title: 14-3-3 proteins GRF6 and GRF8 positively regulate drought tolerance in Arabidopsis thaliana

city: Changchun

first: Liangliang

last: Li

address: 4888 Shengbei Street, Kuancheng District

department: Northeast Institute of Geography and Agroecology

institute: Chinese Academy of Sciences

summary: 14-3-3 proteins constitute a class of highly conserved regulatory proteins present in all eukaryotic cells. In plants, they are involved in diverse biological processes, including responses to abiotic stresses. Here, we investigated the roles of two 14-3-3 proteins, GRF6 and GRF8, in the drought tolerance of Arabidopsis thaliana through comparative transcriptome profiling of wild-type (WT) and grf6 grf8 mutant plants.

overall_design: 12-day-old wild-type (WT) and grf6 grf8 mutant plants were transferred from 1/2 Murashige and Skoog (MS) plates to soil and grown for an additional 12 days. Rosette leaves of these 24-day-old plants were harvested after 0, 2, and 4 hours of dehydration, then immediately frozen in liquid nitrogen. Total RNA was extracted using the TRIzol reagent kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and verified by RNase-free agarose gel electrophoresis. Following total RNA extraction, eukaryotic mRNA was enriched with Oligo (dT) beads. The enriched mRNA was fragmented into short segments using fragmentation buffer and reverse-transcribed into cDNA with the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB#7530, New England Biolabs, Ipswich, MA, USA). Purified double-stranded cDNA fragments underwent end repair, A-tailing, and ligation to Illumina sequencing adapters. The ligation product was purified with AMPure XP Beads (1.0×) and amplified by polymerase chain reaction (PCR). The resulting cDNA library was sequenced on an Illumina NovaSeq 6000 platform.

first: Kaixin

last: Yang

first: Chenbo

last: Zhu

first: Minghui

last: Xing

first: Xiaofan

last: Nie

first: Jiahe

last: Fu

first: Mingming

last: Wang

first: Lam-Son

middle: P

last: Tran

first: Xiaojian

last: Yin

first: Weiqiang


title: Inhibiting LSD1 unlocks retinoid AP-1 programming to activate epithelial immunity and skin tumor suppression [RNAseq_LSD1iRARi]

status: Public on Feb 06 2026

city: Philadelphia

first: Brian

last: Capell

state: PA

address: 421 Curie Blvd

department: Dermatology

country: USA

institute: University of Pennsylvania

type: Expression profiling by high throughput sequencing

summary: Lysine-specific demethylase 1 (LSD1; KDM1A) orchestrates context-dependent chromatin programs, yet its role in epithelial immunity remains largely unknown. Here, we identify LSD1 as a central brake on retinoid- and AP-1–driven enhancer activation in epidermis and demonstrate that its inhibition induces anti-tumor immunity. While epidermal LSD1 is required during development, acute loss or topical inhibition in adult skin is tolerated and triggers coordinated expression of retinoic acid signaling, lipid remodeling, and chemokine induction pathways. CUT&RUN profiling reveals that LSD1 occupies enhancer regions enriched for AP-1 motifs at retinoid metabolism, lipid homeostasis, and immune genes. LSD1 loss increases H3K4me1/2 and gene activation at these sites, licensing a poised AP-1–retinoid program. Single-cell spatial analyses show that discrete keratinocyte subsets initiate retinoid signaling to recruit dendritic cells and activate CD4⁺ T cell responses. Topical LSD1 inhibition suppresses cutaneous squamous cell carcinoma in two models while amplifying keratinocyte–immune crosstalk. Functional perturbations reveal that retinoid signaling partially contributes, whereas CD4⁺ T cells are essential for tumor control. These findings define LSD1 as a master repressor of epithelial immune competence and nominate LSD1 inhibition as a therapeutic strategy to activate retinoid–AP-1 enhancer circuits and drive CD4-dependent tumor immunity in skin cancer.

