title: Differential 5-FU responses of crypt-derived organoids embedded in collagen versus Matrigel, and their transcriptomic comparison with villus-derived organoids

city: Tokyo

first: Shiro

last: Yui

address: 1-5-45, Yushima, Bunkyo-ku

department: Center for stem cell and Regenerative medicine

institute: Institute of Science Tokyo

summary: We compared the effects of 5-fluorouracil (5-FU) on gene expression profiles of mouse crypt-derived intestinal organoids embedded in either Matrigel (MG) or collagen type I (COL). Distinct sets of genes were upregulated in COL-embedded organoids following 5-FU treatment, suggesting that collagen-based ECM uniquely modulates inflammatory and DNA-damage responses. We further compared COL-embedded crypt-derived organoids with COL-embedded villus-derived organoids (V-organoids) and found that their transcriptional profiles were highly similar. These results indicate that collagen embedding promotes the emergence of revival stem cell–like signatures regardless of the cellular origin of the organoids.

overall_design: To evaluate how ECM environments influence responses to chemotherapeutic stress, organoids in each ECM condition (MG and COL) were either left untreated (Control) or treated with 5-fluorouracil (5-FU). This resulted in a 2 × 2 factorial design consisting of four experimental groups: MG_Control, MG_5FU, COL_Control, and COL_5FU, with three biological replicates per condition (n = 3). We established villus-derived intestinal organoids (V-organoids) from mouse small intestine and compared them with crypt-derived organoids cultured under two distinct extracellular matrix (ECM) conditions: Matrigel (MG) and collagen type I (COL-cWRNE).

first: Sakura

last: Kirino

first: Kensuke

last: Miyake


title: Heterogeneity and plasticity of the naive CD4+ T cell compartment

status: Public on Jan 09 2026

city: Lebanon

first: J. Louise

last: Lines

state: NH

address: DHMC

country: USA

institute: Dartmouth College

type: Expression profiling by high throughput sequencing

summary: While naive CD4+ T cells have historically been considered a homogenous population, recent studies provide evidence that functional heterogeneity exists. Using scRNAseq, we identified five transcriptionally distinct naive CD4+ T cell subsets that emerge within the single positive stage in the thymus: a quiescence cluster (TQ), a memory-like cluster (TMEM), a TCR-reactive cluster (TTCR), an IFN-responsive cluster (TIFN), and an undifferentiated cluster (TUND). Elevated expression of transcription factors KLF2, Mx1, and Nur77 within the TQ, TIFN, and TTCR clusters, respectively, enabled enrichment of these subsets for functional validation. Sorted subsets displayed distinctive functional responses upon stimulation. Furthermore, treatment of mice with inflammatory stimuli reduced cytokine production of naive T cells ex vivo. In SLE patients, IFN-induced gene expression was markedly elevated within the TIFN cluster. Our data demonstrate that naive T cells are shaped by their host environment, with functional biases manifesting upon activation. These findings highlight the importance of understanding how naive T cell heterogeneity may be distorted in cancer, autoimmunity, and infectious diseases.

overall_design: naive CD4 T cells were enriched from mouse spleen, LN or blood by fluorescence activated cell sorting, and then were processed for scRNAseq.

first: Alia

last: Sajani

first: Evelien

last: Schaafsma

first: Walburga

last: Croteau

first: Mohamed

last: ElTanbouly

first: Elizabeth

middle: C

last: Nowak

first: Chao

last: Cheng

first: Christopher

middle: M

last: Burns

first: Mary Jo

last: Turk

first: Randolph

middle: J

last: Noelle

first: Louise


title: Proteasome activity maintains cell type-specific gene expression

city: Bethesda

first: Achim

last: Werner

state: MD

address: 30 Convent Dr. MSC 4340

department: NIDCR

institute: NIH

summary: Regulated proteolysis in eukaryotes relies on the ubiquitin-proteasome system, which is critical to myriad cellular functions, including protein quality control, cell cycle regulation, and DNA repair. Here, we provide evidence that proteasome activity is also essential for maintaining cell identity. This discovery was made by evaluating the impact of losing the ubiquitin-binding activity of proteasome substrate receptor hRpn10/PSMD4. The most dysregulated proteins in cells that express hRpn10 truncated before its ubiquitin interaction motifs (ΔUIM) are transcriptionally altered, with striking overrepresentation of proteins canonically restricted in expression to specific tissues. This dysregulation is partly driven by accumulation of the proteasome substrate anddeubiquitinase OTUD5, a known chromatin and transcriptional regulator. Our results thus identify previously unrecognized proteasome-dependent mechanisms to safeguard cell-type specific gene expression programs and expand proteasome biology to include governance of cell identity.

overall_design: RNA-seq profiling of HCT116 cells (Either gene-edited to have a truncated version of rpn10 which is missing its ubiquitin interacting motif or WT), which were transfected with an OTUD5 siRNA or a scrambled control.

first: Xiuxiu

last: Lu

first: Vasty

last: Osei-Amponsa

first: German

last: Michelis

first: Rithik

last: Castelino

first: Hitendra

last: Negi

first: Sudipto

last: Das

first: Julianna

last: King

first: Jesse

last: Turner

first: Daniel

last: Martin

first: Yan

last: Wang

first: Thorkell

last: Andresson

first: Raj

last: Chari

first: Kylie

last: Walters


title: Nucleolar Expansion Drives 3D Genome Reorganization and Transcriptional Repression in Cancer

city: Beijing

first: Qing

last: Fang

state: Haidian District

address: YiHeYuan Road

department: School of Life Sciences

country: China

institute: Peking University

summary: The nucleolus plays a pivotal role in organizing the three-dimensional chromatin architecture through the sequestration and regulation of heterochromatin, yet how such structure remodeling contributes to tumorigenesis remains poorly understood. Analysis of TCGA data revealed widespread upregulation of nucleolar components across multiple human cancers, indicating nucleolar expansion during tumor progression, which was further validated by immunofluorescent imaging. To investigate this phenomenon, we developed Nucleo-seq and Nucleo-C, a pair of highly sensitive methods for high-resolution profiling of nucleolus‑associated chromatins (NADs) and their three-dimensional (3D) interactions from low-input isolated nucleoli. Applying these approaches to normal (MCF10A) and cancer (MDA-MB-231) breast cell lines, we uncovered global architectural alterations: cancer cells exhibited increased NAD signal intensity and enhanced 3D connectivity within NADs, closely correlated with nucleolar enlargement. This NAD reorganization was strongly associated with transcriptional repression of NAD-resident genes, which results in silencing of key tumor-suppressive pathways, including cell adhesion, negative regulation of cell proliferation, and Wnt signaling. Our findings establish a novel paradigm in which nucleolar architectural expansion in cancer drives NAD reorganization and gene dysregulation, offering new 3D genome-related biomarkers for cancer prognosis and therapeutic intervention.

overall_design: Nucleo-seq, Nucleo-C and in situ Hi-C by high throughput sequencing, as well as additional datasets including NAD-seq and RNA-seq

first: Yangyinghui

last: Yu

first: Cheng

last: Li


title: Peripheral glia constitute a pro-reparative niche triggering skin wound healing [Spatial Transcriptomics]

city: Zurich

first: Adrian

last: Salas-Bastos

address: Winterthurerstrasse 190

department: Institute of Anatomy

country: Switzerland

institute: University of Zurich

summary: Skin repair is a complex, dynamic process involving multiple cell types. Using multiplex imaging, spatial transcriptomics, and single-cell RNA sequencing, we show that peripheral nerves —containing repair glia— form a pro-reparative niche closely interacting with macrophages and proliferating fibroblasts in acute skin wounds. Repair glia function as critical early-stage regulators of wound healing by initiating the inflammatory response throughsecretion of monocyte chemoattractant proteins, such as CCL2, which recruit monocyte derived macrophages. Accordingly, depletion of repair glia as well as glia-specific deletion of CCL2 reduces the number of macrophages, leading to impaired fibroblast proliferation and diminished fibroblast to myofibroblast transition. These findings position repair glia as a central component of the pro-reparative niche, coordinating the early immune response and orchestrating the temporal progression of wound healing.

overall_design: spatial transcriptomics profiling of two regenerating skin wounds of Plp1-CreERT2:tdTomato control mouse at 4 days follwing injury.

first: Lukas

last: Sommer

first: Salome

last: Stierli


title: Gene expression profiles of intestinal organoids under collagen/Matrigel culture and 5-fluorouracil treatment in human

first: Shiro

last: Yui

country: Japan

summary: In this study, we examined how 5-fluorouracil (5-FU) treatment modulates transcriptional programs in human intestinal organoids cultured in Matrigel or collagen matrices. By comparing Matrigel-embedded and collagen-embedded organoids under control and 5-FU–treated conditions, we identified ECM-dependent and treatment-specific gene expression changes associated with cellular stress responses, epithelial regeneration, and matrix-driven phenotypic differences.

overall_design: To evaluate how extracellular matrix (ECM) environments influence chemotherapeutic stress responses, organoids in each ECM condition were either left untreated (CTRL) or treated with 5-fluorouracil (5-FU). This generated a 2 × 2 factorial design comprising four experimental groups: MG_Control, MG_5FU, COL_Control, and COL_5FU, with three biological replicates per condition (n = 3).

first: Sakura

last: Kirino

first: Kensuke

last: Miyake


title: Single-nucleus transcriptomics identifies dysregulation of cell cycle control and synaptic pathways during the progression of murine proneural gliomas.

city: New York

first: Patrizia

last: Casaccia

state: NY

address: 85 St Nicholas Terrace

department: Neuroscience

institute: ASRC-CUNY

summary: Here, we injected  in vitro transformed OPCs (with Trp53 deletion and PDGF-BB overexpression, (BB-p53n) into the subcortical white matter of syngeneic mice, and then performed single-nucleus RNA sequencing on brain samples isolated from the murine brains at early and late  time points in order to study the transcriptional changes occurring during the progression from early- to late-stage gliomas. Compared to normal brain, the early- and late- stage samples were characterized by clusters of cells bearing an “OPC-like” transcriptional phenotype. These cells expressed high levels of OPC genes, including Pdgfra and Olig2, but also expressed genes that are frequently upregulated in glioma cells, including Sox2, Nestin and CD44. Compared to early-stage tumor samples, late-stage glioma samples were characterized by the expansion of cells with a transcriptional signature enriched in genes regulating cell cycle and synaptic assembly. Pseudo-time analysis of the early and late samples identified a trajectory of decreasing cell cycle checkpoint regulation and increasing synaptic signaling. Together these data suggest that the progression from transformed OPCs to proneural gliomas includes transcriptional changes that favor proliferation and interactions with neurons.

overall_design: Gliomas were induced in normal mouse brains by the injection of tumorigenic BB-p53n OPCs. Tumors were removed at early- and late-stages and processed for 10X snRNA-seq 3'  Gene expression

first: Dennis

last: Huang

first: Angeliki

last: Mela

first: Hye-Jin

last: Park

first: Peter

last: Canoll


title: Neuronal Activity Suppresses NEUROD-Dependent Transcription and Genome Organization [Hi-C]

city: Evanston

first: Yue

last: Yang

state: IL

address: 2200 Campus Dr

department: Neurobiology

institute: Northwestern University

summary: Neuronal activity induces gene expression programs associated with plasticity in the brain, but it also transiently suppresses gene expression through poorly understood mechanisms. Here, we report that neuronal activity downregulates NEUROD-dependent gene expression in cerebellar granule neurons in adult mice. Using NEUROD1/2 double conditional knockout mice of both sexes, we show that NEUROD binds and activates its target gene enhancers and establishes a three-dimensional genome architecture that facilitates transcription when neuronal activity is low. Moreover, NEUROD antagonizes activity-dependent gene expression programs, including those regulated by MEF2. However, when granule neurons are activated, NEUROD-dependent transcriptional and genome organizing functions are disrupted. Together, these findings reveal NEUROD as a key regulator of transcriptional programs associated with inactive neurons. Our study provides new insights into how neuronal activity reshapes gene transcription and genome architecture in the brain.

