summary: Progesterone, acting through its isoform receptors PGR-A and PGR-B, is essential for regulating uterus function. The ratio between these isoforms is dynamic and context-dependent, driving distinct transcriptional programs. The upstream mechanisms controlling the PGR isoform balance and their functional impact in the uterus remain poorly understood. In this study, we identified two distal PGR enhancers in endometrial stromal cells located 60 kb and 220 kb upstream of the PGR transcription start site. We found that in comparison to PGR promoter-targeting which favors a PGR-B dominant profile, activation of these enhancers shifts the isoform ratio by increasing PGR-A and decreasing PGR-B expression. Bulk RNA-sequencing revealed that the isoform balance alters the progesterone-regulated transcriptome. PGR-A/B-equivalent cells displayed pro-inflammatory gene signatures, whereas PGR-B-dominant cells exhibited suppression of inflammatory signaling and cell cycle activity. Cut&Run assays identified differential genomic binding patterns associated with each isoform profile, and integration with chromatin interaction maps suggest direct regulation of distinct gene subsets. Upstream, ESR1 indirectly activated the PGR-A isoform dominantly, potentially through cooperation with FOXO1 at the U2 enhancer. This study identifies a novel enhancer-driven mechanism regulating PGR isoform dynamics and sheds light on how their imbalance in the endometrial stroma may reshape progesterone signaling in health and disease.

overall_design: RNA-seq profiling of control THESC cells or two THESC cell-lines with activated PGR isoforms at different ratios, treated with vehicle or 1uM Medroxyprogesterone Acetate (MPA) for 72 hours.

first: Skylar

middle: G

last: Montague Redecke

first: Austin

last: Bell-Hensley

first: MyeongJin

last: Yi

first: Shuyun

last: Li

middle: J


title: Enhanced Expression of the Female-Biased Gene Gsta2 in Male Proximal Tubule Cells Improves Renal Resilience to Ischemia-Reperfusion Injury

city: Los Angeles

first: Shun-Yang

last: Cheng

state: CA

address: 1425 San Pablo St

department: USC Stem cell - Eli and Edythe Broad Center for Regenerative Medicine

institute: University of Southern California

summary: Background: Genetic sex is an important determinant of renal injury and repair, with female kidneys typically exhibiting greater resilience to acute kidney injury (AKI). Among the sexually dimorphic genes in mouse proximal tubule cells (PTCs), Gsta2, an NRF2-regulated antioxidant enzyme, is strongly enriched in females. Here, we hypothesized that augmenting Gsta2 in male PTCs would enhance resistance to ischemia-reperfusion injury (IRI).

overall_design: we performed whole-kidney bulk RNA sequencing (bulk RNA-seq) on kidney tissues 4 hours post ischemia-reperfusion injury (IRI) to capture early events in the injury response by comparing transcriptional profiles between wild-type (WT) and transgenic (TG) kidneys

first: Jinjin

last: Guo

first: Taylor

last: Simonian

first: Paolo

last: Caldarelli

first: Andrew

middle: P

last: McMahon


title: Single cell RNA sequencing of the healthy Human Spinal Enthesis

first: Davide

last: Simone

address: Roosevelt Drive

department: Kennedy Institute of Rheumatologt

summary: We used single-cell RNA-sequencing to investigate the cellular basis of immunomodulation in the human spinal enthesis by phenotyping all cells from the bony and the soft tissue part of the entheseal organ

overall_design:
Spinal ligament samples from four individuals with non inflammatory spinal conditions were obtained during spinal surgery, were disstected by their anatomical region of origin (perientheseal bone and entheseal soft tissue); cells were isolated mechanically and enzymatically and sequenced using the 10x Chromium single-cell RNA-seq protocol.

NB The integrated single cell data object is deposited in Zenodo (accession number 15090823)

first: Moustafa

last: Attar

first: Jin

last: Liying

first: Stephen

last: Sansom


title: A genome-wide CRISPR screen identifies the TNRC18 gene locus as a novel regulator of inflammatory signaling

first: Fedik

last: Rahimov

overall_design: To investigate the downstream effects of the TNRC18 and WIPI2 gene knockouts on global gene expression in differentiated U937 myeloid cells, and the effects of the Finnish-enriched risk variant rs748670681 at the WIPI2/TNRC18 gene locus associated with immune-mediated diseases. Genes were knocked out using the CRISPR Cas9 or Cas12a enzymes. The risk alelle T of the rs748670681 SNP was inserted into U937 cells by the HDR-mediated CRISPR/Cas9 editing method. We performed gene expression profiling analysis using data obtained from RNA-seq of U937 cells treated with LPS or vehicle for 24 hours. Comparative gene expression analysis of RNA-seq data were performed between WT and KO cells, and rs748670681-TT and rs748670681-CC cells.

first: Jing

last: Wang

first: Apexa

last: Modi

first: Joshua

last: Stender


title: RNA-seq of ME-HESC in SRF-deficient vs Normal Conditions

first: Francesco

last: DeMayo

summary: The transcription factor SRF plays known roles in mitogenic cell response and muscle cell development, and potential roles in endometrial stromal decidualization have been reported. After RNAi-based SRF knockdown in menstrual effluent-derived primary human endometrial stromal cells (ME-HESC), SRF-deficient cells exhibited disrupted cytoskeletal remodeling, cell cycle, and extracellular matrix genes. These data show that SRF is important for endometrial stromal cell homeostasis.

overall_design: Primary menstrual effluent-derived human endometrial stromal cells (ME-HESCs) were obtained from deidentified archives that were previously banked after non-invasive isolation from menstrual fluids of healthy volunteers with the NIEHS Clinical Research Unit according to IRB approved, published methods and analyzed by RNA-seq at passage number 6. Cells from three distinct human donors were treated with control non-targeting siRNA or siSRF to analyze the impact of SRF deficiency on the transcriptome.

first: Ryan

middle: M

last: Marquardt

first: Peter

middle: F

last: Lais

first: San-Pin

last: Wu


title: RNA-seq of THESC in SRF-deficient vs Normal Conditions

summary: The transcription factor SRF plays known roles in mitogenic cell response and muscle cell development, and potential roles in endometrial stromal decidualization have been reported. After RNAi-based SRF knockdown in hTERT-immortalized human endometrial stromal cells (THESC), SRF-deficient cells exhibited disrupted cytoskeletal remodeling, cell cycle, and extracellular matrix genes. These data show that SRF is important for endometrial stromal cell homeostasis.

overall_design: THESCs (American Type Culture Collection #CRL-4003) were treated with control non-targeting siRNA or siSRF in triplicate followed by RNA-seq to analyze the impact of SRF deficiency on the transcriptome.


title: Multi-organ transcriptome changes in response to acute cold exposure

city: Barcelona

first: Francesc

last: Villarroya

state: Barcelona

address: Diagonal 643

department: Biochemistry and Molecular Biomedicine

country: Spain

institute: University of Barcelona

summary: Cold adaptation is a canonical model to study the induction of thermogenic and mitochondrial activity in adipose tissues. Yet, the effects of thermal adaptation in other metabolic-relevant tissues, such as the liver, are poorly explored. Here, we present a dataset that allows us to contrast the effects of acute cold exposure (24h, 4C) in a multi-tissue RNA sequencing approach.

overall_design: C57BL/6 adult mice were put under thermoneutral (30C) or cold (4C) conditions for 24 hours. Mice were sacrificed for tissues and blood samples to be collected. Interescapular brown adipose tissue, inguinal white adipose tissue, and liver were snap-frozen. From these tissues, RNA was purified and processed for sequencing.