overall_design: RNA-sequencing of adult mouse dorsal epidermis isolated from DMSO, LSD1i, LSD1i + RARi, or RARi treated mice harvested after 15d of treatment

first: Nina

last: Kuprasertkul

first: Carina

middle: A

last: D'souza

first: Sijia

last: Huang

first: Julia

last: Chini

first: Shaun

last: Egolf

first: Alyssa

middle: F

last: Moore

first: Laura

middle: V

last: Pinheiro

first: Alison

last: Jaccard

first: Claudia

middle: T

last: Magahis

first: Lydia

last: Bao

first: Yann

last: Aubert

first: Cyria

last: Olingou

first: Stephen

middle: M

last: Prouty

first: Donna

last: Brennan-Crispi

first: David

last: Hill

first: John

last: Seykora

first: Kathryn

middle: E

last: Wellen

middle: C


title: Enhancer-Mediated Regulation of HIF-2α by the SOCS3–JAK1–STAT3 Axis in Renal Cancer

city: Boston

first: Jeremy

last: Simon

state: MA

address: 450 Brookline Ave

department: Department of Data Science

institute: Dana-Farber Cancer Institute

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Clear cell renal cell carcinoma (ccRCC) is characterized by frequent loss of the von Hippel–Lindau (VHL) tumor suppressor, leading to pathological accumulation of hypoxia-inducible factor 2α (HIF-2α). Although direct HIF-2α inhibitors such as Belzutifan have shown clinical efficacy, therapeutic resistance remains a major challenge. To uncover additional regulatory mechanisms governing HIF-2α activity, we performed a genome-wide CRISPR screen in VHL-deficient ccRCC cells. This revealed the TREX mRNA export complex as a positive regulator and Suppressor of Cytokine Signaling 3 (SOCS3) as the most significant negative regulator of HIF-2α. We demonstrate that SOCS3 suppresses HIF-2α expression through inhibition of the JAK1–STAT3 signaling axis. Loss of SOCS3 activates STAT3, which directly binds to enhancers upstream of the EPAS1 (HIF-2α) gene, promoting its transcription. SOCS3 overexpression or JAK1/STAT3 inhibition suppresses HIF-2α levels, impairs tumor growth, and reduces metastasis in ccRCC models. These findings define a novel SOCS3–JAK1–STAT3–HIF-2α regulatory axis and identify enhancer-mediated transcriptional control as a critical mechanism of HIF-2α regulation. Targeting this pathway offers a promising strategy to overcome resistance to current HIF-2α inhibitors and improve therapeutic outcomes in ccRCC.

overall_design: ChIP-seq of STAT3 in 786-O cells, and STAT3, H3K4me1, H3K4me3, and H3K27ac in HKC8 cells, in biological duplicate with input and IgG controls. H3K27ac HiChIP in 786-O with two technical replicates pooled during analysis

first: Jun

last: Fang

first: Qing

last: Zhang


title: Single cell RNAseq on rheumatoid arthritis-related interstitial lung disease

city: Los Angeles

first: Kensuke

last: Suga

state: California

address: 8700 Beverly Blvd

department: Department of Rheumatology

institute: Kao Autoimmunity Institute

summary: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovitis and/or joint deterioration. Interstitial lung disease (ILD) is one of extra-articular manifestations of RA characterized by inflammation and/or fibrosis. Clinically relevant rheumatoid arthritis-related interstitial lung disease (RA-ILD) occurs in 10% of RA patients and is associated with poor prognosis. Although a poor prognosis has been reported, little is known about RA-ILD. Here, we report the first detailed transcriptomic analysis of the epithelial and immune cell populations in RA-ILD lungs at the single-cell level.

overall_design: We performed single-cell RNA sequencing on FACS-sorted epithelial cells (CD45-CD31-EPCAM+) and immune cells (CD45+CD31-EPCAM-) from lung explants of four controls, three Non-RA Connective tissue disease (CTD)-ILD patients, and five RA-ILD patients.

first: Nunzio

last: Bottini


title: Weaning-Driven Gut Microbiome Shapes Intestinal Stem Cell Epigenetics to Train Immunological Memory (WGS)

city: Houston

first: Lanlan

last: Shen

state: Texas

address: 1100 Bates Ave

department: Pediatrics

institute: Baylor College of Medicine

summary: During weaning, the shift from milk to solid food is accompanied by a rapid expansion and diversification of the gut microbiome. This triggers the weaning reaction, a programmed immune response important for long-lasting mucosal immunity that is poorly understood. Here, we characterize the impact of the gut microbiome on the DNA methylome and transcriptome of intestinal stem cells (ISCs) from weaning to young adulthood in mice. We identify a weaning- IFN-g-TET3 axis that reprograms epithelial MHC-II signaling via enhancer demethylation in ISCs, establishing a lasting transcriptional memory into adulthood. Disruption of this axis by IFN-g blockade or low-dose penicillin attenuates the microbiota-mediated epigenetic control of epithelial MHC-II signaling, leading to impaired mucosal immunity. By engaging immune crosstalk and metabolite production, the weaning microbiome drives epigenetic reprogramming, thereby establishing a microbiome-induced regulatory pathway that couples developmental cues to lifelong epithelial immune resilience.