overall_design: Hi-C libraries were prepared from the ADCV of NEUROD1/2 double conditional knockout or control mice. Libraries were sequenced on the Illumina NextSeq 550 platform to obtain 37 bp paired-end reads. Three biological replicates were performed for all conditions.

first: Pamela

last: Valnegri

first: Tomoko

last: Yamada


title: Neuronal Activity Suppresses NEUROD-Dependent Transcription and Genome Organization [RNA-Seq]

overall_design: RNA-seq libraries were prepared from the ADCV of NEUROD1, NEUROD2, or NEUROD1/2 conditional knockout mice or control mice. Additional RNA-seq libraries were prepared from the ADCV of optostimulated or homecage control mice using the chromatin fraction. Libraries were sequenced on the Illumina NextSeq 550 platform to obtain 37 bp paired-end reads. Two to five biological replicates were performed for all conditions.


title: Peripheral glia constitute a pro-reparative niche triggering skin wound healing

overall_design: scRNAseq profiling of regenerating skin wounds of Plp1-CreERT2:tdTomato:Sox10flx/flx (Sox10 cKO) and control mice at 4 days follwing injury.


title: Mimicking opioid analgesia in cortical pain circuits

city: Philadelphia

first: Benjamin

middle: C.

last: Reiner

state: PA

address: 125 S 31st St

institute: University of Pennsylvania

summary: We performed single nuclei transcriptomics on the anterior cingulate cortex of mice cohorts that received either neuropathic pain (sciatic nerve injury) or sham surgery, followed by varying posto-injury periods (3 days, 3 weeks, or 3 months). Five minutes prior to tissue collection, pain active genes were stimulated via one second static fiber (0.16g) to each hind paw (1x/30sec/5min).

overall_design: Five minutes after touch stimulus to the hindpaws, brains were rapidly dissected, the bilateral anterior cingulate cortex was punch, and the punches for each animal were flash frozen. Frozen punches were used to generate single nuclei suspensions and the nuclei were processed for the 10x Genomics 3' gene expression assay (v3.1).

first: Gregory

last: Corder


title: EPOP and MTF2 Activate PRC2 Activity through DNA-sequence specificity

city: Miami

first: sanxiong

last: liu

state: FL

address: 1501 NW 10th Avenue

department: Human Genetics

institute: University of Miami Miller School of Medicine

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The Polycomb repressive complex 2 (PRC2) establishes transcriptionally silent chromatin by catalyzing histone H3 lysine 27 trimethylation (H3K27me3). Although cofactors such as MTF2 and JARID2 are known to direct PRC2 to nucleation sites and stimulate its catalytic activity, the role of the PRC2-associated factor EPOP has remained unclear and even controversial. Here, we combine biochemical reconstitution and a cellular EED-rescue system to define EPOP’s contribution to initial PRC2 recruitment and de novo H3K27me3 deposition. We show that EPOP, like MTF2, directly stimulates PRC2 histone methyltransferase activity in vitro. In vivo, EPOP is dispensable for initial PRC2 recruitment but promotes de novo H3K27me3 deposition in cooperation with MTF2 and JARID2. Mechanistically, electrophoretic mobility shift assays and reconstituted chromatin substrates reveal that EPOP and MTF2 enhance PRC2–nucleosome binding in a sequence- and chromatin-dependent manner: EPOP preferentially potentiates activity on GA-rich dinucleosomes, whereas MTF2 favors GCN-rich substrates. Together, these findings position EPOP as a positive regulator of PRC2 function during the establishment of H3K27me3 domains, and reveal a paradigm in which distinct cofactors confer DNA-sequence-specific control of de novo H3K27me3 deposition.

overall_design: To dissect the contribution of PRC2 accessory factors to initial PRC2 recruitment and de novo H3K27me3 deposition, we used a mouse embryonic stem cell (mESC) system in which the core PRC2 subunit EED is knocked out, eliminating PRC2 chromatin binding and H3K27me3 after passaging. Re‑expression of HA‑tagged EED (EED‑rescue) was induced and profiled at 0 h (pre‑rescue baseline) and 24 h (early de novo phase). We performed ChIP‑seq for HA‑EED (proxy for PRC2 chromatin recruitment) and ChIP‑seq for H3K27me3 (histone mark deposition) across a factorial panel of genotypes: wild‑type accessory factor background and targeted knockouts of MTF2, JARID2, EPOP, as single KOs and double KOs (MTF2/JARID2, EPOP/MTF2, EPOP/JARID2).This design enables direct comparison of initial PRC2 recruitment (HA‑EED ChIP‑seq) and de novo H3K27me3 deposition (H3K27me3 ChIP‑seq) kinetics in the presence or absence of individual cofactors during the onset of Polycomb domain formation.

first: Jeffrey

last: Granat

first: Sanxiong

last: Liu

first: Danny

last: Reinberg


title: Transcriptomic characterization of adipose tissues in cold-exposed mice

city: Changsha

first: Jianlin

last: Zhou

state: Hunan

address: 36 Lushan road

department: College of Life Science

institute: Hunan Normal University

summary: Cold exposure has been shown to induce white adipose tissue (WAT) browning and alter WAT gene expression and lipid metabolism. To understand the transcriptomic profiles of WAT and brown adipose tissue (BAT) upon cold exposure, we performed RNA-seq analysis of inguinal WAT (iWAT) and BAT from mice maintained at either room temperature (RT, 22 °C) or cold (6 °C).

overall_design: RNA-seq analysis of iWAT and BAT from mice maintained at either at 22 °C or 6 °C.

first: Xing

last: Zhang

first: Qiuyan

last: Zhu


title: scRNA-seq of induced pluripotent stem cell derived decidual natural killer cells

city: La Jolla

first: Virginia

middle: Chu

last: Cheung

state: CA

address: 2880 Torrey Pines Scenic Dr.

department: Pathology

institute: Univeristy of California, San Diego

summary: Decidual natural killer (dNK) cells are the most abundant immune cells at the maternal-placental interface in early gestation. The exploration of dNK cell heterogeneity and function is currently an active area of research, particularly as these differ across gestation and different regions of the placenta. We applied proteomic and transcriptomic single cell definitions of dNK cells and subtypes to characterize these cells at the chorioamniotic membranes (CAM) and basal plate (BP, maternal surface) regions of the placenta at term. We found that there is reduced abundance of low-effector-function dNK1 and increased abundance of high-effector-function dNK3 in term decidual compartments. At term, in comparison to BP-dNK, CAM-dNK had greater abundance of moderate-effector-function dNK2, decreased abundance of dNK3, and lower expression of inhibitory receptor CD9. At the same time, using our knowledge of dNK heterogeneity, we developed a protocol to differentiate induced pluripotent stem cells (iPSC) into dNK cells. In this manuscript, we detail a protocol to differentiate iPSC into dNK-like cells as characterized by scRNA-seq analyses and CD45+CD56brightCD16- protein expression. We further developed the protocol to enrich for dNK2 – the most abundant dNK cell type in first trimester and term CAM – while inducing expression of dNK marker proteins CD9 and CD103 with the addition of TGFß. This study reveals the shift in dNK subtypes underlying functional changes in dNK cells between first trimester and term and detail a protocol with which we can methodically differentiate iPSC into dNK cells and specifically induce the dNK2 subtype for mechanistic perturbation.

overall_design: iPSC-dNK cells grown in suspension culture were collected, filtered, counted and loaded on the 10x Genomics chip.

first: Mana

last: Parast


title: Single-cell RNA-sequenceing analysis of mouse intestinal organoids cultured in Matriel or type I collagen

first: Shiro

last: Yui

summary: Intestinal epithelial regeneration depends on cellular plasticity under inflammatory conditions. Two major forms of plasticity have been described: spatial plasticity, in which mature cells dedifferentiate into crypt base columnar (CBC) stem cells, and fetal reversion, where cells adopt a fetal-like transcriptional state. To investigate the cellular mechanisms underlying these processes, we performed single-cell RNA sequencing on mouse intestinal epithelial organoids cultured in either Matrigel or collagen-based matrices. Combined single-cell transcriptomic and functional analyses revealed bidirectional interconversion between CBCs and revival stem cells (revSCs), as well as the reprogramming of absorptive enterocytes toward revSC-like states. This dataset provides a high-resolution atlas of epithelial stem cell states and plasticity transitions, facilitating further investigation of regenerative hierarchies in the mouse intestine.

overall_design: Cells isolated from standard budding organoids cultured in Matrigel and cystic spherical organoids cultured in collagen Type I established WT mouse small intestune were analyzed by scRNA-seq.

first: Sakura

last: Kirino

first: Kensuke

last: Miyake

first: Fumiya

last: Uefune


title: Fetal reversion from diverse lineages sustains the intestinal stem cell pool and confers stress resilience.

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: The Drosophila proventriculus lacks stem cells but compensates for age-related cell loss via endoreplication-mediated cell growth

city: Boston

first: Yanhui

last: Hu

state: MA

address: 77 Avenue Louis Pasteur

department: Genetics

institute: Harvard Medical School

summary: Drosophila’s proventriculus, a bulb-shaped organ between the foregut and midgut, aids ingestion, peritrophic membrane synthesis, and immune defense. We created a single cell transcriptional atlas of the proventriculus using single nucleus RNAseq (snRNAseq)to to identify molecular markers and use it as the foundation for for further molecular study in various proventriculus cell types.

overall_design: Proventriculus from control and cachectic flies were sequenced separately, then bioinformatically pooled to make an atlas of the tissue.

first: Ben

last: Ewen-Campen

first: Norbert

last: Perrimon


title: Single-cell RNA-sequenceing analysis of human intestinal organoids cultured in Matriel or type I collagen

summary: Intestinal epithelial regeneration depends on cellular plasticity under inflammatory conditions. Two major forms of plasticity have been described: spatial plasticity, in which mature cells dedifferentiate into crypt base columnar (CBC) stem cells, and fetal reversion, where cells adopt a fetal-like transcriptional state. To investigate the cellular mechanisms underlying these processes, we performed single-cell RNA sequencing on human intestinal epithelial organoids cultured in either Matrigel or collagen-based matrices. This dataset provides a high-resolution atlas of epithelial stem cell states and plasticity transitions, facilitating further investigation of regenerative hierarchies in the human intestine.

overall_design: Cells isolated from standard budding organoids cultured in Matrigel and cystic spherical organoids cultured in collagen Type I established from human small intestine were analyzed by scRNA-seq.


title: Neuronal Activity Suppresses NEUROD-Dependent Transcription and Genome Organization

first: Yue

last: Yang


title: Neuronal Activity Suppresses NEUROD-Dependent Transcription and Genome Organization [ChIP-Seq]

overall_design: ChIP-seq libraries using the H3K27ac antibody were prepared from the ADCV of NEUROD1/2 double conditional knockout or control mice. ChIP-seq libraries using the NEUROD1 or NEUROD antibody were prepared from the cerebellum. Libraries were sequenced on the Illumina NextSeq 550 platform to obtain 37 bp paired-end reads. Two biological replicates were performed for all conditions.