first: Tania

last: Quesada-López

first: Albert

last: Mestres-Arenas

first: Joan

first: Marta

last: Giralt


title: Glycosylated Sterols Enhance Cold Tolerance in Tomato via Membrane Stabilization and Jasmonate Signaling

city: Cerdanyola del Vallès

first: ANTONI

last: GARCIA-MOLINA

address: Campus UAB - CRAG Building

department: Plant Development and Signal Transduction

institute: Centre for Research in Agrigenomics

summary: Free and glycosylated sterols play a central role in maintaining the structural integrity and proper functioning of plasma membrane, which serves as the primary sensor of cold and initiates signaling cascades to mitigate chilling-induced damage. Here we characterize the cold response of tomato (Micro-Tom) mutants with opposite glycosylated to free sterol ratio resulting from the overexpression and silencing of the sterol glycosyltransferases SlSGT2 and SlSGT1. A high ratio in the SlSGT2 overexpressing mutants increases cold tolerance, membrane stability, and oxidative stress responses, while a low ratio in the SlSGT1-silenced mutants causes the opposite phenotypes. The different cold sensitivity in these mutants is due to the altered membrane sterol profiles, rather than to a different capacity to adjust sterol levels during cold exposure. Changes in the glycosylated to free sterol ratio also trigger distinct transcriptional programs that establish a preconditioned stress-responsive state under basal conditions and a more efficient response under cold exposure in the SlSGT2 overexpressing mutants and compromise the capacity to withstand the effects of cold stress in the SlSGT1-silenced mutants. The SlSGT2 overexpressing mutants accumulate higher basal levels of jasmonates and display enhanced biosynthetic capacity under cold stress compared to SlSGT1-silenced and control plants. The elevated glycosylated to free sterol ratio in SlSGT2 overexpressing mutants facilitates JA biosynthesis and signaling, leading to the activation of cold-responsive genes, including those of the CBF–COR pathway, antioxidant defenses and polyamine biosynthesis. Our findings provide key insights into the mechanisms by which glycosylated sterols improve cold tolerance in tomato.

overall_design: Tomato (S. lycopersicum cv. Micro-Tom) plants were grown in pots containing a substrate mixture of peat (Klasmann TS2), perlite, and vermiculite (3:1:1) under controlled environmental conditions. The growth chamber was set to a 16-h photoperiod (light intensity: 200 μmol m⁻² s⁻¹) and an 8-h dark period, with a day/night temperature of 25/22°C. When plants were one-month-old, half of the them were transferred to a growth chamber maintained at 4°C while retaining the same light/dark regime, whereas the remaining plants continued under control conditions. Leaf samples (third and fourth leaves) were collected at different time points from both control and cold-treated plants, and either immediately analyzed or frozen in liquid nitrogen and stored at -80°C for further processing.

first: Cuiyun

last: Deng

first: Antoni

last: Garcia-Molina

first: Aurelio

last: Gomez-Cadenas

first: Vicente

last: Vives-Peris

first: Ruben

last: Alcazar

last: Ferrer

first: Teresa

last: Altabella


title: A genome-wide CRISPR screen identifies the TNRC18 gene locus as a novel regulator of inflammatory signaling [CUT&Tag]

first: Fedik

last: Rahimov

overall_design: CUT&Tag was performed on two biological replicates per condition using the CUT&Tag-IT Assay Kit (Active Motif, 53172) following manufacturers recommendation. Briefly, 450,000 cells per replicate per condition were bound to the prepared Concanavalin A beads (+spike-in control, Active Motif, 53173) and incubated overnight at 4°C in a rotator with primary antibodies [1 µg of mouse anti-H3K27Ac (1:50, Active Motif, 91193), 1 ug of positive control mouse anti-H3K4me3 (1:50, Active Motif, 91263)]. The samples were then incubated with Rabbit Anti-Mouse secondary antibody (1:100, Active Motif, 53160) for 1 hour at room temperature on a rotator, followed by washes in Dig-Wash buffer. The beads were resuspended with CUT&Tag-IT Assembled pA-Tn5 Transposons and incubated at room temperature on a rotator, followed by washes with Dig-300 buffer. The beads were finally resuspended in tagmentation buffer, and the reactions incubated at 37°C for 60 minutes. The DNA was extracted as detailed in the manufacturer's instructions. Libraries were PCR-amplified using i5 and i7 indexed primers (14 cycles) and primer dimers were removed with AmpureXP bead (Beckman Coulter, A63881) purification. The final libraries were evaluated on Agilent 4200 Tapestation (quality) and Thermo Qubit (quantity) and sequenced on the Illumina NextSeq550 platform (75-bp paired-end reads). Data analysis was performed using HOMER and DeepTools. Briefly, raw FASTQ read pairs were pre-processed where adapter, low-quality sequences, duplicates were removed using cutadapt, rmdup. Preprocessed reads were aligned to human genome (hg38) using Bowtie2 and Drosophila genome (spike in control) to generate the normalization factor. Bedtools were used to sort and index, and HOMER to generate TagDirectory, peak calls and bedgraph for visualization.

first: Rahimov

last: Fedik

first: Ghosh

last: Sujana

first: Shi

last: Manman

first: Stender

last: Joshua


title: Effect of pressure-overload on cardiac endothelial cells in Balb/c mice

city: Rouen

first: Ebba

last: Brakenhielm

address: Inserm U1096

summary: We performed single-cell RNA-seq analysis of FACS-sorted cardiac blood vascular and lymphatic endothelial cells in the heart of Balb/c female mice in a model of pressure-overload.

overall_design: Cardiac CD31+ CD45neg endothelial cells, and Lyve1/Podoplanin-expressing lymphatic endothelial cells (LEC) were extracted from hearts of mice 8 weeks after pressure-overload or sham surgery in a murine model of transaortic constriction, and analyzed by single-cell RNA-sequencing. Briefly, cardiac single cell suspensions were sorted by FACS (ARIA II, BD Biosciences). BECs were defined as live CD45-/CD31+/Lyve1- cells, whereas LECs were identified as CD45-/CD31+/Lyve1+/Pdpn+ cells. A 1:3 mix of cardiac LECs and BECs were included in each sample prepared using the 10X Genomics Chromium Next Gem Single Cell 3' kit. For each group 1-2 reactions were performed using each time pooled cells from 10 mouse hearts. Samples were sequenced using Illumina NextSeq550 at an estimated sequencing depth of 20 000 reads per cell. Reads were aligned to the mouse reference genome (GRCm39.111) using STARsolo RNA-seq aligner (v2.7) to produce raw UMI count matrix. Data filtering, normalization, integration, principal component-based unsupervised clustering, and differential analysis were performed using the Seurat package (version 5.0.2) in R (version 4.1.2). For low-quality data filtering, genes expressed by <10 cells, and cells expressing <500 genes, were excluded, as were cells with UMI numbers <500 or cells with >10% mitochondria-derived UMI counts.  Among 3908 total sequenced cells, 3436 cardiac cells remained post-filtering, including 1932 cells from sham-operated BALB/c controls and 1504 from post-TAC mice.

first: Coraline

last: Heron

first: Theo

last: Lemarcis

first: Oceane

last: Laguerre

first: Benjamin

last: Bourgeois

first: Corentin

last: Thuilliez

first: Chloe

last: Valentin

first: Anais

last: Dumesnil

first: Manon

last: Valet

first: Gaetan

last: Riou

first: Sophie

last: Candon

first: Celine

last: Derambure

first: Caroline

last: Berard

first: Helene

last: Dauchel

first: Virginie

last: Tardif

first: Brakenhielm

last: Ebba


title: RNA-seq of PgrCre/+Srfflox/flox  and Srfflox/flox mouse uterus at gestation day 3.5

first: Francesco

last: DeMayo

summary: Upon conditional ablation of Srf in the Pgr-expressing cells of the mouse uterus, PgrCre/+Srff/f (SrfKO) females were infertile, displaying uterine fibrosis and failure to decidualize or support embryo implantation. RNA-seq analysis demonstrated dramatic changes in gestation day 3.5 SrfKO uterine tissues including dysregulation of cytoskeletal remodeling and growth response genes in addition to strong induction of proinflammatory cytokine signaling. These data show that SRF is key for endometrial function and fertility through regulation of inflammatory homeostasis.

overall_design: Four mice of each genotype were staged at gestation day 3.5 (3 days after finding vaginal plug), and RNA was isolated from uterine tissues for sequencing.