overall_design: Metagenomic shotgun sequencing was performed on mice housed under specific pathogen-free (SPF) or low-dose penicillin (LDP) conditions from 2 to 12 weeks of age. In addition, gut microbiota transfer (GMT) experiments were conducted at various time windows, including 3–15 weeks, 8–15 weeks, 12–15 weeks, 12–20 weeks, as well as from SPF donors at 15 and 20 weeks of age.

first: Li

last: Yang


title: Ketone body β-hydroxybutyrate restores Tau proteostasis via metabolism-independent mechanism

city: La Jolla

first: Leigh-Ana

last: Rossitto

state: CA

address: 9500 Gilman Drive

department: Neurosciences

institute: UC San Diego

summary: Alzheimer’s disease (AD) and related tauopathies are marked by brain accumulation of pathological Tau aggregates and associated metabolic dysfunction. Metabolic interventions including ketogenic diets, caloric restriction, and exercise have shown promise in ameliorating cognitive impairments in AD, but variable efficacy and issues of compliance and side effects mar their translational potential. A shared molecular feature between these interventions is the induction of ketosis, a metabolic state marked by elevated circulating levels of β-hydroxybutyrate (βHB). Previous studies show βHB administration directly or in the form of a precursor can reproduce and even improve upon the benefits of metabolic interventions, but the molecular mechanism underlying the neuroprotective properties of βHB in AD remains to be elucidated. In the current study, we find that a βHB precursor diet is sufficient to ameliorate Tau pathophysiology in the PS19 mouse model at both the pathological and single-cell levels. This GEO record is for the hippocampal single nucleus RNA sequencing data generated for our diet paradigm.

overall_design: Male mice, either PS19 hTau+ or WT littermate controls, were switched from standard vivarium chow to either a ketone ester diet (KE) or a macro-controlled diet (CTL) at six months of age until about 9.5 months of age. At this time, mice were perfused with cold PBS, and hippocampi were dissected on ice, flash frozen, and stored at -80C until nuclei extraction. Three mice per group were included in this sequencing experiment for a total of 12 samples.

first: Jamie

last: Foo

first: Julia

last: Di Silvestri

first: Leanne

last: Lehmann

first: Melina

last: Brunelli

first: Yuhang

last: Nie

first: Daniel

last: Muñoz-Mayorga

first: Suborno

last: Jati

first: Qiang

last: Xiao

first: Anna

last: Dai-Liu

first: Meghan

last: Rossi

first: Richard

last: Daneman

first: Jeffery

middle: W

last: Kelly

last: Newman

first: Samuel

last: Myers

first: Xu

last: Chen


title: Inhibiting LSD1 unlocks retinoid AP-1 programming to activate epithelial immunity and skin tumor suppression [CutRun_LSD1i48h]

first: Brian

last: Capell

overall_design: Cut&Run for H3K4me2 of adult mouse dorsal epidermis isolated from H2O or LSD1i treated mice harvested after 48h of treatment

first: Nina

last: Kuprasertkul

first: Carina

last: D'souza

first: Sijia

last: Huang

last: Chini

first: Shaun

last: Egolf

first: Alyssa

last: Moore

first: Laura

last: Pinheiro

first: Alison

last: Jaccard

first: Claudia

last: Magahis

first: Lydia

last: Bao

first: Yann

last: Aubert

first: Cyria

last: Olingou

first: Stephen

last: Prouty

first: Donna

last: Brennan-Crispi

first: David

last: Hill

first: John

last: Seykora

first: Kathryn

last: Wellen


title: Neutrophils and CD8+ T cells in Muscle-invasive Bladder Cancer (MIBC)