first: Pamela

last: Valnegri

first: Tomoko

last: Yamada


title: Activating and repressive FOX family proteins regulate HSV-1 chromatin and the lytic-latent balance through epigenetic modulators

city: Bostin

first: Don

last: Coen

address: 25 Shattuck St

summary: Herpes simplex virus 1 (HSV-1) switches between lytic and latent infections in neurons, yet the switch mechanisms remain obscure. Here we identify forkhead box (FOX) family proteins that can strongly activate or repress HSV-1 replication particularly in neuronal cells. Expression of activating FOX genes (Foxa, Foxc, Foxe, Foxf) is ordinarily low and expression of repressive FOX genes (Foxk) is high in neurons. Lytic HSV-1 infection and other stresses can increase activating FOX gene expression. Such overexpression or knockout of endogenous FOXK1 promotes reactivation from latency. These FOX proteins broadly associate with the viral genome and regulate viral gene transcription through epigenetic modulators. FOXF1 associates with histone acetyltransferases CBP and P300 to open viral chromatin. FOXK1 collaborates with SIN3A, a known cofactor of histone deacetylation, and MAX to suppress HSV-1 replication and antagonize activating FOX proteins. Thus, HSV-1 uses FOX family members with opposing effects to regulate the lytic-latent balance.

overall_design: RNA-seq profiling of human iNGN3 iPSC derived neuronal cells mock or HSV-1 infected under lytic conditions.

first: Dongli

last: Pan

first: Donald

first: David

last: Knipe

first: Biswajit

last: Das

first: Yao Yu

last: Yeo

first: Jean

last: Pesola

first: Pritha

last: Sen


title: A Glycolytic Shunt via the Pentose Phosphate Pathway is a Metabolic Checkpoint for Nervous System Sensory Homeostasis and Axonal Regeneration

city: London

first: Yayue

last: Song

address: Burlington Danes, Hammersmith Hospital, Imperial College London

country: United Kingdom

institute: Imperial College London

summary: Homeostasis and repair in the nervous system are thought to rely on distinct molecular programs. Here, we uncover an unexpected role for the pentose phosphate pathway (PPP) in peripheral sensory axons, where it supports both homeostatic mechanosensation and injury-induced axonal regeneration. We show that the PPP is enriched and active in sciatic nerve axoplasms, where it maintains redox balance via NADPH production, enabling normal mechanical sensation. Following sciatic nerve injury, the PPP is required for regeneration by fueling ribonucleotide synthesis through ribose-5-phosphate production. In contrast, this pathway remains inactive after spinal cord injury (SCI), contributing to regenerative failure. Reactivation of the PPP, through neuronal transketolase overexpression or oral ribose supplementation, promotes metabolic reprogramming, restores sensory and motor axonal growth, and improves neurological recovery after SCI. These findings reveal a dual role for the PPP as metabolic rheostat in sensory neuron physiology and regeneration and highlight its therapeutic potential for central nervous system repair.

overall_design: RNA sequencing profiles of 8 weeks C57BL/6J mice with either AAV-GFP or AAV-Tkt virus injected through scaitic nerves for 4 weeks, L4-6 dorsal root ganglia neurons were extracted 72hr following SCI. RNA sequencing profiles of 8 weeks C57BL/6J mice L4-6 dorsal root ganglia neurons were extracted 72hr following SCI with either 10% ribose or vehicel treatment.

first: Simone

last: Di Giovanni


title: EZH2 PROTACs are unable to abolish EZH2 activator function but inhibit certain prostate cancer by rapid senescence induction

city: Atlanta

first: Jindan

last: Yu

state: GA

address: E330, 1760 Haygood Dr NE

department: Urology

institute: Emory University

summary: Enhancer of Zeste Homolog 2 (EZH2) is the enzymatic subunit of the Polycomb Repressive Complex 2 (PRC2) that catalyzes H3K27 methylation for epigenetic silencing. EZH2 up-regulation or mutations contribute to the progression of various cancers, including prostate cancer (PCa). Enzymatic EZH2 inhibitors (EZH2i) exert limited efficacy in PCa, partially due to PRC2-independent roles of EZH2 in activating androgen receptor (AR) expression. Several proteolysis-targeting chimera (PROTAC) degraders of EZH2 have been developed, which, surprisingly, continued to have limited efficacy in PCa cells. Here, we designed and developed a series of EZH2 PROTACs using EZH2i EPZ-6438 as a ligand and identified PROTAC-6272 as a potent lead compound. We demonstrated that 6272 effectively degraded EZH2 and other PRC2 subunits across diverse PCa cell lines. Interestingly, EZH2 PROTACs were consistently unable to decrease AR and p300 due to their inability to engage and target EZH2 outside of the PRC2 complex. Regardless, EZH2 PROTAC-6272 exhibited greater anti-proliferative activities than EPZ-6438 in some PCa cell lines, wherein it rapidly induced cellular senescence and p21 expression independently of H3K27 methylation. In summary, we report that EZH2i-based PROTACs could benefit certain PCa but are unable to target the EZH2 activator function, shedding significant light on further drug development.

overall_design: To investigate the transcriptional effects of EZH2 degradation versus inhibition, we performed RNA-seq analysis in VCaP and 22Rv1 prostate cancer cells treated with DMSO, the VHL-inactive control compound 6286, the EZH2 PROTAC degrader PROTAC-6272, or the EZH2 enzymatic inhibitor EPZ-6438 for 2days.

first: Wanqing

last: Xie

first: Qi

last: Chu

first: Xiaodong

last: Lu

first: Jonathan

last: C. Zhao


title: Targeted Degradation of G-Quadruplex Binding Cellular Proteins Using G4-Ligand-Based PROTACs

city: Cambridge

first: Xuan

last: He

address: Lensfield Road

department: Chemistry

institute: University of Cambridge

summary: Targeted protein degradation is a cutting-edge approach in drug discovery, especially for addressing disease-related proteins that have been difficult to target with traditional methods. Here, we present a conceptually distinct strategy utilizing G-quadruplex (G4) ligand-based PROTACs (G4L-PROTACs) to selectively degrade proteins associated with DNA and RNA G-quadruplexes. Our method leverages G4 ligands that bind endogenous G4s and recruit E3 ubiquitin ligases to promote the degradation of cellular G4-binding proteins. We demonstrate degradation of a range of G4-specific binding proteins, including transcription factors and chromatin remodelers such as FUS, SMARCA4, and ATRX. These proteins play crucial roles in diverse cellular processes like transcription regulation and DNA repair, making them important therapeutic targets. Our results highlight the potential of G4L-PROTACs as a versatile tool for selectively degrading G4-binding proteins, offering new possibilities for therapeutic protein degraders, especially in diseases characterized by aberrant G4 activity, such as cancer.

overall_design: Genome binding/occupancy profiling by CUT&Tag experiments and high throughput sequencing for U2OS cells treated with PDS as control and PROTACs.

first: Zixuan

last: Wang

first: Xiaoyun

last: Zhang

first: Jochen

last: Spiegel

first: Sean

last: Flynn

first: Shankar

last: Balasubramanian


title: Silencing mitochondrial gene expression in living cells

city: Sherbrooke

first: Luis

middle: Daniel

last: Cruz-Zaragoza

state: Quebec

address: 2500 boul. de l'Université

department: Biology

country: Canada

institute: Université de Sherbrooke

summary: Mitochondria fullfill central functions in cellular metabolism and energy supply. They express their own genome, which encodes key subunits of the oxidative phosphorylation system. However, central mechanisms underlying mitochondrial gene expression remain enigmatic. A lack of suitable technologies to target mitochondrial protein synthesis in living cells has limited experimental access. Here, we silence the translation of specific mitochondrial mRNAs in living cells by delivering synthetic peptide-morpholino chimeras. This approach allows comprehensive temporal monitoring of cellular responses. Our study provides insights into mitochondrial translation, its integration into cellular physiology, and represents an innovative strategy to address mitochondrial gene expression in living cells. The tools that we present can readily be applied to analyze the mechanisms and pathophysiology of mitochondrial gene expression in a broad range of cellular model systems.

overall_design: HEK293T cells were treated for 48 hours with chimeras targeting different mtDNA-encoded mRNAs. After discarding the media, cells were harvested in PBS, and resuspended in Trizol reagent. The RNA was purified using RNA Clean & Concentrator kit (Zymo Research) following the manufacturer’s instructions. RNA quality control was assessed in a Fragment Analyzer. The NovaSeq X Plus sequencing platform (Illumina, USA) was used to perform 50 bp single‐end sequencing on the samples with 9 G raw data per sample.

first: Luis Daniel

first: Drishan

last: Dahal

first: Mats

last: Koschel

first: Angela

last: Boshnakovska

first: Aiturgan

last: Zheenbekova

first: Mehmet

last: Yilmaz

first: Marcel

last: Morgenstern

first: Jan-Niklas

last: Dohrke

first: Julian

last: Bender

first: Anusha

last: Valpadashi

first: Kristine

middle: A

last: Henningfeld

first: Silke

last: Oeljeklaus

first: Laura

middle: S

last: Kremer

first: Mirjam

last: Breuer

first: Oliver

last: Urbach

first: Michael

last: Lidschreiber

first: Stefan

last: Jakobs

first: Bettina

last: Warscheid

first: Peter

last: Rehling


title: APEX-seq Maps Transcriptome-wide Subcellular RNA Localization in Living Cells

city: Houston

first: Surbhi

last: Sharma

address: 1840, Dynamic way

institute: Baylor College of Medicine

summary: Although we know a great deal about the subcellular location of most proteins, our knowledge of where most RNAs localize within cells remains limited. As RNA subcellular localization determines the fate, function, and regulation of both coding and non-coding RNAs , there has been substantial interest in developing new scalable approaches to study the location of RNAs in their native context. Furthermore, many locations, such as membrane-bound and membrane-less organelles, have traditionally been challenging to study due to lack of suitable tools to interrogate their constituents. Here, we describe a detailed protocol for APEX-seq that yields transcriptome-wide information of the subcellular address of RNAs that can, in principle, be applied to any subcellular location, membrane, or condensate. APEX-seq utilizes a genetically-encoded engineered ascorbate peroxidase (APEX2) tagged to a specific protein that localizes it to a region of interest. In the presence of biotin-phenol (BP) and hydrogen peroxide, APEX2 catalyzes the biotinylation of RNAs in its vicinity, which can be purified using streptavidin beads and sequenced to reveal the RNA repertoire at that subcellular location. APEX-seq experiments can be carried out by laboratory personnel trained in molecular biology. The analysis of APEX-seq data requires familiarity with standard RNA-seq workflows. With APEX2-expressing cell lines in hand, the entire procedure from labeling reaction to analysis can be completed in one week. We expect this proximity labeling approach to facilitate the unbiased discovery of RNAs localizing to different organelles and to generate hypotheses for the mechanisms and pathways involved in regulating these processes.

overall_design:
APEX-seq was performed in HEK293T cells expressing APEX2 at the outer mitochondrial membrane (OMM). Biotinylated RNA was enriched using streptavidin beads and extracted with TRIzol. RNA-seq libraries were prepared from the enriched RNA using the TruSeq Stranded mRNA Library Preparation Kit. The samples included two labeled targets and two unlabeled controls.
For the ribominus libraries, APEX-seq was performed in HEK293T cells expressing APEX2 in the nucleus (NLS). Biotinylated RNA was enriched using streptavidin beads and released with Proteinase K. RNA-seq libraries were generated using the SMARTer Stranded Total RNA-Seq protocol. These samples also included two labeled targets and two unlabeled controls.

first: Madeline

middle: E

last: Rasband

first: Xuemei

last: Wang

first: Karen

last: Tolentino

first: Sharon

last: Huang

first: Furqan

last: Fazal


title: Single-cell gene expression analysis using lung derived cells from SARS-CoV-2-infected mice in aged and adult mice

city: Kumamoto-city

first: Takushi

last: Nomura

state: Kumamoto

address: 2-2-1 Honjo, Chuo-ku

department: Joint Research Center for Human Retrovirus Infection

institute: Kumamoto University

summary: We conducted single-cell gene expression analysis to elucidate the molecular basis of immune cells in the mouse lung associated with age-dependent severity of COVID-19 infection. In aged infected mice, genes associated with inflammation were more highly expressed in several cell populations compared to adult infected mice.