first: Ryan

last: Marquardt

first: Peter

last: Lais

first: San-Pin

last: Wu

first: John

last: Lydon


title: Decreased CD4+ T Cell Counts Drive Aberrant B Cell Repertoire Alterations in People Living with HIV

city: Hefei

first: Lina

last: Huang

state: 安徽

address: Hefei in Anhui Province

institute: university of science and technology in China

summary: Understanding the evolution of broadly neutralizing antibody (bNAb) activity in people living with HIV is crucial for vaccine design and immunization strategies. It has been proposed that antibody cross-reactive activity is associated with lower CD4+ T cell counts during people living with HIV, but the underlying mechanisms remain unclear. To further explore the correlation between antibody reactivity and CD4+ T cell counts, we recruited people living with HIV with varying CD4+ T cell counts: (i) CD4+ T cell <= 50 cells/mL, (ii) 50 cells/mL < CD4+ T cell <= 200 cells/mL, (iii) 200 cells/mL < CD4+ T cell <= 500 cells/mL, (iv) 500 cells/mL < CD4+ T cell. We constructed four SOSIPs.664 trimers and evaluated the antigen-specific antibodies in serum. Immune repertoire sequencing was used to characterize the BCR repertoire of these individuals. The evaluation of antigen-specific antibodies with different SOSIP.664 trimers showed enhanced reactivity in individuals with low CD4+ T cell counts compared to those with high/normal CD4+ T cell counts. Analysis of antibody gene repertoires through BCR high throughput sequencing revealed an increased proportion of IgG with heavy chain complementarity-determining region 3 (CDRH3) loops exceeding 20 amino acids in individuals with CD4+ T cell counts below 50 cells/mL. Notably, the IGHV1-46 and IGHV4-34 germlines, which result in most polyreactive B cells, were preferentially used in individuals with low CD4+ T cell counts. These results suggest that limited engagement of CD4+ T cells could facilitate the survival of aberrant B cell repertoire with long CDRH3 regions.

overall_design: High-throughput sequencing (HTS) to investigate the BCR repertoire in people living with HIV with different CD4+T cell counts

first: Xiangyu

last: Zhang

first: Yu

last: Shi

first: Rishen

last: Liang

first: Qianqian

last: Chen

first: Jing

last: Yang

first: Xiaoni

first: Anning

last: Fang

first: Qian

first: Chengchao

last: Ding

first: Jiabin

first: Jianjun

first: Yong

last: Gao


title: HOXC4 Promotes Proliferation of Endometriotic Stromal Cells via the SLIT2-ROBO1 Axis

city: Shanghai

first: Yuqi

address: No. 826 Zhangheng Road, Pudong New District, Shanghai

department: Department of Pharmacology

institute: School of Pharmacy, Fudan University

summary: Current interventions for endometriosis mainly involve hormone therapies but have limited efficacy and unacceptable side effects, due to the lack of selectivity to distinguish between endometriosis and endometrial tissues. Elucidating the molecular mechanism underlying rapid growth of endometrial-like stromal cells, one of the main components of endometriotic lesions, will pave a path for more effective treatment of endometriosis. In the current study, we utilized transcriptome sequencing to compare the transcriptional profiles of endometrial-like stromal cells from endometriosis and endometrial tissues and demonstrated that Homeobox C4 (HOXC4) is preferentially expressed in endometriotic lesions. HOXC4 is indispensable for the proliferation of stromal cells from endometriosis, but not those from endometrial tissues. Mechanistically, HOXC4 acts as a transcription factor to promote the expression of Slit Guidance Ligand 2 (SLIT2) and thereby, increases the p38 MAPK activity via the SLIT2 receptor Roundabout Guidance Receptor 1 (ROBO1). Considering the essential role of the p38 MAPK activity in facilitating the development of ectopic endometrium, our findings strongly support the idea of HOXC4, as well as the SLIT2-ROBO1 axis, being as potential therapeutic targets for endometriosis.

overall_design: RNA-seq profiling of primary endometriotic stromal cells, endometrial stromal cells , and HOXC4 knockdown primary endometriotic stromal cells from endometriosis patients.

first: Ding

first: Qingquan

last: Li


title: Neuroprotective effect of CrAT overexpression in mice RGC

city: Sendai

first: Kota

last: Sato

state: Miyagi

address: 1-1, Seiryo-machi, Aoba-ku

department: Ophthalmology

country: Japan

institute: Tohoku University

summary: AAV2-CrAT prevent RGC loss against optic nerve crush and NMDA injury. in vitro study demonstrated that CrAT overexpression could prevent ferroptosis in oxidative stress in neuronal cells.

overall_design: We performed AAV2-CrAT intravitreal injection, and optic nerve crush in mice one month later after AAV2 injection. RGC was purified by FACS and gene expression changing was examined (duplicate).

first: Dong

last: Xiaoxuan

first: Michiko

last: Ohno-Ohishi

first: Daisuke

last: Saigusa

first: Ritsumi

last: Saito

first: Akira

last: Uruno

first: Amane

last: Fujioka

first: Shigeto

last: Maekawa

first: Sarah

last: Erp

first: Satoru

last: Noguchi

first: Seiya

last: Kanno

first: Chiaki

last: Yamaguchi

first: Sakura

last: Shiina

first: Masayuki

last: Yamamoto

first: Toru

last: Nakazawa


title: A comprehensive tRNA pseudouridine map dependent on   human stand-alone pseudouridine synthases

city: Oxford

first: Linzhen

last: Kong

address: University of Oxford Old Road Campus Research Building

department: Nuffield Department of Medicine

institute: Ludwig Institute for Cancer Research

summary: Pseudouridine (Ψ) is one of the most abundant RNA modifications in human cells, which is introduced post-transcriptionally by pseudouridine synthases (PUS). Despite its prevalence, the biological functions of Ψ remain poorly understood, largely due to the limited knowledge linking specific PUS enzymes to their targets. To address this gap, we systematically knocked out or knocked down 9 stand-alone PUS in HCT116 cells and mapped their Ψ profiles using 2-bromoacrylamide-assisted cyclization sequencing (BACS). Through this approach, we uncovered previously unknown targets of several PUS enzymes, including RPUSD1, RPUSD2, PUS3, PUSL1, and PUS7L. Additionally, we revealed that TRUB1 and PUS10 function redundantly to catalyze the highly conserved Ψ55 modification in cytosolic tRNAs. Intriguingly, we found that RPUSD3 and TRUB2 do not exhibit noticeable enzymatic activities in human cells. By integrating these findings with earlier results for TRUB1, PUS7, and PUS1, we constructed a comprehensive map of stand-alone PUS-dependent Ψ modifications across human tRNAs. Using this map, we further demonstrated that different PUS enzymes introduce Ψ modifications at distinct stages of pre-tRNA processing.

overall_design: Pseudouridine detection using BACS in HCT116/HeLa WT cells, TRUB2 KD, PUSL1 KO, RPUSD1 KO,RPUSD2 KO,RPUSD3 KO,RPUSD4 KD, PUS10 KO,PUS3 KO and PUS7L KO cells. Two monoclones for each KO were selected for BACS.

first: Haiqi

last: Xu

first: Mengjie

first: Giuseppina

last: Pisignano

first: Jingfei

last: Cheng

first: Feng

last: Feng

first: Parinaz

last: Mehdipour

first: Chun-Xiao

last: Song


title: Disruption of transcriptional programming by CDK9 inhibition suppresses triple-negative breast cancer and is enhanced by EGFR inhibition

city: Leiden

first: Bob

last: van de Water

state: Leiden

address: Einsteinweg 55

country: Netherlands

institute: Leiden University

summary: CDK9, in complex with cyclin T1 or T2, is essential for mRNA transcription by enabling paused RNA polymerase II to proceed into elongation. Increasing evidence highlights CDK9’s involvement in transcriptional addiction in cancer. In this study, we explored the efficacy of novel CDK inhibitors and demonstrated that targeting CDK9 significantly impairs proliferation and induces apoptosis in various triple-negative breast cancer (TNBC) cell lines. Transcriptomic analysis revealed that CDK9 inhibition caused downregulation of genes governing transcription, cell cycle progression, and oncogenic signalling pathways, such as TGF-β and Wnt/β-catenin signalling. This disruption of transcriptional programs was further enhanced by combining CDK9 and EGFR inhibitors.