middle: Qi Rong

last: Wong

address: 6670 Bertner Ave

institute: Houston Methodist

summary:
Biopsies in FFPE blocks from MIBC patients who responded (Responded) or did not respond (Non-Responder) to gemcitabine-cisplatin neoadjuvant chemotherapy were subject to Xenium spatial transcriptomics analysis.
While local immunological effects of chemotherapy-induced tumor cell death are well studied, its systemic impact on distant bone marrow—a site essential for immune cell maturation—remains underexplored. Here, we show that gemcitabine chemotherapy induces inflammatory caspase-1 activation in epithelial cancer cells (epiCaspase-1), triggering pyroptotic cell death. Despite its inflammatory nature, epiCaspase-1 mediated cell death is non-immunogenic. Clinically, cancer patients with high expression of a newly derived epiCaspase-1 gene signature show poorer outcomes. Mechanistically, epiCaspase-1 induces noncanonical release of IL-1α through NINJ1 lytic pores, which remotely skews bone marrow hematopoiesis towards granulocyte-monocyte progenitors and mature neutrophil output. This systemic alteration leads to a heightened neutrophil-to-lymphocyte ratio (NLR) in both peripheral blood and the tumor microenvironment. Notably, pharmaceutic inhibition of caspase-1 blocks this cascade, normalizes hematopoiesis, and recalibrates NLR to favor intratumoral CD8+ T cell infiltration and activation—ultimately improving chemotherapeutic efficacy. These findings underscore that inflammatory pyroptosis is not inherently immunogenic, instead, it reshapes systemic immune landscape towards neutrophil-dominant inflammation in the chemotherapy context.

overall_design: Patient FFPE blocks were sectioned and processed for downstream Xenium spatial transcriptomics according to manufacturer's protocol.

first: Yung Hsing

middle: Q

first: Ethan

middle: J

last: Subel

first: Hongbo

last: Gao

first: Fotis

last: Nikolos

first: Keith

middle: S

last: Chan


title: Inhibiting LSD1 unlocks retinoid AP-1 programming to activate epithelial immunity and skin tumor suppression [Xenium tumor]

first: Brian

last: Capell

overall_design: Xenium spatial transcriptomics of tumors from Fyn mouse model treated with either DMSO or LSD1i for 15d

first: Nina

last: Kuprasertkul

first: Carina

last: D'souza

first: Sijia

last: Huang

first: Julia

last: Chini

first: Shaun

last: Egolf

first: Alyssa

last: Moore


title: Inhibiting LSD1 unlocks retinoid AP-1 programming to activate epithelial immunity and skin tumor suppression [Xenium CreERT]

overall_design: Xenium spatial transcriptomics of adult mouse dorsal whole skin from K14 Cre-ERT WT/Control or K14 Cre-ERT Lsd1 KO mice harvested 6 weeks post tamoxifen induction (ie 13-14 wks of age)

first: Laura

last: Pinheiro

first: Alison

last: Jaccard

first: Claudia

last: Magahis

first: Lydia

last: Bao

first: Yann

last: Aubert

first: Cyria

last: Olingou

first: Stephen

last: Prouty


title: Inhibiting LSD1 unlocks retinoid AP-1 programming to activate epithelial immunity and skin tumor suppression

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

overall_design: Refer to individual Series


title: Rewiring miR-22/SNAI1 via CRISPR-Based Edge Editing Destabilizes the Epithelial Phenotype

city: Richardson

first: John

middle: Tran

last: Nguyen

state: TX

address: 800 W Campbell Rd

department: Bioengineering

institute: The University of Texas at Dallas

summary: Epithelial-to-Mesenchymal Transition (EMT) is a critical biological process by which cells acquire enhanced migratory and invasive properties. A key signaling pathway involved in EMT phenotypes includes transforming growth factor β (TGFβ) and transcription factors (TFs) such as Snail, Zeb, and Twist. Additionally, microRNAs (miRNAs) – small, non-coding molecules that regulate gene expression by targeting mRNA transcripts – directly regulate genes central to the EMT process. Notably, miR-22 has been identified as a significant regulator of EMT through direct inhibition of EMT drivers like SNAI1 and indirect regulation of upstream genes. In this study, we performed CRISPR-based network rewiring by selectively removing an edge—the connection between two nodes—to investigate its impact on EMT dynamics. Specifically, we disrupted the connection between miR-22 and Snail1 without affecting other interactions involving miR-22 or Snail1 and examined the resulting effects on EMT. We demonstrate that the removal of the miR-22 target site from the SNAI1 gene renders cells more sensitive to TGFβ-mediated EMT. This finding highlights the critical importance of the direct regulatory connection between miR-22 and SNAI1 in modulating EMT, distinct from miR-22's effects on other targets or indirect pathways. More generally, our results underscore the importance of CRISPR-mediated edge ablation for exploring the interactions that govern biological networks and highlight an underexplored opportunity to develop edge-based therapeutic modalities.