overall_design: We isolated live CD45-positive cells from the lungs of representative mice from aged and adult SARS-CoV-2-infected groups, as well as their uninfected control groups, and subjected them to 10x single-cell gene expression, TCR, and BCR analysis.

first: Rise

last: Kurokawa

first: Chatherine

last: Mtali

first: Innocent

last: Daniel

first: Thorbjorg

last: Einarsdottir

first: Omnia

last: Reda

first: Nobuko

last: Irie

first: Sakhor

middle: W

last: Binti

first: Koki

last: Niimura

first: Mitsuyoshi

last: Takatori

first: Kenji

last: Sugata

first: Chanidapa

last: Tye

first: Yorifumi

last: Satou

first: Masahiro

last: Ono


title: Lysophosphatidic acid synergizes with 1,25-dihydroxyvitamin D to promote fibroblast growth factor-23 synthesis via MAPK signaling and induction of the IL12A gene

city: BOSTON

first: Birol

last: Ay

state: Massachusetts

address: 50 BLOSSOM STREET

institute: MASSACHUSETTS GENERAL HOSPITAL

summary: Dysregulated actions of the bone-derived phosphaturic hormone, fibroblast growth factor-23 (FGF23), underlie the pathophysiology of several diseases. FGF23 is synthesized primarily in osteocytes in response to various endogenous molecules; however, the mechanisms governing FGF23 production are incompletely understood. Glycerol-3-phosphate (G3P), a glycolytic by-product originating from the kidney, critically controls skeletal FGF23 synthesis via its conversion in bone to lysophosphatidic acid (LPA), which stimulates osteocyte FGF23 production. The bioactive vitamin D, 1,25-dihydroxyvitamin D (1,25D), also promotes FGF23 production in osteocytes. We herein demonstrated that LPA requires 1,25D action to raise FGF23 levels in mouse bone explants and mice. RNA sequencing of osteocyte-like Ocy454 cells identified differentially expressed genes (DEGs) uniquely induced by LPA/1,25D co-treatment. These unique DEGs were enriched for the ribosome biogenesis pathway. DEGs concurrently induced by individual LPA and 1,25D treatments were enriched for MAPK signaling, and inhibiting this pathway obliterated LPA/1,25D-induced FGF23 production. DEGs following LPA/1,25D co-treatment were enriched for the cytokine-cytokine interaction pathway. Moreover, LPA/1,25D co-treatment, but not individual LPA and 1,25D treatments, rapidly induced the expression of Il12a, the gene encoding the proinflammatory cytokine interleukin-12 alpha-subunit, which responded solely to 1,25D at later times and required MAPK-ERK1/2 signaling. Inhibiting cytokine signaling or knocking down Il12a inhibited, while overexpressing Il12a enhanced LPA/1,25D-induced FGF23 production. However, challenging Ocy454 cells with recombinant bioactive interleukin-12 failed to enhance FGF23 production, suggesting that Il12a plays a non-canonical role. Our results reveal a mechanism of skeletal FGF23 synthesis involving cooperative actions of LPA and 1,25D, advancing our understanding of FGF23 regulation.

overall_design: RNAseq data of Lysophosphatidic acid (LPA) and 1,25-dihydroxy vitamin D (1,25D) treated Ocy454 cells to promote skeletal fibroblast growth factor-23 (FGF23) synthesis and explore the involved signaling pathways at 2 and 8 hours.

first: Sajin

last: Cyr

first: Yorihiro

last: Iwasaki

first: Elizabeth

last: Hill

first: Eugene

middle: P

last: Rhee

first: Nevil

last: Singh

first: Petra

last: Simic

first: Murat

last: Bastepe


title: DNA Replication Dynamics within the Centromeres of Chromosome Y and Chromosome 21 in Human Neural Progenitor Cells Overexpressing Tau Proteins

city: Bethesda

first: Mirit

middle: I

last: Aladjem

address: 37 Convent Dr. Building 37, Room 5068D

department: DNA Replication Group

institute: National Cancer Institute

summary: Aging somatic cells are characterized by specific chromosome aneuploidy. This work examines the role of DNA replication within the centromeres of chromosomes Y (ChrY) and 21 (Chr21). The aneuploidy of these two chromosomes is associated with Alzheimer's Disease (AD) pathology. Using human neural progenitor cells engineered to overexpress wild-type (wt) and pseudo-hyper-phosphorylated (php) Tau proteins, we developed a novel method to analyze replication dynamics in centromeric regions. Our findings reveal overexpression reduces the replication initiation events that are active within alpha satellite sequences in control condition. Mass spectrometry analysis on immunoprecipitated Tau identified nuclear interactors of Tau, particularly in its php form, which might directly influence chromatin architecture and gene expression. This research provides critical insights into the molecular mechanisms of aneuploidy in tauopathies.

overall_design: Nascent strands were purified with the lambda exonuclease methods from CIMH cells

first: Bhushan

middle: L

last: Thakur

first: Carl

last: Schildkraut

first: Elisa

last: Balzano


title: Glucocorticoid-dependence and independence of the circadian liver transcriptome

city: Manchester

first: Robert

last: Maidstone

address: Michael Smith Building, Oxford Road

institute: University of Manchester

summary: The liver, a critical metabolic organ, shows clear day-night variation in patterns of gene and protein expression, and in metabolic pathway activity. Liver cells possess molecular circadian clocks, but systemic factors also contribute to the circadian rhythmicity of liver gene expression. Here, we examine the role of glucocorticoid signalling. Glucocorticoids are essential hormones, with a strong circadian oscillation, which control multiple cellular processes via their action on the glucocorticoid receptor (GR). We first compare clock factor and GR binding at promoters and enhancers linked to rhythmic genes, and find increased clock factor binding at those elements where GR is also present. We find that genes with GR binding at the promoter are more likely to show rhythmic expression. We go to test the role of GR more directly, by examining circadian rhythms of gene expression in mice with and without hepatocyte-targeted GR deletion. Notably, we observe only a small effect of GR deletion, with the vast majority of rhythmic genes showing unchanged rhythmic expression, despite evidence for GR binding in the vicinity of these genes. We do find a small number of genes to show lost or gained rhythmicity with GR deletion, and genes which lose rhythmicity do associate with GR binding sites. However, GR appears redundant for conveying timing information to most of the rhythmic liver transcriptome, a conclusion which is supported by our re-analysis of an independent published dataset.

overall_design: Cre-mediated recombination was induced by injecting mice with 0.1mg tamoxifen i.p. (T5648, Sigma-Aldrich) each day for 5 days. 14 days after the first injection, at ZT0, all mice were transferred to light-tight cabinets, and maintained in constant darkness. After a further 24 hrs (CT0), tissue collection was commenced, with one Cre- (‘WT’) and one Cre+ (‘GR-LKO’) mouse being euthanised every 2 hrs for 48 hrs. As far as possible, the sex of the mice euthanised was alternated at each time point (e.g. males at CT0, CT4, CT8; females at CT2, CT6, CT10).

first: Louise

last: Hunter

first: Mudassar

last: Iqbal

first: Nicola

last: Begley

first: David

last: Bechtold

first: Matthew

last: Baxter

first: Andrew

last: Loudon

first: Magnus

last: Rattray

last: Ray


title: The expression of Welander distal myopathy-related TIA1 mutation exacerbates the dynamics of P-bodies and stress granules under oxidative stress

city: Cantoblanco

first: Juan

middle: Carlos

last: Oliveros

state: Madrid

address: Darwin 3

country: Spain

institute: CNB, CSIC

summary: T-cell intracellular antigen 1 (TIA1) is an RNA-binding protein that is primarily involved in the post-transcriptional control of the cellular fate of RNAs. Moreover, it is a key component of stress granules (SGs), RNA and protein aggregates that are formed in response to stressful stimuli to reduce cellular activity as a survival mechanism. TIA1 p.E384K mutation is the genetic cause of Welander distal myopathy (WDM), a late-onset muscular dystrophy whose pathogenesis has been related to modify SGs dynamics. In this study, we have focused on the differential transcriptomic and proteomic characterization of purified wild-type (WT) and WDM TIA1-dependent SGs by affinity immunopurification followed by mass spectrometry and RNA seq analysis under oxidative stress.  The results have revealed small differences in specific components of cellular machineries involving in regulating splicing, RNA degradation and/or translation linked to WDM TIA1 expression under oxidative stress. Our findings suggest a potential regulatory linking among WDM TIA1-dependent SGs and the number, size and colocalization/overlapping with P-bodies under oxidative stress.  Taken together, these findings allow us to expand our knowledge on the role of Welander’s TIA1 mutation in interfacing SGs and P-bodies dynamics by involving potential regulatory impacts on cell machineries of RNA decay/turnover and/or translation.

overall_design: Stress granules cores were enriched from FT293-TIA1aWT and FT293-TIA1aWDM cells stably transfected with plasmids for GFP-TIA1aWT and GFP-TIA1aWDM expression (generated in-house) as described previously (Sánchez-Jiménez et al. (2015) Cell Death Dis., 6, e1669; Carrascoso et al. (2019) Mol. Cell. Biol., 39, e0029918). Briefly, FT293 cells expressing either GFP-TIA1aWT or GFP-TIA1aWDM were subjected to 0.5 mM NaAsO2 for 1 h and snap-frozen on dishes. Cells were scraped, washed once with DMEN alone, snap-frozen with liquid nitrogen and stored at -70ºC. Cellular pellets were thawed on ice at 4ºC for 10 min and processed using lysis buffer (50 mM Tris HCl, pH 7.4, 100 mM KOAc, 2 mM MgOAc, 0.5 mM DTT, 50 µg/ml heparin, 0.5% NP-40, and protease inhibitors cocktail) and passed through a 25G x 5/8” (0.5mm x 16 mm) (BD Microlance 3) needle. The lysate was centrifuged at 1,000 g for 5 min, and the supernatant from this spin was centrifuged at 16,873 g for 20 min. The pellet containing SG cores was resuspended in the lysis buffer and incubated with GFP-Trap magnetic agarose beads (Chromotek) for 4 h. Beads were washed three times with washing buffer 1 (20 mM Tris HCl and 200 mM NaCl, pH 8.0), once with washing buffer 2 (20 mM Tris HCl and 500 mM NaCl, pH 8.0), and once with washing buffer 3 (lysis buffer containing 2 M urea). The proteins were eluted in 2x Laemmli sample buffer at 95ºC for 10 min. The total RNAs were purified using TRIzolR Reagent (Ambion by Life technologies).

first: Beatriz

last: Ramos-Velasco

first: José

last: Alcalde

last: Izquierdo


title: Reconstruction of Extracellular Matrix by Grhl3 Modulates Cell Fate Commitment from Fibroblasts to Induced Cardiomyocytes

city: Wuhan

first: Xin

last: Wu

address: 115 Donghu Road, Wuchang District

institute: Wuhan university

summary: Direct cardiac reprogramming provides a promising therapeutic strategy for heart regeneration by leveraging the endogenous fibroblast population to restore myocardial structure and function. While cardiac transcription factors such as Mef2c, Gata4 and Tbx5 (MGT) drive the generation of induced cardiomyocyte (iCM), the role of extracellular matrix (ECM) components during this process remain largely unexplored. Here, we investigated ECM dynamics during iCM reprogramming and identified integrin subunit alpha 8 (Itga8) as a critical ECM component that suppresses iCM conversion. A loss-of-function screen revealed grainyhead-like protein 3 homolog (Grhl3) as a transcriptional regulator of ECM, including Itga8. Multi-omics analyses demonstrated that Grhl3 efficiency remodels ECM composition to a cardiomyocyte-like state, enhancing iCM reprogramming efficiency and functional maturation. In a mouse model of myocardial infarction, removing Grhl3 enhanced in vivo cardiac reprogramming, and contributed to reduced scar size and improved heart function. These findings establish ECM remodeling as a critical determinant of cardiac reprogramming and identify Grhl3 as a promising therapeutic target to advance myocardial repair strategies.