overall_design: We performed RNA-sequencing to analyse the effects of 6 hours treatment with the CDK9 inhibitors I-73, Y3-21, D10-81 and combined CDK9 and EGFR inhibition by I-73 and lapatinib on gene expression in the Hs578T TNBC cell line.

first: Ronan

last: McLaughlin

first: Vera

middle: E

last: van der Noord


title: Transcriptomic analysis of Hela Ezrin Ko cells

city: Pozzuoli

first: Rossella

last: De Cegli

address: Via Campi Flegrei 34

country: Italy

institute: Tigem

summary: Ezrin (EZR), a cell membrane to cytoskeleton linker protein,  plays key roles in maintaining cellular homeostasis. Its deregulation at various levels strongly correlates with cancer progression, metastasis and higher patient mortality.  However, the precise mechanistic basis for its role remains unknown. Recently, we demonstrated that Ezrin participates in EGFR/AKT signaling pathways. Importantly, transitions between active (phosphorylated open) and inactive (dephosphorylated closed) forms of Ezrin is crucial for its function in cancer. Gene expression analysis suggested dysregulation of genes that are functionally linked to cell energetic metabolism with potential implications for cell proliferation, survival and apoptosis. Total RNA was extracted from n=4 for each experimantal group: Transcriptomic analysis was performed on HeLa WT and HeLa Ezrin KO cells.

first: Ivan

last: Conte

first: Giuliana

last: Giamundo


title: Mouse Spinal Cord Transcriptome Data

city: Stockholm

first: Keying

last: Zhu

state: -- select state --

address: L8:04, Center for Molecular Medicine, Karolinska University Hospital

country: Sweden

institute: Karolinska Institutet

summary: To compare the transcriptomic profile of mouse spinal cord homegenates from different conditions using the NanoString nCounter Mouse Glial Profiling Panel with additional customized probes.

overall_design: In this study, we extracted mRNA from mouse spinal cord homogenates from the following groups: wild-type 1-year-old female (WT-OF), wild-type 1-year-old male (WT-OM), wild-type 2–3-month-old female (WT-YF), wild-type 2–3-month-old male (WT-YM), 1-year-old female 10 days post-microglial Tgfbr2 deletion (KO-OF-D10), 1-year-old female 20 days post-microglial Tgfbr2 deletion (KO-OF-D20), 1-year-old female 30 days post-microglial Tgfbr2 deletion (KO-OF-D30), 1-year-old male 30 days post-microglial Tgfbr2 deletion (KO-OM-D30), 2–3-month-old female 30 days post-microglial Tgfbr2 deletion (KO-YF-D30), and 2–3-month-old male 30 days post-microglial Tgfbr2 deletion (KO-YM-D30). The samples were hybridized to the nCounter Mouse Glial Profiling Panel CodeSet (XT-CSO-M-GLIAL-12, NanoString). This CodeSet includes 757 probes for genes of interest, 13 probes for internal reference genes, and 10 additional customized probes for Smad4, Smad7, Nrros, Tgfbr2, Tgfbr3, Smurf1, Tgfb2, Cxcr4, Adgre4, and Clec4b1. A total of 3–5 mice were included for each group.

first: Robert

last: Harris

first: Harald

last: Lund


title: A sperm-enriched 5’fragment of tRNA-Valine regulates preimplantation embryonic transcriptome and development

city: Santa Cruz

first: Upasna

last: Sharma

state: California

address: 1156 High St

department: MCD Biology

institute: UCSC

summary: Sperm small RNAs have been implicated in intergenerational epigenetic inheritance of paternal environmental effects; however, their biogenesis and functions remain poorly understood. We previously identified a 5’ fragment of tRNA-Valine-CAC-2 (tRFValCAC) as one of the most abundant tRNA fragments in mature mouse sperm. We previously reported that tRFValCAC is specifically enriched in sperm during post-testicular maturation in the epididymis, and extracellular vesicles secreted by the epididymis epithelial cells can deliver tRFValCAC to sperm. Here, we investigated the mechanistic basis of tRFValCAC delivery from epididymis to sperm and its functions during early embryonic development. We show that tRFValCAC interacts with an RNA binding protein, heterogeneous nuclear ribonucleoprotein A/B (hnRNPAB), in the epididymis and that hnRNPAB regulates the levels of tRFValCAC in the vesicles secreted by epididymis epithelial cells. Inhibition of tRFValCAC in preimplantation embryos altered the transcript abundance of genes involved in RNA splicing and mRNA processing. Importantly, tRFValCAC-inhibited embryos showed altered mRNA splicing, including alternative splicing of various splicing factors and genes important for proper preimplantation embryonic development. Moreover, our data suggest that tRFValCAC regulates its target genes via interaction with RNA-binding proteins. Finally, we find that inhibition of tRFValCAC in zygotes delayed preimplantation embryonic development. Together, our results reveal a potential function of a sperm-enriched tRF in regulating alternating splicing and preimplantation embryonic development and shed light on the mechanism of sperm small RNA-mediated epigenetic inheritance.

overall_design: We examined the regulatory roles of tRFValCAC in early preimplantation embryos by inhibiting tRFValCAC using an antisense locked-nucleic acid (LNA) containing oligo targeting tRFValCAC (Val_LNA). Embryos were microinjected with either H3.3-GFP mRNA with LNA-containing scramble oligo (Ctrl_LNA) (control group) or H3.3-GFP mRNA plus tRFValCAC antisense LNA oligo (Val_LNA) (experimental group). GFP-positive embryos were cultured until the late 2-cell stage for mRNA-seq (28 hours post-IVF).

first: Simeiyun

last: Liu

first: Andrew

middle: D

last: Holmes

first: Sol

last: Katzman


title: RB modulates chromatin organization by regulating cohesin-dependent loops and enhancer-promoter interactions [ChIP-seq]

city: Boston

first: Hanjun

last: Lee

state: Massachusetts

address: 77, Avenue Louis Pasteur

department: Department of Genetics

institute: Harvard Medical School

summary: The retinoblastoma protein (RB) is a well-characterized repressor of E2F transcriptional activity that controls genes involved in cell proliferation during the G1-phase. Here, we examine the effect of RB on chromatin organization and uncover a non-canonical role for RB in which it promotes the removal of cohesin from insulators and thereby induces the expression of adjacent genes. We identify that RB colocalizes with cohesin across the human genome. During mitosis, RB facilitates the removal of cohesin from CTCF sites, with this effect persisting into early G1, impacting loop formation and extrusion at topologically associating domain (TAD) boundaries. Chromosome conformation capture assays revealed that RB reduces insulation at TAD boundaries and promotes enhancer-promoter interactions marked by histone 3 lysine 27 acetylation. In this way, RB enhances the transcription of hundreds of genes beyond the E2F transcriptional program, expanding our understanding of this key tumor suppressor and cell cycle regulator.

overall_design: SMC3 ChIP-seq for RB WT or RB KO hTERT1-RPE1 cells that are synchronized in G1 or mitotic phase

first: Ioanna-Maria

last: Gkotinakou

first: Michael

middle: S

last: Lawrence

first: Ioannis

last: Sanidas


title: Chromatin accessibility (ATAC-seq) analysis on inner ear organ of Corti from WT and Bmi1 KO neonatal mice

city: SHANGHAI

first: yunzhong

last: zhang

state: SHANGHAI

address: 83 fenyang road, xuhui district

department: Department of ENT Institute and Otorhinolaryngology

institute: Eye & ENT Hospital of Fudan University

summary: Clarify changes in chromatin accessibility regions after Bmi1 knockout by performing ATAC-seq on Bmi1 KO and WT organ of Corti from neonatal mice, providing a foundation for integrated analysis of epigenetics and transcriptome sequencing"

overall_design: ATAC-seq data was extracted from OC of WT and Bmi1-/- neonatal mice organ of corti.


title: Transcriptome sequencing samples of the Bmi1 knockout and wildtype neonatal mouse organ of Corti.