overall_design: RNA-seq profiling of wildtype A549 cells and the miR-22/SNAI1 edge knockout derivative cultured in the precense or absence of EMT induction agent TGFβ (at 10 ng/mL) for 5 days.

first: Lijia

first: Herbert

last: Levine

first: Mingyang

last: Lu

first: Leonidas

last: Bleris


title: Galns-deficiency enhances lysosomal exocytosis and alters cartilage development in zebrafish

city: Greenwood

first: Heather

last: Flanagan-Steet

state: South Carolina

address: 113 Gregor Mendel Cir.

department: J.C. Self Research Institute

institute: Greenwood Genetic Center

summary: Lysosomes represent a heterogenous population of organelles whose optimal function is essential for early tissue development. The importance of this is evidenced by the number of inherited disorders, collectively called the lysosomal storage disorders (LSDs), caused by lysosomal dysfunction. While it is clear that macromolecular accumulation can adversely impact tissue development, the full breadth of downstream pathways driving pathology in these disorders is yet to be elucidated. Recent studies support the existence of mechanisms beyond lysosomal storage that profoundly influence early tissue formation. Among these, impaired mitochondrial function, oxidative stress, inflammation, and abnormal growth factor signaling have been linked to early pathology in multiple LSD. Studies in zebrafish models of mucolipidosis II and sialidosis identified a mechanism whereby inappropriate secretion of lysosomal cathepsins disrupts the TGF-ß related signaling pathways that control skeletal formation. Here we demonstrate that loss of galns (N-acetyl galactosamine-6-sulfatase) also enhances lysosomal exocytosis in developing cartilage of mutant zebrafish. Along with changes in the activity of several cathepsin proteases, enhanced exocytosis disrupts TGFß and BMP signaling altering cartilage and bone development in galnsm/m mutant larvae. Together these data suggest a role for altered growth factor signaling in MPSIVA skeletal pathology.

overall_design: RNAseq profiling of homozygous galns mutants (galns m/m) compared to wild type laboratory strain (TLAB) zebrafish at 8 dpf.

first: Jen-Jie

last: Lee

first: Maxwell

middle: B

last: Colonna

first: Richard

last: Steet


title: Crip2 preserves hematopoietic stem and progenitor cell production through inhibition of Notch signals

city: Trieste

first: UDAY

last: RANGASWAMY

state: Trieste

address: via bonomea 265

department: NEUROSCIENCE

country: Italy

institute: SISSA

summary: To decipher the mechanisms underlying the cripDM phenotype, single cell RNA sequencing was performed on sorted, kdrl:mCherry+ cells at 30 hpf. The analysis revealed an upregulation of genes essential for vascular development.

overall_design: 10X single-cell RNA sequencing from sorted mCherry+ endothelial cells from whole zebrafish embryos at 30 hours post fertilization comparing wild-type and crip mutant samples.

first: Angelika

middle: G

last: Aleman

first: Uday

last: Rangaswamy

first: Remo

last: Sanges

first: Kimara

middle: L

last: Targoff


title: Inhibiting LSD1 unlocks retinoid AP-1 programming to activate epithelial immunity and skin tumor suppression [CreERTRNAseq]

first: Brian

last: Capell

overall_design: RNA-sequencing of adult mouse dorsal epidermis isolated from K14 Cre-ERT WT/Control or K14 Cre-ERT Lsd1 KO mice harvested 6 weeks post tamoxifen induction (ie 13-14 wks of age)

first: Nina

last: Kuprasertkul

first: Carina

last: D'souza

first: Sijia

last: Huang

first: Julia

last: Chini

first: Shaun

last: Egolf

first: Alyssa

last: Moore

first: Laura

last: Pinheiro

first: Alison

last: Jaccard

first: Claudia

last: Magahis

first: Lydia

last: Bao

first: Yann

first: Donna

last: Brennan-Crispi

first: David

last: Hill

last: Seykora

first: Kathryn

last: Wellen


title: Inhibiting LSD1 unlocks retinoid AP-1 programming to activate epithelial immunity and skin tumor suppression [CreERTCutRun]

overall_design: Cut&Run for H3K4me2 of adult mouse dorsal epidermis isolated from K14 Cre-ERT WT/Control or K14 Cre-ERT Lsd1 KO mice harvested 6 weeks post tamoxifen induction (ie 13-14 wks of age)