overall_design: RNA-seq profiling of  cardiac fibroblasts and iCMs treated with shNT/shGrhl3 lentivirus for 5 days;CUT&Tag (H3K27ac)  profiling of  iCMs treated with shNT/shGrhl3 lentivirus for 5 days

first: Li

last: Wang


title: Single-cell RNAseq of from murine pancreas tumor (KPC) and chronic pancreatitis (KC)

city: Woodbury

first: Jonathan

last: Preall

state: NEW YORK

address: 500 Sunnyside Blvd

institute: Cold Spring Harbor Laboratory

summary: Pancreatic ductal adenocarcinoma (PDA) is characterized by a desmoplastic extracellular matrix (ECM) that is produced and remodeled by myofibroblastic cancer-associated fibroblasts (myCAFs), thus promoting tumor progression and resistance to therapy. Thus, identification of the regulatory pathways that drive desmoplasia is a pressing clinical need. Transcriptional profiling of tumor resident fibroblasts across PDA progression identified a CAF subset, termed WNT-sensing myCAFs, in both mouse and human PDA. These surround WNT7A/B+ cancer cells and express genes associated with ECM deposition and remodeling. Pharmacological inhibition of ligand-dependent WNT signaling or genetic ablation of Wnt7b in PDA cells depleted the number of WNT-sensing CAFs subpopulation and reduced collagen deposition in vivo, while ectopic Wnt7b expression enhanced desmoplasia. WNT7B overexpression also led to increased metastasis, resulting in a metastatic niche presenting similar WNT-sensing myCAF architecture. Our findings establish WNT7 as a critical regulator of myCAF function and offer a targetable axis to reduce tumor desmoplasia.

overall_design: Pancreatic tumors from KPC (Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre), or chronic pancreatitis tissue from KC (Kras+/LSL-G12D; Pdx1-Cre) mice were minced and enzymatically digested in DMEM supplemented with Collagenase D, Dispase, DNase I, and soybean trypsin inhibitor at 37°C. KPC tumor suspensions were then stained for CD45,CD31, EPCAM, and FACS sorted. Fibroblasts were enriched by sorting viable (DAPI-) cells from the population negative for all three markers (triple-negative, "TN"). Alternatively, all viable cells were enriched from the DAPI- fraction ("DN").  KC pancreata were harvested for single-cell RNAseq without sorting.  For Wnt7b targeting, Cas9-expressing mT6LOH organoid lines were first established by spinfection with LentiV-Cas9-puro vector (Addgene #108100). The single-guide RNA (sgRNA) cassette (sgRNA1-scaffold-bovineU6-sgRNA2) was synthesized as a gene block (IDT) and cloned into LRG2.1-Blast. sgRNAs targeting Wnt7b were sgRNA1= CTCGCGGAACTTAGGTAGCG,  sgRNA2= GTGCAACCGCACCTCGCCGG. For control, sgRNA1=GAAGATGGGCGGGAGTCTTC, targeting the Rosa26 locus and sgRNA2= AGATCATCATATAAGCTTTA, targeting a safe harbor region on chromosome 10 (28474669). KPC-derived mT6LOH organoids were tranduced in culture and single clones selected and verified for loss-of-function mutations by PCR. Orthotopic tumors were grown by injecting 500 cells in the pancreatic parenchyma of C57BL/6J mice, and harvest for scRNA-seq as above.

first: Youngkyu

last: Park

first: Sandeep

last: Nadella

first: Mojdeh

last: Shakiba

first: Jennifer

last: Thalapillil

first: Giuseppina

last: Caligiuri

first: Juliene

last: Hinds

last: Tuveson


title: Single Cell Transcriptome of Highly Heterogenous Hepato-Hematopoietic Lineages in Fetal Liver Organoids from Human Pluripotent Stem Cells

city: Cincinnati

first: Kentaro

last: Iwasawa

state: OH

address: 3333 BURNET AVENUE

department: Division of Gastroenterology, Hepatology and Nutrition

institute: Cincinnati Children's Hospital Medical Center

summary: To investigate the differentiation of hematopoietic fetal liver organoids (hFLOs) from induced pluripotent stem cell, we performed single cell RNA-Sequencing on different culture days.

overall_design: FLO cultures on day 10 (prior to hematopoietic growth cocktail) and hFLO cultures on days 13, 16 and 20 (after hematopoietic growth cocktai, total n=4 samples) were singularized prior single cell-RNA sequencing

first: Milad

last: Rezvani

first: Kyle

last: Lewis

first: Konrad

last: Thorner

first: Norikazu

last: Saiki

first: Takanori

last: Takebe


title: Regulation of the type I interferon response by chromatin organization (RNA-seq of HoxB8-FL pDC activation)

city: New York City

first: Eduardo

last: Esteva

state: New York

address: 550 1st Ave

institute: NYU Grossman School of Medicine

summary: Antiviral type I interferons (IFN-I) including IFN-β and multiple subtypes of IFN-α are encoded within a single locus. Whereas most cell types produce primarily IFN-β, plasmacytoid dendritic cells (pDCs) respond to viruses by rapidly producing all IFN-I subtypes. We show that during pDC differentiation, the IFN-I locus translocates from the nuclear periphery and undergoes reorganization of topologically associated domains (TADs). Accordingly, IFN-I production by pDCs was strictly dependent on the TAD-organizing cohesin complex. Promoters of Ifna genes in pDCs were poised, which was imparted by the pDC-enriched transcription factor IRF8. Finally, we identified two distal regulatory regions that facilitated the expression of adjacent IFN-I genes. Thus, the IFN-I response is controlled by multilevel chromatin organization of the IFN-I locus, including its unique poised state in pDCs.

overall_design: RNA-seq was performed on purified HoxB8-FL pDCs stimulated with CpG-A for 6 hours and 24 hours, as well as unstimulated pDCs at the same timepoints as a control (3 replicates per stimulation condition and timepoint).

first: N

last: Adams

first: I

last: Tiniakou

first: E

first: A

last: Khodadadi-Jamayran

first: B

last: Reizis


title: An Integrated Analysis of the Heat Response in Wheat Reveals Ploidy-Dependent Effects on Transcriptomic Reprogramming.

status: Public on Jan 08 2026

city: Valdivia

first: Javier

last: Canales

state: Los Ríos

address: Campus Isla Teja s/n

department: Instituto de Bioquimica y Microbiologia

country: Chile

institute: Universidad Austral de Chile

summary: Understanding heat stress (HS) responses across wheat species with different ploidy is crucial for breeding climate-resilient varieties. We combined field experiments with RNA sequencing to compare diploid (T. monococcum), tetraploid (T. turgidum), and hexaploid (T. aestivum) wheat during early grain filling. Under severe HS, grain yield declined most drastically in the diploid (74%) and moderately in the hexaploid (37.8%), while the tetraploid showed the greatest resilience (19% reduction). Transcriptome profiling revealed ploidy-dependent reprogramming, with the hexaploid exhibiting the largest set of differentially expressed genes (2,227 vs. 859 and 757 in diploid and tetraploid, respectively). Alternative splicing patterns also diverged; notably, we detected species specific, heat-induced exon skipping of the NF-YB transcription factor exclusively in hexaploid wheat. Gene co-expression analysis identified 12 modules linked to grain traits, underscoring the relationship between transcriptional control and phenotype. Together, these results indicate that genome complexity shapes HS responses, with polyploid species exhibiting greater resilience supported by more intricate regulatory networks. These insights provide valuable information for breeding heat-tolerant wheat varieties and understanding the role of polyploidy in stress adaptation.

sample_organism:
Triticum aestivum
Triticum monococcum
Triticum turgidum

overall_design: Expression profiling by high throughput sequencing

first: Arenas-M

last: Anita

first: Miño

last: Ignacio

first: Uauy

last: Cristóbal

first: Calderini

last: Daniel

first: Canales

last: Javier


title: Essential roles for Sgo2 regulating 2-cell like genes in embryonic stem cells.

city: Tianjin

first: Jie

last: Li

address: Wei Jin Road Nankai District No. 94

department: Department of Cell Biology and Genetics

institute: Nankai University

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

overall_design: Refer to individual Series


title: Regulation of the type I interferon response by chromatin organization


title: Gene expression profiling reveals two overarching types of anaplastic large cell lymphoma with distinct targetable biology: an LLMPP study

city: Rochester

first: Surendra

last: Dasari

state: MN

address: 200 First Street SW

department: Quantitative Health Sciences Research

institute: Mayo Clinic

summary: Anaplastic large cell lymphomas (ALCLs) are CD30+ T-cell lymphomas that share pathologic features but differ in presentation, outcome, and genetics.  Current classification incorporates clinical presentation and ALK status, but inadequately addresses molecular heterogeneity and therapeutic vulnerabilities.  We studied 689 ALCLs in the Lymphoma/Leukemia Molecular Profiling Project and performed expert consensus review, genetic subtyping (ALK, DUSP22, TP63, and triple-negative),  and immunohistochemistry for phospho-STAT3Tyr705.  RNAseq with unsupervised gene expression profiling (n=393) identified 2 main molecular types of ALCL that could be predicted with 91% accuracy based on the presence (Type I) or absence (Type II) of phospho-STAT3Y705 expression (P<0.0001).  Type I ALCLs included ALK+ ALCL and a distinct subset of triple-negative ALCLs (TN-I); Type II ALCLs included tumors with DUSP22 and/or TP63 rearrangements and the remaining triple-negative ALCLs (TN-II).  Type I ALCLs were enriched for JAK-STAT3 (FDR<0.0001), whereas Type II ALCLs were enriched for non-tyrosine kinase pathways, particularly epigenetic regulators (FDR=0.003).  EZH2 was the top gene (FDR=1.3×10-97) and EZH2 and H3K27me3 were overexpressed by immunohistochemistry (P<0.0001).  Prognosis in systemic ALCL (N=257) was favorable for DUSP22-rearranged ALCL (5-year OS, 95%) and ALK+ ALCL (88%), intermediate for triple-negative ALCL (TN-I, 52% and TN-II, 37%), and poor for TP63-rearranged ALCL (0%; P<0.0001).  We introduce an integrated molecular classification that preserves currently diagnosed ALCL entities but identifies 4 molecularly distinct ALK− ALCL subtypes (DUSP22-rearranged, TP63-rearranged, TN-I, and TN-II).  This classification can be easily implemented on paraffin tissue in routine practice or clinical trials and stratifies ALCL into diagnostically, prognostically, biologically, and potentially therapeutically relevant subtypes.

overall_design:
A total 393 anaplastic large cell lymphoma samples were subjected to gene expression profiling.