summary: Clarify the transcriptome changes in the inner ear cochlea after Bmi1 knockout

overall_design: Collect  the organ of Corti  from Bmi1 knockout newborn mice and WT mice for RNA extraction and transcriptome sequencing


title: ZIKV infection of a hiPSC-NPC line derived from a Congenital ZIKV Syndrome (CZS)-affected patient

city: Southampton

first: Rob

last: Ewing

state: Hampshire

address: University Rd, Southampton

department: School of Biological Sciences

institute: University of Southampton

summary: Zika virus (ZIKV) is a mosquito-borne flavivirus consisting of historical African and epidemic-associated Asian lineages. The latter can access the central nervous system and cause microcephaly in the developing foetus through infection and depletion of neural stem and progenitor cells. The molecular mechanisms involved in this depletion response are grossly unknown for cells arising from CZS-affected children. Here, we sought to investigate the molecular mechanisms underlying the neuropathology of ZIKV infection in NPCs.

overall_design: The Neural precursor cell line NPC-608-1 was cultured under monolayer conditions and infected for 12 and 24 hours with Zika virus (Strain KU365779.1)

first: Matthew

last: Sherwood

first: Thiago

last: Mitsugi

first: Brandon

last: Coke

first: Yilu

last: Zhou


title: H3K27me3 ChIP sequencing on inner ear organ of Corti from WT and Bmi1 KO neonatal mice

summary: We conducted ChIP sequencing for H3k27me3 mark on inner ear organ of Corti from WT and Bmi1 KO neonatal mice, thereby clarifying the epigenetic mechanisms by which Bmi1 impacts auditory cell fate.

overall_design: Comparative CUT&Tag analysis was performed by pulling down with an anti-H3K27me3 antibody and comparing this with a nonspecific negative control Input.


title: Histone methyltransferase PRDM9 promotes survival of drug-tolerant persister cells in glioblastoma [RNA-seq]

city: Camperdown

first: George

middle: Louis

last: Joun

state: NSW

address: John Hopkins Drive

department: School of Medical Sciences

country: Australia

institute: The University of Sydney

summary: Chemotherapy often kills a large fraction of cancer cells but leaves behind a small population of drug-tolerant persister cells. These persister cells survive drug treatments through reversible, non-genetic mechanisms and cause tumour recurrence upon cessation of therapy. Here, we report a drug tolerance mechanism regulated by the germ-cell-specific H3K4 methyltransferase PRDM9. Through histone proteomic, transcriptomic, lipidomic, and ChIP-sequencing studies combined with CRISPR knockout and phenotypic drug screens, we identified that chemotherapy-induced PRDM9 upregulation promotes metabolic rewiring in glioblastoma stem cells, leading to chemotherapy tolerance. Mechanistically, PRDM9-dependent H3K4me3 at cholesterol biosynthesis genes enhances cholesterol biosynthesis, which persister cells rely on to maintain homeostasis under chemotherapy-induced oxidative stress and lipid peroxidation. PRDM9 inhibition, combined with chemotherapy, resulted in strong anti-cancer efficacy in preclinical glioblastoma models, significantly enhancing the magnitude and duration of the antitumor response by eliminating persisters. These findings reveal a previously unknown role of PRDM9 in promoting metabolic reprogramming that enables the survival of drug-tolerant persister cells.

overall_design: PRDM9 Knock-out RKI1 glioblastoma stem cell line was treated with +/- 10µM CMPD1 for 72 hours. The KO cell line was compared to Non-Targetted Control (NTC) RKI1 glioblastoma stem cell line, also treated with +/- 10µM CMPD1 for 72 hours, of the same transduction batch and passage number within each biological replicate. There are three biological repeats. We excluded RKI1 n=2 NTC CMPD1 (Sample006) from downstream analysis due to the sample not clustering with the other two NTC CMPD1 repeats.

first: Lenka

last: Munoz

first: Emma

last: Kempe


title: CD8 T cells are primed by cDC1 and exacerbate tau-mediated neurodegeneration

city: SAINT LOUIS

first: Hao

last: Hu

state: MO

address: 4370 Duncan Ave

department: Neurology

institute: Washington University School of Medicine

summary: There are changes in adaptive immunity in Alzheimer’s disease (AD) and increases in activated CD8 T cells in brain correlate with tau pathology. However, which cells mediate T cell priming in tau-mediated neurodegeneration remains unclear. In different conditions such as cancer, viral infections, and autoimmune diseases outside the CNS, conventional type-1 dendritic cells (cDC1) perform antigen cross-presentation to prime CD8 T cells. We demonstrate that tauopathy mice deficient in cDC1 are markedly protected against tau-mediated neurodegeneration and display a selective decrease in brain CD8 T cell infiltration and glial reactivity. The remaining CD8 T cells showed an antigen inexperienced status with less clonal expansion, indicating suboptimal T cell priming. We confirm that brain derived antigens are presented in the secondary lymphoid tissues to prime CD8 T cells. Our study identifies cDC1 as critical for CD8 T cell priming outside of the CNS. This priming is required for a large increase in activated CD8 T cells in the brain which promotes tau-mediated neurodegeneration.

overall_design: Single cells were prepared from brains pooled from each strain of mice. Cells were stained with propidium iodide, CD45, and CD11b antibodies to identify live CD45hi cells. These cells were sorted by flow cytometer in DPBS supplemented with 20% FBS and the fresh cells were submitted to for single cell sequencing librabry preparation and sequencing

first: Hu

last: Hao

first: Zeng

last: Carisa

first: Artyomov

last: Maxim N

first: Ulrich

last: Jason D

first: Holtzman

last: David M


title: Chromatin landscape and ChIP-seq profiling of Anopheles stephensi MSQ43 cell line

city: Novosibirsk

first: Andrey

last: Popov

address: Academician Lavrentiev Avenue, 10

country: Russia

institute: Institute of Cytology and Genetics, SB RAS

summary: Anopheles mosquitoes are known as dominant vectors of malaria parasites and other viral and bacterial pathogens. Fine-tune interactions between host and pathogens modulate the global distribution of mosquito-borne diseases. The way of those interactions may vary between different Anopheles species providing biological advantages for ones and limitations for others. Transmitting ability of mosquitoes can also differ between species, raising the challenging but principal questions. Deciphering the genomes has opened a new era of research and allowed to uncover in depth several molecular mechanisms involved in pathogen transmission. Nowadays there is a lack of appropriate model systems to study specific vector-pathogen interactions at molecular level. Even inbred mosquito colonies represent a heterogeneous population of cells and require special care facilities. Insect cell cultures could serve as a useful alternative due to the ease of handling and maintaining, especially for genomic studies requiring millions of cells per experiment. Here, we provide chromatin contacts heatmap and a comprehensive dataset of epigenetic characteristics, including histone marks profiles as well as RNA-seq data, for MSQ43 - established earlier cell line, initially obtained from Anopheles stephensi larva, known as a keynote malaria vector in Southeast Asia. We assume that our genomic data will be of interest for scientists who can potentially/alternatively use MSQ43 as a model system for mosquito genomic studies.

overall_design: ChiP-seq and RNA-seq experiments

first: Varvara

last: Lukyanchikova

first: Miroslav

last: Nuriddinov

first: Anna

last: Khabarova

first: Polina

last: Belokopytova

first: Igor

middle: V

last: Sharakhov

first: Veniamin

last: Fishman


title: RNA-Seq of hippocampus in founder mice

city: Bar Harbor

first: Gary

last: Churchill

state: Maine

address: 600 Main St

institute: The Jackson Laboratory

summary: To understand the genetic regulation of gene expression and patterns of gene co-expression, we sequenced the transcriptome of the hippocampus of founder mice of both sexes. Mice were sacrificed between 6-8 weeks of age and hippocampus dissected. Total hippocampal RNA was isolated using a TRIzol Plus RNA purification kit (Life Technologies) and mRNA sequencing library was prepared using a TruSeq kit (Illumina), both according to manufacturer's protocols. Paired-end 100bp reads were obtained using the Illumina HiSeq 2000.