last: Aubert

first: Cyria

last: Olingou

first: Stephen

last: Prouty

first: John


title: Uptake of polystyrene nanoplastics by cumulus-oocyte complexes impairs oocyte and embryo development

city: Utrecht

first: Jorke

last: Kamstra

state: Non United States Or Canada

address: PO Box 80177

department: Faculty of Veterinary Medicine

country: Netherlands

institute: Utrecht University

summary: Nanoplastics (NPs), plastic particles smaller than 1 μm, have gained increasing attention due to their potential health effects. Experimental studies in animals have shown that NPs can pass biological barriers, reach the female reproductive tract, and enter the ovary. Their presence in the ovary might have profound consequences for reproductive endpoints, such as oocyte development. This study aimed to investigate the uptake and associated effects of polystyrene (PS) NPs on oocyte and embryo development using a bovine in vitro fertilization (IVF) model. Bovine cumulus-oocyte complexes (COCs) were exposed to pristine PS-NPs (50 nm or 200 nm) at different doses during in vitro maturation (23 hours), followed by IVF and embryo culture. RNA sequencing was performed to investigate mechanisms involved in possible effects on oocyte maturation. Confocal microscopy confirmed the internalization of 50 nm and 200 nm PS-NPs into cumulus cells, however, only 50 nm particles were able to transfer into oocytes. Additionally, exposure to 50 nm PS-NPs at 3 μg/mL significantly delayed oocyte nuclear maturation and early embryo development. At this dose, RNA sequencing did not identify differentially expressed genes in the COCs, however, gene set enrichment analyses did indicate effects on mitochondrial processes. Nevertheless, subsequent analysis on mitochondrial activity remained unaffected.

overall_design: IVM was performed using groups of 50 bovine cumulus-oocyte complexes (COCs)with or without the presence of 50 nm polystyrene nanoplastics (3 μg/mL). Experiments were performed with 6 independent replicate samples, derived from 3 batches of bovine material. Replicates within each batch were prepared independently from one another.

first: Jiayi

last: Yang

middle: H

first: Richard

last: Wubbolts

first: Kaylee

last: Nieuwland

first: Nikita

last: Korpel

first: Juliette

last: Legler

first: Hilde

last: Aardema


title: Inhibiting LSD1 unlocks retinoid AP-1 programming to activate epithelial immunity and skin tumor suppression [Xenium embryo]

first: Brian

last: Capell

overall_design: Xenium spatial transcriptomics of whole skin or whole mount embryos from constitutive Krt14Cre LSD1 KO mouse model harvested at E17.5

first: Nina

last: Kuprasertkul

first: Carina

middle: A

last: D'souza

first: Sijia

last: Huang

first: Julia

last: Chini

first: Shaun

last: Egolf

first: Alyssa

last: Moore

first: Laura

last: Pinheiro

first: Alison

last: Jaccard


title: Extracellular Small RNAs in Breast Milk: Molecular Profiles and Biological Implications

city: Wilmington

first: Uciel

last: Chorostecki

state: Delaware

address: 251 Little Falls Drive

institute: Kresko RNA Tech

type: Non-coding RNA profiling by high throughput sequencing

summary: Small RNA sequencing of human and bovine milk revealed abundant tRNA-, yRNA-, and rRNA-derived fragments. These exRNAs were validated and shown to promote cell survival under stress, suggesting a functional role in milk’s biological effects.

sample_organism:
Homo sapiens
Bos taurus

platform_organism:
Homo sapiens
Bos taurus

overall_design: Skim milk samples were collected from healthy humans (n=3) and cows (n=3). RNA was extracted and sequenced with Illumina TruSeq Small RNA libraries on a NovaSeq. Reads were quality filtered, aligned to curated sRNA references, and analyzed with custom scripts.