***************************************************************
Raw files for human/patient samples were not submitted to GEO due to concerns about submitting personally identifiable sequence data for open access.
***************************************************************

first: Andrew

last: Feldman


title: Essential roles for Sgo2 regulating 2-cell like genes in embryonic stem cells

summary:
In order to study the regulation of Sgo2 on 2c gene, we established Sgo2 KO cell lines .
We then performed gene expression profiling  with the data obtained from the RNA-seq .

overall_design: Comparative gene expression profiling analysis of RNA-seq data for WT cell line and  KO cell lines


title: Antitumor effects of anti-CD4-depleting antibody in lung fibrosis through CD8+ T cell activation and regulatory T cell inhibition in mice

city: Kashihara-shi

first: Toshihiro

last: Ito

state: Nara

address: 840, Shijocho

department: Immunology

institute: Nara Medical University

summary:
Background
Lung cancer (LC) as a complication worsens the prognosis of patients with idiopathic pulmonary fibrosis (IPF); however, the underlying mechanisms are not fully understood. This study aimed to investigate the immunological characteristics of IPF-LC.
Methods
We created a murine IPF-LC complication model by intravenous administration of Lewis lung carcinoma cells into bleomycin-induced pulmonary fibrosis model mice. We administered anti-CD4 monoclonal antibody (mAb) to the mice and evaluated the effects on tumor growth, T-cell phenotype in the lungs and lymph nodes, and survival rate. T-cell phenotype was analyzed by flow cytometry and survival analysis was performed using the Kaplan-Meier method.
Results
Tumor number and size were both significantly increased in this model. CD8+ T cells were significantly decreased, while regulatory T cells (Tregs) were significantly increased in the lungs and lymph nodes. Anti-CD4 mAb treatment significantly increased the number and antitumor gene expression of CD8+ T cells, suppressed tumor growth in fibrotic lungs, reduced the number of Tregs, and improved survival.
Conclusions
These results indicate that anti-CD4 mAb treatment inhibits tumor growth by enhancing the number and antitumor activity of CD8+ T cells in IPF-LC mice. Anti-CD4 mAb may thus be a promising clinical therapeutic option in patients with IPF-LC.

overall_design: RNA-seq profiling of CD8+ T cells from CD4+ Tcell-depleted/control IPF-LC/LC mice

first: Tatsuki

last: Nishioka

first: Takehiro

last: Sakabe

first: Masahiro

last: Kitabatake

first: Noriko

last: Ouji-Sageshima

first: Atsushi

last: Hara

first: Ichiro

last: Sato

first: Nobuhiro

last: Fujioka

first: Ryutaro

last: Furukawa

first: Tomoko

last: Kasamatsu-Nishimura

first: Shigeto

last: Hontsu

first: Shigeo

last: Muro


title: KLHDC4 serves as a novel prognostic biomarker and drives tumor progression via PI3K/AKT signaling in clear cell renal cell carcinoma

city: Beijing

first: Qingjiang

last: Xu

address: Senior Department of Urology, Chinese PLA General Hospital, Beijing, China.

institute: Chinese PLA General Hospital

summary: The clinical significance and molecular mechanisms of KLHDC4 in malignancies, particularly in clear cell renal cell carcinoma (ccRCC), remain poorly understood. In this study, we integrated multi-omics data—including tran-scriptomic, proteomic, and single-cell RNA sequencing—to systematically evaluate the clinical relevance and func-tional role of KLHDC4, followed by experimental validation. Our results demonstrated that KLHDC4 is significantly upregulated in multiple cancer types and serves as an independent prognostic biomarker for poor survival in ccRCC. KLHDC4 expression was correlated with immune cell infiltration, tumor mutational profiles, and immune check-point expression. Furthermore, KLHDC4 levels predicted response to drug therapies, including immunotherapy and tyrosine kinase inhibitors. Functional experiments showed that KLHDC4 knockdown suppressed proliferation, mi-gration, and invasion in vitro, as well as tumor growth in vivo, whereas its overexpression promoted malignant phenotypes. Mechanistic investigations through RNA sequencing and Western blot analysis indicated that KLHDC4 activates the PI3K/AKT signaling pathway. Rescue assays confirmed that KLHDC4-driven oncogenic effects are partially mediated through this axis. Additionally, molecular docking identified ledipasvir as a potential KLHDC4 inhibitor. Collectively, our findings establish KLHDC4 as a novel prognostic biomarker and therapeutic predictor, highlighting the KLHDC4/PI3K/AKT axis as a potential target for ccRCC treatment.

overall_design: RNA-seq profiling of OSRC-2 cells and their knockdown derivatives(shKLHDC4) at Day2


title: DAXX governs the silencing of LINE1 during spermatogenesis in mice [ChIP-seq]

first: shen

last: jin

state: Hubei

address: No. 299, Bayi Road, Wuchang District, Wuhan City

summary: The suppression of transposable elements in germline is critical for safeguarding fertility. Here we identify that the DAXX, a potent transcription repressor and an H3.3 chaperone, is indispensable for transposable elements silencing during spermatogenesis. Male mice with conditional knockout of Daxx in germ cells mediated by Ddx4-cre display delayed meiotic progression, deformed and reduced production of sperms, and an age-dependent decline in fertility. The ablation of DAXX results in activation of evolutionarily young LINE1 and ERVs subfamilies. Further study found that reduced H3K9me3 modification and DNA methylation of young LINE1, especially L1Md_A, L1Md_Tf and L1Md_Gf.In summary, this study identifies DAXX as a previously unknown regulator of spermatogenesis and demonstrates that it functions as an epigenetic regulator to maintain the silencing of the young LINE1

overall_design: Genome-wide H3K9me3 ChIP-seq profiling was conducted in meiotic cells and round spermatids from adult wild-type and knockdown mice to assess changes in heterochromatin organization, including at transposable element–associated loci.

first: Zejia

first: Chaoyang

last: Xiong

first: Jin

last: Shen

first: Chen

last: Tan

first: Dupeng

last: Ma

first: Jun

last: Chen

first: Rong

last: Liu

first: Qi

last: Sun

first: Weihao

last: Gong

first: Wenming

last: Yuan

first: Mengya

last: Huang

first: Li

first: Yueqiu

first: Guohong

first: Mengcheng

last: Luo


title: Single nucleus transcriptomic analysis of osimertinib-induced cardiac dysfunction in the mouse heart

city: Duarte

first: June-Wha

last: Rhee

address: 1500 E Duarte Rd

department: Division of Cardiology, Department of Medicine

institute: City of Hope

summary: We performed single nuclei RNA sequencing (snRNA-seq) analysis of mouse left ventricular tissues to investigate transcriptomic changes caused by osimertinib treatment.

overall_design: Eight mouse snRNA-Seq libraries were prepared from four treatment groups (n=2 per group) – Sham, Osimertinib (Osi), Transverse aortic constriction (TAC), and TAC-Osi – using Next GEM 10x Genomics Chromium Single Cell 3' Reagent Kit v3.1 chemistry, and sequenced on Illumina NovaSeq 6000 at Translational Genomics Research Institute (TGen), an affiliate of City of Hope.


title: Daphnetin sensitizes hepatocellular carcinoma cells to chemotherapeutics and suppresses malignant phenotypes through modulating DDIT3-associated stress-response

city: Hangzhou,

first: Guangyue

last: Li

state: Zhejiang

address: No.600 Dunyu Rd., Xihu District

institute: westlake university

summary: Many natural products can both suppress tumor growth and promote human health. Daphnetin (DAP) is a natural coumarin and possesses suppressive activities against various cancers, but the molecular mechanisms underlying its anticancer action remain poorly understood. In the treatments of hepatocellular carcinoma (HCC), many generic chemotherapeutic agents, such as doxorubicin (DOX) and cisplatin (DDP), manifest significant side effects on patients. In the present study, we first investigated the cytotoxic effects of DAP alone or in combination with DOX or DDP in HCC HepG2 and SMMC-7721 cells, and in normal liver HL-7702 cells. Based on isobologram analysis, DAP synergistically enhanced cytotoxic activities of DDP and DOX in HCC cells but not in HL-7702 cells. RNA-seq analysis of DAP-treated HepG2 cells identified 484 protein-coding DEGs involved in multiple pathways, such as PI3K-AKT signaling, amino acid deprivation, and unfolded protein response. Especially, DAP treatment upregulated a stress-responsive pro-apoptotic transcription factor DDIT3. Consistently, DDIT3 knockdown desensitized HCC cells to DAP, while constitutively active AKT did not rescue DAP-induced defective phenotypes. Collectively, we demonstrate DAP as a multi-pathway stress-modulating agent that can both attenuate malignant phenotypes of HCC cells and sensitize the cells to clinical chemotherapeutics.

overall_design: HepG2 cells were subjected to treatment with DAP and DMSO, followed by RNA-seq analysis to elucidate the underlying molecular mechanisms

first: Guangchao

last: Sui

first: RAZA

last: ALI


title: Glucocorticoid-dependence and independence of the circadian liver transcriptome [ChIP-seq]

first: Robert

last: Maidstone

overall_design:
Mice were treated acutely with an intraperitoneal injection of dexamethasone or vehicle, and liver tissue harvested one hour later at ZT18. Data from ZT6 is previously published and avaliable from Array Express: E-MTAB-10224
This study includes re-analysis of samples from ArrayExpress E-MTAB-10224/ERP127680

first: Louise

last: Hunter

first: Mudassar

last: Iqbal

first: Nicola

last: Begley

first: David

last: Bechtold

first: Matthew

last: Baxter

first: Andrew

last: Loudon

first: Magnus

last: Rattray

last: Ray


title: Potassium ion homeostasis modulates mitochondrial function

city: South San Francisco

first: Adam

middle: James

last: Waite

address: 1170 Veterans Blvd

institute: Calico Life Sciences LLC


title: Lactate-Mediated Cross-Talk between KRT15 and GPR68 drives Type 2 Immunity in ECRSwNP

city: Jinan

first: Hongjie

last: Du

address: No. 324, Jingwu Road, Huaiyin District

department: Shandong Provincial Hospital

institute: Shandong First Medical University

summary: Eosinophilic chronic rhinosinusitis with nasal polyps (ECRSwNP) is a refractory inflammatory disease driven by type 2 immunity, yet the mechanistic link between epithelial stress and immune activation remains elusive. Here, we identify a pathogenic expansion of KRT5⁺KRT15⁺ basal epithelial cells that undergo a metabolic switch to aerobic glycolysis, resulting in lactate overproduction and microenvironment acidification.

overall_design: This acidic niche activates the proton-sensing receptor GPR68 on infiltrating CD4⁺ T cells, triggering a Gq–IP1–IKK/ERK signaling cascade that promotes Th2 differentiation and cytokine release. Genetic ablation of GPR68 in CD4⁺ T cells or its pharmacological inhibition with a natural antagonist, 3-Bn-6-Bnid-DKP, ameliorated type 2 inflammation and pathology in vivo. Thus, we define a lactate-GPR68 axis that metabolically couples epithelial dysfunction to adaptive type 2 immunity in ECRSwNP

first: Ming

last: Xia


title: Potassium ion homeostasis modulates mitochondrial function I

summary: Age-associated decline in mitochondrial membrane potential (MMP) is a ubiquitous aspect of eukaryotic organisms and is associated with many aging-related diseases. However, it is not clear whether this decline is a cause or consequence of aging, and therefore whether interventions to reduce MMP decline are a viable strategy to promote healthier aging and longer lifespans. We developed a screening platform in S. cerevisiae to identify mutations that slowed or abrogated the age-associated decline in MMP. Characterization of the longest-lived mutant revealed that reduced internal potassium increased MMP and extended lifespan. Distinct interventions improved cellular MMP and lifespan: deleting a potassium transporter; altering the balance between kinases and phosphatases that control potassium transporter activity; and reducing available potassium in the environment. Similarly, in isolated mitochondria, reducing the concentration of potassium was sufficient to increase MMP. These data indicate that the most abundant monovalent cation in eukaryotic cells plays a critical role in tuning mitochondrial function, consequently impacting lifespan.

overall_design: RNA-seq profiling of WT prototrophic cells grown in complete synthetic media (SC) and sampled either during exponential growth (young) or after 24 hours in a ministat aging device (Hendrickson, 2018).

first: Beiduo

last: Rao

first: Elizabeth

last: Schinski

first: Nathaniel

middle: H

last: Thayer

first: Manuel

last: Hotz

first: Austin

last: Lefebvre

first: Celeste

last: Sandoval

first: Daniel

last: Gottschling


title: Large-Scale Comparative Analysis of Canine and Human Osteosarcomas Uncovers Conserved Clinically Relevant Tumor Microenvironment Subtypes

first: Christina

last: Mazcko

state: MD

address: 10 Center Drive Room 1B58

department: CCR

institute: NCI

summary: Analyze clinically annotated bulk tumor transcriptomic datasets of canine and patients with human osteosarcoma to identify potentially conserved patterns of disease progression

overall_design: The study conducted a large-scale, cross species comparative analysis of canine and human osteosarcoma samples using genomic and tumor-microenviornment profiling to idenity conserved, clinically relevant molecular subtypes.