overall_design: transcription profiling by high-throughput sequencing design

first: Candice

middle: N

last: Baker


title: Analysis of 3-dimensional genome of exhausted CD8+ T cells reveals a critical role of IRF8 in their terminal differentiation in cancer [ChIP]

city: Beijing

first: Ruifeng

last: Li

address: haidian

institute: Tsinghua University

summary: Exhausted CD8+ T cell differentiation is accompanied by extensive changes in epigenome. However, whether and how higher-order chromatin organization is involved in exhausted CD8+ T cell differentiation is unclear. Here, we showed extensive changes in the three-dimensional genome during exhausted CD8+ T cell differentiation, associated with changes in gene transcription. Moreover, we identified Interferon regulatory factor 8 (IRF8) as an essential transcription factor in re-organization of spatial chromosomal interactions. Irf8 deficiency inhibits the development of terminal exhausted CD8+ T cells and promotes tumor growth. Mechanistically, IRF8 bound to genes associated with exhausted CD8+ T cells differentiation and promoted the formation of chromosomal loops. At the loop anchor regions, IRF8 recruited CTCF to form active chromosomal structure to regulated exhaustion-associated genes. These results thus identify a critical role of IRF8-dependent chromatin topology in gene transcription during exhausted CD8+ T cell differentiation.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChlP-seg) for IRF8 and CTCF  of Irf8fl/fl vs Irf8-/- CD8+ T cells activated in vitro.

first: Kun

last: Wei

first: Chen

last: Dong


title: The causative effect of CXCR7 on experimental autoimmune prostatitis injury and fibrosis

first: Yi

last: Zhang

address: Jixi Road 218, Shushan District, Hefei City 230022, Anhui Province, People’s Republic of China.

institute: the First Affiliated Hospital of Anhui Medical University

summary: The purpose of this study is to further understand the changes in mRNA expression in the prostate tissue of EAP mice after the use of VUF11207. After establishing the EAP mouse model, the experimental group was intraperitoneally injected with VUF11207, while the control group was intraperitoneally injected with an equal amount of DMSO solvent. After continuous injection for 14 days, the mRNA expression in the mouse prostate was sequenced and analyzed. Using RNA-Seq technology, a large number of differentially expressed genes were identified at the whole-genome level. Our study suggests that compared to the control group, under the action of VUF11207, CXCR7 can cause prostate epithelial cell damage and fibrosis.

overall_design: The mice used to establish the EAP model were divided into two groups. The experimental group was injected intraperitoneally with VUF11207 (5mg/kg/day), while the control group was injected intraperitoneally with an equal amount of DMSO. After continuous intraperitoneal injections for 14 days, the mice were euthanized, and the prostate tissues were collected for RNA-Seq analysis of mRNA expression levels.

first: Rui

last: Feng

first: Sixu

last: Chen

first: Zhengbin

last: Wang

first: Cong

last: Huang

first: Li

first: Jing

first: Chaozhao

last: Liang


title: 3D genome organization by CTCF prevents exTreg differentiation to control autoimmunity peripheral tolerance and immunotherapy outcomes [scRNAseq_scTCRseq]

city: Guangzhou

first: Xiaoling

state: Guangdong

address: No. 195, West Dongfeng Road

institute: Guangzhou Medical University

summary: Regulatory T cells (Tregs) can undergo transformation into exTregs by downregulating Foxp3 expression, yet the underlying mechanisms and functional implications remain incompletely understood (1, 2). Here, we show that PD-1 blockade induces significant alterations in chromatin accessibility at CTCF binding sites within tumor-infiltrating Tregs. CTCF plays a crucial role in maintaining Treg identity and preventing autoimmune disorders in mice through the three-dimensional organization of key genes regulating Treg stability. Moreover, acute deletion of Treg-specific CTCF enhances T cell responses and promotes the differentiation of CD40L-expressing exTregs within tumors, without disrupting overall immune homeostasis in murine models. The single-cell T cell receptor sequencing analysis of colitis tissues from melanoma patients treated with anti-PD-1 and anti-CTLA-4 antibodies reveals a notable emergence of exTregs, elucidating the paradoxical increase in both Tregs and cytotoxic CD8+ T cells (3) (3, 4). Our findings underscore the therapeutic potential of manipulating exTreg differentiation to enhance cancer immunotherapy and mitigate associated adverse effects.

overall_design: Parallel single-cell RNA sequencing (scRNA-seq) and single-cell TCR sequencing (scTCR-seq) of CD45+ cells sorted from tumors on day 25 post-subcutaneous inoculation of wild-type MC38 cells into control (ctr) or tamoxifen-inducible Ctcf knockout (cko) mice

first: Qibin

last: Leng

first: Guangshuai

last: Jia

first: Shengbao

last: Suo


title: 3D genome organization by CTCF prevents exTreg differentiation to control autoimmunity peripheral tolerance and immunotherapy outcomes [Hi-C]

overall_design: FACS-sorted Tregs isolated from the spleen and lymphoid nodes of mice (CtrlCre vs CtcfCre, or Control vs CtcfiKO) were used for Hi-C library


title: Agilent LncRNA profiles in AD

city: Changsha

first: Liang

last: Tang

address: No 1501, Leifeng road

institute: Changsha Medical University

type:
Expression profiling by array
Non-coding RNA profiling by array

summary: to investigate the lncRNA profiles between AD and control mice models.

overall_design: AD vs. Control


title: Identification of lncMB3-dependent transcriptome in MYC-amplified D283 Med cells.

city: Napoli

first: Francesco

last: Migliaccio

address: Via Pietro Castellino 111

department: DIETI / IAC

institute: Federico II / IAC

summary: Medulloblastoma (MB) stands as the most prevalent malignant pediatric tumor affecting the central nervous system. Categorized into four primary subgroups, each with distinct biological and clinical features, MB presents unique therapeutic implications. Group 3 (G3) and 4 (G4) MBs collectively account for the majority of cases (60%) and exhibit the highest degree of aggressiveness. However, these subgroups remain poorly characterized from a biochemical perspective. Therefore, it is crucial to unveil the fundamental molecular mechanisms underlying G3 e G4 MBs to deepen our overall comprehension of these specific conditions and to pave the way for precision medicine-driven approaches. Compared to normal human cerebella, the MYC-dependent lncRNA MB3 was found to be induced in G3-derived cell lines expressing the proto-oncogene and upregulated in G3-MB primary tumors. Consistently, LncMB3 is downregulated upon MYC inhibition and in vitro studies reveal its potent anti-apoptotic activity. To dissect the molecular network through which LncMB3 operates in MB carcinogenesis, we initiated the identification of its target genes by analyzing through RNA-Sequencing the alteration of the transcriptome following LncMB3 knockdown in G3 MB cell lines, achieved through transfections of antisense Locked Nucleic Acid (LNA) oligonucleotides (GapmeRs). Here we provide the data of three polyA+ RNA samples derived from lncMB3 knockdown (KD) and 3 control samples from the MYC-amplified D283 Med cell line that were subjected to RNA-Seq analysis.

overall_design: Three polyA+ RNA samples derived from lncMB3 knockdown (KD) and 3 control samples from the MYC-amplified D283 Med cell line were subjected to RNA-Seq analysis.

first: Alessia

last: Grandioso

first: Paolo

last: Tollis

first: Annamaria

last: Carissimo

first: Pietro

last: Laneve


title: 3D genome organization by CTCF prevents exTreg differentiation to control autoimmunity peripheral tolerance and immunotherapy outcomes [bulkRNAseq_ATACseq_CUT_Tag]

overall_design: ATAC-seq analyses on the following samples: (1) Treg cells (2-3 × 104 per biological replicate) isolated from MC38 subcutaneous tumors of Foxp3YFP-Cre mice treated with PBS (3 biological replicates) or anti-PD-1 antibody (2 biological replicates); (2) Treg cells (5 × 104 per biological replicate) purified from the spleen and lymph nodes of CtrlCre and CtcfcKo mice (3 biological replicates for each group); (3)Treg cells (5 × 104 per biological replicate) purified from the spleen and lymph nodes of Control and CtcfiKO mice (3 biological replicates for each group).RNA was extracted from FACS-sorted CD4+CD25+YFP+ Treg cells isolated from the spleen and peripheral lymph nodes (PLNs) of mice. Each sample consisted of pooled cells from 2-4 mice as a biological replicate.