first: Sushant

last: Patkar

first: Joshua

last: Mannheimer

first: Stephanie

last: Harmon

last: Ramirez

middle: N

first: Peter

last: Choyke

first: Gregory

middle: T

last: Brown

first: Baris

last: Turkbey

first: Amy

middle: K

last: LeBlanc

first: Jessica

last: Beck


title: KDM5 protein mediated cisplatin resistance in ovarian cancer. [ChIP-Seq]

city: Incheon

first: So Hee

last: Kwon

address: 85, Songdogwahak-ro, Yeonsu-gu

department: Pharmacy

country: South Korea

institute: Yonsei University

summary: High recurrence and relapse rates of ovarian cancer (OVC) make it one of the deadliest gynecological cancers and the 5th most common cause of death in women. It is well recognized that acquired resistance is accompanied by abnormal alteration of epigenetic regulation, yet there is a lack of in-depth mechanistic study on cisplatin-resistant OVC in epigenetic context. Herein, we investigate the role of lysine lysine demethylase 5 (KDM5) and how its abnormal overexpression correlates with cisplatin resistance and OVC tumor progression.

overall_design: ChIP-seq profiling of changes in H3K4me3 observed in cisplatin sensitive ovarian cancer cells (SKOV-3) and cisplatin-resistant ovarian cancer cells (SKOV-3 CDDP), SKOV-3 CDDP KDM5B knockout cells.

first: Jung

last: Yoo


title: Investigating Trophoblast Invasion and Angiogenesis Expression Changes in a Caloric Deficient Mouse Model of Fetal Growth Restriction

city: Aurora

first: JAMES

middle: R

last: BARDILL

state: CO

department: Surgery

institute: UNIVERSITY OF COLORADO-ANSCHUTZ MED

summary: Fetal growth restriction (FGR) is a severe pregnancy complication often caused by placental insufficiency. Proper trophoblast invasion is essential for placental development and function, ensuring adequate nutrient and oxygen supply to the developing fetus. Dysregulation impairs placental perfusion, leading to FGR. This study uses a calorie-restricted mouse model to investigate genes/molecular mechanisms regulating trophoblast invasion across gestational timepoints.  Pregnant mice received either a standard or 50% calorie-restricted diet from E8.5. Placentas and invasion sites were analyzed at E10.5, E12.5, E14.5, E16.5, and E17.5. mRNA sequencing and RT/qPCR examined trophoblast invasion-related genes (Mmp2, Mmp9, Efna1, Rac1, Rras, Ascl2, Tfap2c, Prl7b1) and angiogenesis genes (Vegfa, Vegfb, Pdgf, Akt3). Immunohistochemistry of trophoblast cells (cytokeratin 8, CK8) and endothelial cell markers (endomucin, CD31, CCD105, VEGFR2) was performed.   Statistical analysis used Student’s t-test.  Caloric restriction significantly reduced fetal/placental weights from E12.5, with persistent growth restriction at E16.5, and E17.5. IHC at E17.5 showed reduced decidual depth, trophoblast invasion distance, and trophoblast quantity within the decidua. This impaired growth was accompanied by reduced expression of trophoblast invasion genes (Mmp2, Mmp9, Efna1, Rac1, Rras, Ascl2, Tfap2c, Prl7b1) in FGR placentas, with a reduction in CK8 trophoblast staining.  Angiogenesis reduction in FGR was demonstrated with reduced Vegfa, Vegfb, and Akt3 and supported by reduced CD31, CD105, and VEGF2 endothelial cell markers A caloric-restriction mouse model replicates key FGR pathophysiology, including reduced fetal/placental growth, downregulation of trophoblast invasion genes, impaired trophoblast invasion into the decidua, and reduced placenta angiogenesis. These findings offer molecular insights into placental insufficiency that merits further exploration regarding FGR pathogenesis.

overall_design: PolyA-RNA sequencing of whole mouse placentas at embryonic age E12.5.  There are 5 control (healthy) placentas and 5 FGR placentas.

first: Bardill

last: James

first: Derderian

last: Chris (Sarkis)


title: Lesion-remote astrocytes govern microglia-mediated white matter repair IV

city: Los Angeles

middle: Evan

last: Burda

state: CA

address: 127 S San Vicente Blvd

department: Biomedical Sciences, Center for Neural Science and Medicine

institute: Cedars Sinai Medical Center

summary: Visium Spatial Transcriptomics was applied to spared tissue regions both rostral and caudal in a mouse model of incomplete spinal cord injuryt (ISCI) at multiple timepoints after injury (3,7,28dpi) to understand spatially distinct  changes occuring in spared, yet reactive, spinal tissue after ISCI.

overall_design: Mice were perfused at 3,7,and 28 days post injury, spinal cords dissected and immediately fresh frozen. Tissue was embedded in OCT and 2 10um sections were then collected both above and below the injury site. Equivalent tissue was taken from uninjured mice as controls

first: Sarah

last: McCallum

first: Keshav

middle: B

last: Suresh


title: Lesion-remote astrocytes govern microglia-mediated white matter repair III

summary: Single Nuclei RNA seq was used to examine gene expression of all spinal cord cell types from rostral and caudal spared regions of the injured spinal cord in a mouse model of incomplete spinal cord injury (iSCI, lateral hemisection) at multiple timepoints after injury (3, 7, 28 dpi). A focus of this study was to characterize spatiotemportal lesion-remote astrocyte gene expression via integration with paired spatial transcriptomics data.

overall_design: Cohorts of WT mice were perfused at 3, 7, or 28 days post injury, spinal cords dissected out and 3mm chunks of spared spinal cord tissue rostral and cuadal to the injury site were dissected out, flash frozen and processed for nuclei isolation. Equivalent tissue was taken from uninjured healthy control mice.

first: Keshav B.

first: Joshua E.


title: Lesion-remote astrocytes govern microglia-mediated white matter repair


title: DAXX governs the silencing of LINE1 during spermatogenesis in mice [BiSulfite-seq]

first: shen

last: jin

institute: Wuhan university

type: Methylation profiling by high throughput sequencing

overall_design: This study uses whole-genome bisulfite sequencing (WGBS) to characterize DNA methylation dynamics in the testes of P8 wild-type and knockdown mice, with a specific emphasis on methylation changes across genomic regions and transposable element families.

first: Zejia

last: Li

first: Chaoyang

last: Xiong

first: Jin

last: Shen

first: Chen

last: Tan

first: Dupeng

last: Ma

first: Jun

last: Chen

first: Rong

last: Liu

first: Qi

last: Sun

first: Weihao

last: Gong

first: Wenming

last: Yuan

first: Mengya

last: Huang

first: Li

first: Yueqiu

first: Guohong

first: Mengcheng

last: Luo


title: Lesion-remote astrocytes govern microglia-mediated white matter repair II

first: Joshua

summary: RNA sequencing was applied to examine changes in gene expression in astrocytes in spared tissue regions both rostral and caudal to the injury site in a mouse model of incomplete spinal cord injury (ISCI) over multiple post-injury time points. The RiboTag method was used to isolate astrocyte ribosome-associated mRNAs from the spinal cord of wild type mice and mice with astrocyte-specific conditional gene deletion (cKO) of a known transcriptional regulators of reactivity, STAT3 (Stat3 knockout: Stat3-cKO).

overall_design: Astrocyte ribosome-associated mRNAs and bulk RNA from IP input were isolated from the rostral and caudal spared tissue regions of the wild type and Stat3-astro-cKO Ribotag mouse spinal cord at 3,7,14,and 28 days post-SCI. Equivalent spinal cord tissues were examined from genotype matched healthy (uninjured) control mice.

first: Sarah

first: Keshav


title: Targeting Galectin-3 Mitigates Chronic Kidney Fibrosis and Preserves Kidney Function

first: Lianjiu

last: Su

address: 500 Landfair Ave.

institute: University of California, Los Angeles

summary: Renal fibrosis is a common pathological hallmark of progressive chronic kidney diseases, characterized by persistent inflammation, immune activation, and excessive extracellular matrix deposition.  Lectins play a role in modulating immune cell recruitment, amplifying pro-inflammatory signaling, and promoting fibrotic cellular phenotypes. Here, we demonstrate, in a population cohort of individuals with CKD, that the Galectin-3 is robustly expressed in diseased human kidneys and strongly correlates with clinical indices of renal dysfunction. Using the models of adenine induced nephropathy and unilateral ureteral occlusion in mice, we find that Galectin-3 upregulated in kidney collecting tubular cell after injury. Renal fibrosis was significantly reduced in Galectin-3-deficient kidneys, accompanied by lower levels of serum creatinine and blood urea nitrogen and decreased expression of myofibroblast marker α-SMA. RNA sequencing revealed that genes associated with the NF-κB signaling pathway were significantly enriched in the Galectin-3 knockout group. Treatment with GB1107, a selective Galectin-3 inhibitor, also markedly attenuated renal fibrosis, improved renal function and suppressed α-SMA expression. Taken together, our findings suggest that Galectin-3 promotes renal fibrosis through enhancement of inflammatory and fibrotic signaling pathways, and that targeting Galectin-3 may offer therapeutic benefit by preserving kidney function and attenuating interstitial fibrosis.

overall_design: This study investigated the role of Galectin-3 in renal fibrosis and its therapeutic potential in chronic kidney disease using an integrated approach combining human CKD cohort analysis, genetic and pharmacological mouse models, and transcriptomic profiling. Galectin-3 expression and its association with renal dysfunction were examined in human kidney biopsies. Renal fibrosis was induced in mice using adenine-induced nephropathy and unilateral ureteral obstruction (UUO), and the functional role of Galectin-3 was assessed using Galectin-3–deficient mice. RNA sequencing was performed to identify Galectin-3–dependent inflammatory and fibrotic pathways. Finally, the therapeutic effects of the Galectin-3 inhibitor GB1107 were evaluated in murine models of renal fibrosis.


title: Lesion-remote astrocytes govern microglia-mediated white matter repair I

last: Burda

summary: A 50 μl spot drop containing 50,000 microglia cells was seeded into the desired wells of a 24-well plate. Cells were cultured for 7 days, with a full media change given on day 1 followed by half media change on day 4. On day 7, cells were either left untreated or treated with CCN1 (Peprotech: 120-25) at 50 ng ml−1 or vehicle (BSA) for 24 h. Each condition was carried out in triplicate for each experiment. Cells were then removed using a cell scraper, replicates pooled, and then processed for RNA-seq.

overall_design: Microglia were cultured for 7 days in defined microglia media under controlled conditions (37C/5%CO2)


title: Characterization of mouse melanocytes reveals ultrastructural and immunological insights into the inner ear function [RNA-Seq]

first: Jing

last: Cai

state: Shandong

address: 4 Duanxing West Road

department: Shandong Provincial ENT Hospital

institute: Shandong University

summary: Melanocytes are important components of the inner ear’s cellular architecture. However, limited morphological research has hindered a complete understanding of inner ear function. This study characterized the morphology of inner ear melanocytes and cells often misidentified as melanocytes. Immunofluorescence, Smart-seq, transmission/field emission scanning electron microscopy, and immunoelectron microscopy were used. Cells with a “black ball” appearance in the vestibule were identified as macrophages. We examined variation in melanocytes or macrophages with age, strain, and cisplatin injury. Kir4.1 expression and the greater noise resistance observed in pigmented mice suggest potential melanocyte functions. Based on melanin distribution in Pou3f4y/- mice, we hypothesize that melanocytes migrate from the modiolus along Reissner's membrane to the stria vascularis, following a base to apex gradient. These findings provide novel ultrastructural and immunological insights into inner ear function.

overall_design: By comparing the results of two sets of sequencing, the possible mechanism of inner ear pigmentation in Pou3f4y/- mice was determined.

first: Lei

last: Xu

first: Yongdong

last: Song

first: Aizhen

last: Zhang

first: Zheng

first: Boqin

first: Haibo

last: Wang


title: Landscape of allele-specific expression in prostate cancer reveals recurrent, stage-specific events in AR signaling and resistance pathways [ChIP-Seq]

city: San Francisco

first: Franklin

address: 513 Parnassus Ave

department: Hematology and Oncology

institute: UCSF

summary: The effects of cis-regulatory alterations in prostate cancer (PCa) are insufficiently characterized, presenting an opportunity to discover driver genes and therapeutic targets. To comprehensively study these effects, we identify genes undergoing allele-specific expression (ASE) in localized PCa and metastatic castration-resistant PCa (mCRPC) samples. By defining recurrent ASE events across prostate tissue and tumor-enriched ASE, we develop CASEDI, a computational framework for prioritizing cancer drivers by integrating ASE and clinical data. CASEDI reveals genes showing recurrent ASE and altered expression in PCa, including AR-regulated and oncogenic ACSM1. mCRPC samples show enrichment of ASE in DNA repair, resistance pathways, and oncogenes and increased frequency of monoallelic expression (MAE) compared to localized tumors.  We define an mCRPC gene signature based on MAE status that identifies a subgroup of localized patients with worse prognosis. Using ASE analysis, we expand the landscape of cis-regulatory events in PCa to inform the identification of additional therapeutic targets.

overall_design: ChIP-seq for H3K27ac in MDAPCA2B cells in both control and synthetic androgen (R1881) treatment conditions

first: Margaret

last: Tsui

first: Rebecca


title: Landscape of allele-specific expression in prostate cancer reveals recurrent, stage-specific events in AR signaling and resistance pathways [RNA-Seq 2]

overall_design: Bulk RNA-seq of LNCaP cells treated with synthetic androgen (R1881) and control cells treated with 1% ethanol


title: Exploring the sex and estrous cycle-dependent differences in microglia response to amyloid-beta pathology in female and male C57BL6/J (WT) and 5xFAD using bulk mRNAseq of isolated microglia.

first: Lia

last: Calcines Rodriguez

state: NY

address: 17 S Goodman St, Apt. 2

department: Neuroscience

institute: University of Rochester

summary: Alzheimer’s disease (AD) presents with a sex bias where women are at higher risk and exhibit worse cognitive decline and brain atrophy compared to men. Microglia play a significant role in the pathogenesis and progression of AD and have been shown to be sexually differentiated in health and disease. Whether and how microglia contribute to the sex differences in AD remains to be elucidated. Herein, we characterize the sex differences in microglial transcriptomic changes that occur in females and males in response to amyloid-beta (Aβ). In females, we focus on two hormonally distinct stages of the rodent estrous cycle: proestrus and diestrus. Our transcriptomic data revealed that female microglia are not overtly different at the proestrus or diestrus stages. However, we found stark sex differences between female and male microglia transcriptomes in the 5xFAD brains, where female 5xFAD microglia upregulated genes involved in glycolytic metabolism, antigen presentation, disease-associated microglia, and microglia neurodegenerative phenotype (DAM/MGnD) compared to male 5xFAD microglia. Interestingly, a significant proportion of differentially expressed genes (DEGs) between female and male 5xFAD microglia were interferon-stimulated genes (ISGs), all of which were upregulated in females.

overall_design: RNAseq profiling of diestrus and proestrus female and male C57BL6/J (BL6) and 5xFAD microglia at 5.5 months of age. Vaginal smears (started 2 weeks prior to sacrifice) and uterus weight (at the time of sacrifice) was used to group females into proestrus (Pro) and diestrus (Die). Cortex was microdissected and homogenized to make a single cell suspension. Myelin was removed using a Percoll density gradient and microglia were stained with fluorescently-conjugated antibodies. Micoglia were isolated via fluorescence-activated cell sorting on the basis of CD11b+CD45low-intermediate expression. TMEM119 and P2RY12 were used to cofirm microglial identity.

first: Calcines-Rodriguez

last: Lia

first: Noyes-Martel

last: Nikita

first: Becker

last: Jennifer

first: Osabutey

last: Mark

first: Trojanczyk

last: Lee

first: Majewska

last: Ania

first: O'Banion

middle: M

last: Kerry

first: Nguyen

last: Sophia


title: Landscape of allele-specific expression in prostate cancer reveals recurrent, stage-specific events in AR signaling and resistance pathways [RNA-Seq 1]

last: Huang

overall_design: Bulk RNA-seq of LNCaP cells overexpressing ACSM1 and LacZ control

first: Lorraine

last: Nuniz


title: Potassium ion homeostasis modulates mitochondrial function II

first: Adam

last: Waite

middle: J

first: Beiduo

last: Rao

first: Elizabeth

last: Schinski

first: Nathaniel

last: Thayer

first: Manuel

last: Hotz

first: Austin

last: Lefebvre

first: Celeste

last: Sandoval

first: Daniel

last: Gottschling


title: Redistribution of RNA localization in early embryos of rainbow trout

city: Vestec by Prague

first: Radek

last: Sindelka

address: Průmyslová 595

country: Czech Republic

institute: Institute of Biotechnology, Czech Academy of Science, v.v.i.

summary: This study investigates spatial transcriptomic dynamics during early embryogenesis in Oncorhynchus mykiss (rainbow trout), a teleost species with large oocytes and prolonged developmental timing. Using TOMO-seq, we profiled RNA localization along the animal–vegetal axis at key embryonic stages, including 0 hpf (freshly fertilized egg/zygote), 1 hpf (early cleavage stage), 1 dpf (early blastula stage, ~32-64 cells), and 3 dpf (mid-blastula, ~1000 cells). Samples were collected in biological triplicates and sectioned into five defined regions (A–E) along the axis to assess spatial distribution. The data were used to analyze maternal RNA localization, transcript stability, and onset of zygotic transcription. Our results reveal spatially distinct transcriptome changes, highlighting both conserved and species-specific mechanisms relative to other teleosts such as zebrafish.

overall_design: To investigate transcriptomic changes during early embryonic development, TOMO-seq was applied to a series of developmental stages: 0 hpf (freshly fertilized egg/zygote), 1 hpf (early cleavage stage), 1 dpf (early blastula stage, ~32-64 cells), and 3 dpf (mid-blastula, ~1000 cells). Biological triplicates were used for each condition. Samples were sectioned along the animal–vegetal axis into five distinct regions, labeled A through E, with region A representing the most animal and region E the most vegetal portion. This approach enabled the analysis of both temporal and spatial transcriptomic dynamics to uncover stage-specific transcriptomic changes occurring during the onset of embryogenesis.

first: Katerina

last: Cihakova

first: Ravindra

last: Naraine

first: Viktoriia

last: Hantzsch

first: Roman

last: Franek

first: Martin

last: Psenicka


title: Lysine lactylation regulates ATF4-mediated stress responses under glucose starvation in canine hemangiosarcoma [CUT&Tag]

city: Sapporo

first: Suzuki

last: Tamami

state: Hokkaido

address: Kita-18-jo, Nishi-9-chome, Kita-ku

department: Department of Clinical Sciences, Faculty of Veterinary

institute: Hokkaido University

summary: Excess lactate is produced in tumor cells by aerobic glycolysis and regulates gene expressions by histone lactylation. However, how histone lactylation functions under glucose-limited conditions remains unknown. Here, we show that lysine lactylation redistributes to transcription start sites (TSSs) during glucose deprivation, thereby altering biological behaviors in canine hemangiosarcoma (HSA) cells. Glucose deprivation significantly decreased global histone lactylation levels, while lactylation peaks were enriched at TSSs of ATF4‑regulated stress‑response, asparagine-synthesis and immune‑related genes. Stress-response gene expressions were upregulated, and ATF4 polyclonal knockout abrogated this activation. [U-13C]glutamine tracing demonstrated that HSA cells synthesized asparagine from glutamine when glucose was scarce, and asparagine supplementation modestly activated cell proliferation. In HSA patient tissues, H3K18la levels were heterogeneous, and M2-like macrophages preferentially infiltrated tumor regions showing low histone lactylation levels. These findings demonstrate that lysine lactylation regulates transcription that supports tumor cell survival and fosters a pro-tumor microenvironment even under glucose-limited conditions.

overall_design: CUT&Tag in canine hemangiosaroma cell lines with or without glucose using pan-Kla, H3K4me3, H3K27ac and RNPII-Ser5 antibodies.

first: Tamami

last: Suzuki

first: Keisuke

last: Aoshima


title: Lysine lactylation regulates ATF4-mediated stress responses under glucose starvation in canine hemangiosarcoma [RNAseq]

overall_design: RNA-seq in canine hemangiosaroma cell lines with or without glucose


title: Spatiotemporal transcriptomic profiling (TOMO-seq) of transcriptomic dynamics during early development of rainbow trout and zebrafish

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

sample_organism:
Danio rerio
Oncorhynchus mykiss

platform_organism:
Danio rerio
Oncorhynchus mykiss

overall_design: Refer to individual Series


title: KDM5 protein mediated cisplatin resistance in ovarian cancer.

first: So Hee

last: Kwon

summary: High recurrence and relapse rates of ovarian cancer (OVC) make it one of the deadliest gynecological cancers and the 5th most common cause of death in women. It is well recognized that acquired resistance is accompanied by abnormal alteration of epigenetic regulation, yet there is a lack of in-depth mechanistic study on cisplatin-resistant OVC in epigenetic context. Herein, we investigate the role of lysine lysine demethylase 5 (KDM5) and how its abnormal ovrexpression correlates with cisplatin resistance and OVC tumor progression.

overall_design: RNA-seq profiling of cisplatin sensitive ovarian cancer cells (SKOV-3) and cisplatin-resistant ovarian cancer cells (SKOV-3 CDDP), including their KDM5A and KDM5B double-knockout derivatives.

first: Jung

last: Yoo

first: Enzhi

last: Luo

first: Junjeong

last: Choi


title: Single cell RNA-seq for AKPS metastatic colon cancer model

first: Robert

last: Piskol

address: 1 DNA Way

department: gRED Computational Sciences

institute: Genentech Inc.

summary: Colorectal cancer, a leading cause of cancer-related mortality due to distant metastases, is largely driven by activating mutations in the WNT and MAPK pathways. Understanding the mechanism underlying the various steps involved in the metastatic process is essential for developing effective treatments. Using serial in vivo orthotopic passaging, we developed an immunocompetent mouse model of metastatic colorectal cancer. We demonstrate that highly metastatic tumor cells exhibit chromosomal amplifications in MAPK pathway genes, leading to increased MAPK activity, which in turn suppresses WNT-associated transcriptional programs, including stem cell genes. Pharmacological inhibition of mutant KRASG12D led to a reduction in the MAPK-high/ WNT-low transcriptional state and effectively decreased metastatic dissemination both to the lung and liver. Analysis of CRC patient data revealed that the metastatic gene signature associated with the MAPK-high/ WNT-low state correlated with poorer survival outcomes. These findings underscore the plasticity of metastasis-initiating cells in CRC that arise due to the opposing roles of MAPK and WNT signaling, despite the apparent synergy of these pathways observed during colon tumorigenesis.

overall_design: We have developed an immuno-competent mouse model of metastatic colorectal cancer by sequentially passaging small intestinal organoids with mutations in Apc, Kras, Tp53 and Smad4 in vivo. Organoids from either Passage 1 (P1, m4) or Passage 5 (P5, m484) were orthotopically injected into the colon of C57BL/6N mice. 5 weeks post orthotopic injection colon tumors were harvested, digested into single cells and live Epcam+ cells sorted. P1 tumors: n=4; P5 tumors: n=4

first: Melanie

last: Heinlein

first: Soufiane

last: Boumahdi

first: Ginny

middle: X

last: Li