title: Analysis of 3-dimensional genome of exhausted CD8+ T cells reveals a critical role of IRF8 in their terminal differentiation in cancer [RNA]

first: Ruifeng

last: Li

overall_design: To globally analyze the genes regulated by IRF8, we performed RNA-seq analysis of Irf8fl/fl vs Irf8-/- CD8+ T cells activated in vitro.

first: Kun

last: Wei

first: Chen


title: α-Asarone promotes tendon-bone healing through regulating Dmp1 transcription via targeting transcription factor PPARG

last: Tong

address: Jingxi

institute: Nanfang Hospital

summary:
Background: The tendon-bone interface (TBI) frequently proves refractory to restoration following surgical reconstruction or even surgical failure,  due to the intricate nature of its structure. α-Asarone (αASA),  an active ingredient derived from the traditional Chinese medicinal plant Calamus,  has been demonstrated to possess beneficial effects in the treatment of inflammatory and osteoporotic conditions. However,  its impact on TBI has yet to be investigated.
Objective: This study aimed to elucidate the therapeutic effects of αASA on TBI in vivo and in vitro,  gain insight into the underlying mechanisms involved,  and assess its potential medicinal value in humans.
Methods: A mouse model and a cellular model of tendon-bone interface (TBI) healing were developed. The impact of αASA on TBI was investigated at both the in vitro and vivo levels through a range of techniques,  including protein gene assays,  cell staining,  biomechanics,  imaging,  and histology. Concurrently,  network pharmacology,  transcriptomics,  molecular docking,  transcription factor prediction,  and experimental validation were employed to conduct a comprehensive investigation of its specific effects and mechanism of action. Subsequently,  In addition,  the clinical medicinal value of αASA was validated by human BMSCs (hBMSCs).
Results: In vivo,  αASA treatment enhanced the biomechanical properties and osseointegration of tendon-bone samples in mice. In vitro,  αASA promoted osteogenic differentiation of BMSCs,  as evidenced by staining and the expression of osteogenic marker genes. Using network pharmacology,  we identified 29 core co-target genes of αASA in the treatment of TBI. The top 20 differentially expressed genes (DEGs) underwent GO,  KEGG,  and GSEA enrichment analyses,  revealing their involvement in tissue mineralization and ossification processes. Additionally,  207 transcription factors (TFs) were predicted for these DEGs,  with 9 identified as core co-target genes. Surface plasmon resonance (SPR) confirmed the strong affinity of αASA for the transcription factor PPARG,  while luciferase assays demonstrated PPARG binding to the Dmp1 promoter to regulate transcription. αASA also enhanced osteogenic differentiation in human BMSCs,  supporting its potential for clinical application.
Conclusion: αASA promotes osteogenic differentiation and improves TBI healing in mBMSCs by targeting and down-regulating the transcription factor PPARG to inhibit its binding to the Dmp1 promoter,  while exerting the same effect in hBMSCs.

overall_design: To explore transcriptional regulation of the therapeutic effect of α-ASA on TBI,  we used mouse BMSCs to induce osteogenic differentiation in osteogenic medium and divided them into two groups with or without α-ASA,  each with 6 biological replicates,  for high-throughput sequencing.We used the RNA-seq data to perform differential gene analysis,  and GO, KEGG,  and GSEA enrichment analyses were performed for the top 20 differential genes.Subsequently,  the specific mechanism of action was deeply explored through the interactive analysis of RNA-seq data and network pharmacology data

first: Tong

first: Kairui

last: Zhang


title: CRISPR/Cas9 mutagenesis reveals an essential role of PI4KB in promoting growth and resisting hemorrhagic disease caused by GCRV-II infection in juvenile grass carp

city: wuhan

first: wu

middle: xiao

last: man

address: Donghu South Road

institute: Institute of Hydrobiology, Chinese Academy of Sciences

summary: Few studies reported for obtaining the grass carp resistant to hemorrhagic disease via gene editing in commercial fish. Here, we demonstrate that the expression and activity of grass carp PI4KB (gcPI4KB) are vital for GCRV-I and GCRV-II replication. Due that obvious cytopathic effect (CPE) in the present available cell lines is only caused by GCRV-I, but GCRV-II is the current popular and fatal strain in grass carp, GCRV-I and GCRV-II are used in cell lines and in grass carp, respectively. The in vitro studies in CIK cells revealed that gcPI4KB interacted with NS80 and VP3 of GCRV-I, and that gcPI4KB was recruited by NS80 for promoting the generation of GCRV VIBs. Since the negative regulatory role of gcPI4KB in GCRV infection was confirmed by in vitro data,we performed gene editing of gcPI4KB in grass carp. We found that PI4KB F0 crispants juvenile grass carp have obvious advantages in promoting growth and in resisting GCRV-II infection. Compared with uninfected WT grass carp, the uninfected PI4KB F0 crispants juvenile grass carp exhibit a higher expression level of many genes involved in growth- and development-related metabolic pathways such as the FoxO signaling pathway and insulin signaling pathway. Compared with WT grass carp without infection, PI4KB F0 crispants juvenile grass carp without infection or WT grass carp infected with GCRV-II, higher expression levels for many genes involved in metabolic diseases and viral infection were observed in the liver from PI4KB F0 crispants juvenile grass carp infected with GCRV-II. Altogether, the present study suggests the mechanism of gcPI4KB in facilitating GCRV replication, the signaling pathways regulated by gcPI4KB, and the possibility to obtain the grass carp resistant to hemorrhagic disease via gene editing of PI4KB.

overall_design: The gcPI4KB F0 crispants were generated using CRISPR/Cas9 for gene-editing gcPI4KB in grass carp. In brief, the single guide RNA(sgRNA) of gcPI4KB was designed using the ZiFiT (http://zifit.partners.org/ZiFiT/). To explore the influence of gcPI4KB mutant on GCRV-II infection in vivo, the 6-month-old individuals of WT, gcPI4KB F0 crispants were challenged with GCRV-II (5μl/g) by intraperitoneal injection. For gene expression analysis, the fish were maintained in aerated tanks in triplicate with 35 individuals per group with supplemental 35 L water at 25 ± 2 °C. The fish were fed with a commercial pelleted diet at 3 % body weight per day through-out the study. The number of surviving fish was counted daily for 14 days. GraphPad Prism 9.0 was used to generate survival curves, and the log-rank test was used to test differences in survival between the WT, gcPI4KB or gcPI4K2B F0 crispants. The liver, intestine and kidney of 6 WT or gcPI4KB F0 crispants grass carp with or without GCRV-II infection were collected at 5 dpi or 16 dpi, and used for Illumina deep sequencing.

first: Jie

first: Li

last: Qiang Zhang

first: Xiao

last: Man Wu

first: Min

last: Hui Tao

first: Yang

last: Chen

first: Ming

last: Xian Chang


title: Targeting glutamylated microtubules protects inter-cardiomyocyte coupling against ischemia/reperfusion injury

city: Hangzhou

first: Jiayin

state: Zhejiang

address: Center for Genetic Medicine, Zhejiang University School of Medicine

institute: Zhejiang University

summary:
Purpose: The glutamylation of MT is regulated by the activities of TTLLs (glutamylases) and CCPs (deglutamylase), enzymes responsible for the addition or removal of glutamate residues on the C-terminal tails of tubulin, respectively. Given the presence of multiple TTLLs and CCPs in mammals, we performed RNA sequencing on mouse hearts to assess the abundance of their expression.
Methods: Heart tissue of C57BL/6J mice at day 9 after birth were collected for RNA sequencing.
Results: We found that the expression levels of TTLLs (glutamylases) and CCPs (deglutamylase) in mouse heart tissue.
Conclusions: TTLL4 and CCP5 are enzymes involved in post-translational modification of microtubule glutamination in cardiomyocytes.

first: Xinjian

last: Wang

first: Peidong

last: Han


title: Y-chromosome clonal random expression produces mosaics in male mice

city: Madrid

first: Alfonso

last: Gutiérrez-Adán

address: Av. Puerta de Hierro 28

department: Animal Reproduction

institute: INIA

summary: Monoallelic expression of X-chromosome inactivation (XCI) genes produces female mosaicism, but it is unknown whether chromosome Y (chrY) mosaicism exists in males. ChrY contains some ubiquitously expressed and evolutionary conserved dosage-sensitive regulator genes, including neighbouring Kdm5d, Ddx3y, Uty, and (in mice) Eif2s3y, that have on chrX counterparts that escape XCI. Here, we have produced a transgenic mice line containing an EGFP gene between Uty and Ddx3y genes. These males have mosaic (tortoiseshell) green fluorescence in the skin, similar to females carrying one copy (hemizygous) of an X-linked EGFP transgene owing to inactivation of one chrX, and show clonal inheritance of the inactive state through cell division. The skin tortoiseshell expression of the Y-GFP indicates that the choice of which chrY remains active is stably maintained and clonally inherited. We have produced 3 Sertoli lines from Y-GFP transgenic male, and we have separated by flow cytometry size homogeneous GFP-positive and GFP-negative cells. To analyse the transcriptomic profile and evaluate whether genes from chrY follow the same EGFP expression pattern and explore the regulatory mechanism of the chrY we performed RNA-seq. The results indicate that GFP-positive and GFP-negative cells have considerable differences in their expression levels at chrY, chrX and autosomal chromosomes. Concretely, 18 chrY genes show under expression in GFP-negative cells, including Ddx3y, Zfy1, Zfy2, Usp9y, Uba1y and 10 genes from the Rbmy family. Moreover, the differences in expression observed brings out the influence of chromosome Y expression on the expression of the rest of the autosomal genes.

overall_design: Total RNA-seq from Sertoli cell lines with positive- and negative-Y-GFP expression (3 replicates per condition)

first: Eva

last: Pericuesta

first: Paula

last: Navarrete-López

first: Maria

last: Maroto

first: Raul

last: Fernández-González


title: Histone methyltransferase PRDM9 promotes survival of drug-tolerant persister cells in glioblastoma [RKI1_DTPs_RNAseq]

first: George

last: Joun

summary: Chemotherapy often kills a large fraction of cancer cells but leaves behind a small population of drug-tolerant persister cells. These persister cells survive drug treatments through reversible, non-genetic mechanisms and cause tumour recurrence upon cessation of therapy. Here, we report a drug tolerance mechanism regulated by the germ-cell-specific H3K4 methyltransferase PRDM9. Through histone proteomic, transcriptomic, lipidomic, and ChIP-sequencing studies combined with CRISPR knockout and phenotypic drug screen, we identified that chemotherapy-induced PRDM9 upregulation promotes metabolic rewiring in glioblastoma stem cells, leading to chemotherapy tolerance. Mechanistically, PRDM9-dependent H3K4me3 at cholesterol biosynthesis genes enhances cholesterol biosynthesis, which persister cells rely on to maintain homeostasis under chemotherapy-induced oxidative stress and lipid peroxidation. PRDM9 inhibition, combined with chemotherapy, resulted in strong anti-cancer efficacy in preclinical glioblastoma models, significantly enhancing the magnitude and duration of the antitumor response by eliminating persisters. These findings demonstrate a previously unknown role of PRDM9 in promoting metabolic reprogramming that enables the survival of drug-tolerant persister cells.

overall_design: RKI1 Glioblastoma Stem Cell line were treated for fourteen days with 25uM CMPD1, to generate drug tolerant persisters (DTP). RNA sequencing was conducted on the persisters, compared to the matched, Day 0 control. N=3 experiments were conducted.

first: Lipin

last: Loo


title: Methicillin-resistant Staphylococcus aureus strain ST398 shows abundant cis-regulatory transcription with limited antisense and intergenic expression upon linezolid stress

city: Würzburg

first: Jakob

middle: Jeremias

last: Jung

state: Bayern

address: Josef-Schneider-Str. 2/Bau D15

department: Institute of Molecular Infection Biology (IMIB)

institute: University of Würzburg

summary: Background: Bacterial small regulatory RNAs (sRNAs) have been implicated in important processes including antimicrobial stress response. However, the full extent of sRNA involvement in antimicrobial response in Staphylococcus aureus, an important pathogen, is incompletely understood. We investigated the transcriptional profiles of a linezolid-resistant, livestock-associated methicillin-resistant S. aureus (LA-MRSA) strain ST398 under conditions of linezolid stress.  Methods: Cells in mid-exponential growth were subjected to low (8 µg/ml) and high (16 µg/ml) dose linezolid treatments followed by high throughput RNA sequencing. Read mapping and differential expression analysis were performed followed by detection and interrogation of various sRNA and mRNA transcripts.  Results: Twenty-three putative regulatory RNA transcripts were expressed under low- and high-dose exposure conditions. Cis-acting regulatory elements, mainly targeting ribosomal biogenesis constituted the majority of transcriptional response with limited antisense and small RNA expression.  Conclusions: This is the first study to investigate linezolid-responsive small RNA transcription in LA-MRSA strain ST398 and the first to query regulatory RNAs on  a background of linezolid resistance. It provides preliminary insights and a basis for interrogating small RNAs in other strains in the quest to understand drug-responsive regulatory RNAs and identify potential anti-staphylococcal therapeutic candidates.

overall_design: Multidrug-resistant strain of S. aureus was subjected to 2 different doses of Linezolid (0, 8 and 16ug/mL) and RNA was sequenced after 10 minutes of exposure. MHB was used as media.

first: Justin

last: Nyasinga

first: Tessa

last: Marciniak

first: Andrew

last: Nyerere

first: Gunturu

last: Revathi

first: Wilma

last: Ziehbuhr


title: Optimization of GAO oxidation and glycosidase digestion for glycoRNA enrichment

city: 苏州市

first: Jianbo

last: Deng

state: 江苏省

address: 江苏省苏州市姑苏区三香路1055号

institute: Second Affiliated Hospital of Soochow University

type: Non-coding RNA profiling by high throughput sequencing

summary: Ribonucleic acid (RNA), essential for protein production and immune function, undergoes glycosylation, a process that attaches glycans to RNA, generating unique glycoRNAs. These glycan-coated RNA molecules regulate immune responses and may be related to immune disorders. However, studying them is challenging due to RNA's fragility. Therefore, a robust method for identifying glycoRNA is important. To address this, we optimized parameters for RNA stability, oxidation, and digestion, thereby enriching and identifying glycoRNAs. This breakthrough paves the way for exploring their potential interactions with immune receptors and tumor suppression. Our approach involved investigating factors such as preservation reagent, enzyme buffer, digestion temperature, oxidant, glycosidase, and incubation time. We successfully optimized digestion conditions, achieving efficient cleavage of both N-linked and O linked glycoRNAs at room temperature using 25 mM ammonium bicarbonate, demonstrating the effectiveness of this method. Additionally, RNA preservation in RNAlater at -80°C allows controlled release of glycoRNAs within hours. While sequential digestion of different glycoRNA types is possible, significant degradation occurs after the first glycosidase step. Thus, we recommend separate harvesting for each type of glycoRNA. These optimized protocols, utilizing SPCgRNA and TnORNA methods, pave the way for further research on N- and O-glycoRNAs in health and disease.

overall_design: We first optimized the SPCgRNA and TnORNA techniques. Following this, we applied the optimized methods to colorectal cancer tissues, successfully enriching for N- and O-glycoRNAs. Subsequently, the enriched samples were sent for samll RNA sequencing, and the raw data were processed and analyzed to identify the types and expression levels of glycoRNAs.

first: Deng

last: Jianbo

first: Miao

last: Xinyu

first: Yang

last: Xiaodong

last: Shuang


title: The functional impact of HGF on cholangiocarcinoma cells.

city: JiNan

first: Anda

last: Shi

address: Culture West Road num 107

institute: Qilu Hospital

summary: The cytokine HGF is a potential signaling molecule in the microenvironment of cholangiocarcinoma. The downstream mechanisms of its action on tumor cells are yet to be studied.

overall_design: Assess the gene expression changes in cholangiocarcinoma cells following stimulation with HGF.

first: Zongli

last: Zhang