first: Clara

last: Claus

first: Carla

last: Borini Etichetti

first: Bruno

last: Costa

first: Julieta

last: B. Grosso

first: Juan P

last: Tosar

first: Silvana V

last: Spinelli


title: Impairment of hippocampal gamma oscillations, mitochondria and neurovascular function in CADASIL

city: Stockholm

first: Michael

last: Vanlandewijck

address: Blickagången 6

department: Department of Medicine

country: Sweden

institute: Karolinska Institute

summary: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a small vessel disease caused by NOTCH3 gene mutations, leading to vascular smooth muscle cell degeneration, arteriopathy, and subcortical ischemic infarcts. Many CADASIL patients, however, also develop cognitive impairment, indicating that neuronal functions are disturbed, but less is known about the cellular and molecular basis of these aspects in CADASIL. In this study, we used a humanized CADASIL mouse model harbouring the R182C mutation (R182C-TgN3), post-mortem human CADASIL brain sections with three different NOTCH3 gene mutations and primary human vascular smooth muscle cells harbouring the R133C NOTCH3 mutation as primary cellular models to characterise the neurovascular contribution to cognitive impairment. To specifically evaluate neuronal, mitochondrial and neurovascular function, we performed ex vivo electrophysiology, immunohistochemistry (confocal and iDISCO+ methods), western blotting, Seahorse assay, quantitative polymerase chain reaction (qPCR), and single-cell RNA sequencing. In CADASIL mice, hippocampal gamma oscillation patterns were impaired along with significant decreases in neuronal fiber length and aberrant neuronal morphology. The latter two phenotypes were also observed in post-mortem human brain tissue from CADASIL patients. Consistent with these findings, we noted significantly lower levels of mitochondrial respiratory complexes in the CADASIL mouse hippocampus, isolated mouse brain vessels and primary human vascular smooth muscle cells. Human vascular smooth muscle cells exhibited reduced oxygen consumption rates leading to reduced ATP production as well as decreased glycolytic capacity in conjunction with increased pro-inflammatory gene expression, suggesting a broader impact on cellular energy metabolism and a neuroinflammatory process. In the CADASIL mice, we also observed extensive accumulation of the NOTCH3 extracellular domain on hippocampal vessels. Light sheet imaging with iDISCO+ clearing demonstrated substantial vascular smooth muscle cell loss and reduced vessel density in the hippocampus at 9 months of age. Additionally, 3D imaging showed increased microglial attachment to vessels and enlargement of the size of the vessel-associated microglia in CADASIL mice. Single-cell RNA sequencing revealed a microglial subcluster expressing genes involved in mitochondria respiration and inflammation. Collectively, our results reveal that exacerbated vascular network damage may mediate cognitive decline observed in the later stages of CADASIL and highlight the critical role of the neurovascular unit. Our findings provide valuable insights into the underlying mechanisms of neuronal dysfunction and pave the way for future research and potential therapeutic strategies.

overall_design: Using a microvascular isolation protocol (PMID 37056377), single cell suspensions of adult murine brain vasculature were extracted. The following mouse models were used: A CADASIL mouse model (Tg-N3-R182C) and a matched control mouse (Tg-N3)

first: Wenchao

last: Shao

first: Daniel

last: Oliveira

first: Luana

last: Naia

first: Yue

last: Li

first: Katrine

last: Dahl Bjørnholm

first: Arturo

last: Isla

first: Per

last: Uhlén

first: Raj

last: Kalaria

first: Saskia

last: Lesnik Oberstein

first: Urban

last: Lendahl

first: Luis

last: Enrique Arroyo-García

first: Chao-bo

last: Jin

first: Helena

last: Karlström


title: Inflammatory epiCaspase-1 dampens immunogenic cell death by remotely skewing bone marrow hematopoiesis to drive systemic neutrophil-dominant inflammation

first: Stephen

last: Wong

summary: While local immunological effects of chemotherapy-induced tumor cell death are well studied, its systemic impact on distant bone marrow—a site essential for immune cell maturation—remains underexplored. Here, we show that gemcitabine chemotherapy induces inflammatory caspase-1 activation in epithelial cancer cells (epiCaspase-1), triggering pyroptotic cell death. Despite its inflammatory nature, epiCaspase-1 mediated cell death is non-immunogenic. Clinically, cancer patients with high expression of a newly derived epiCaspase-1 gene signature show poorer outcomes. Mechanistically, epiCaspase-1 induces noncanonical release of IL-1α through NINJ1 lytic pores, which remotely skews bone marrow hematopoiesis towards granulocyte-monocyte progenitors and mature neutrophil output. This systemic alteration leads to a heightened neutrophil-to-lymphocyte ratio (NLR) in both peripheral blood and the tumor microenvironment. Notably, pharmaceutic inhibition of caspase-1 blocks this cascade, normalizes hematopoiesis, and recalibrates NLR to favor intratumoral CD8+ T cell infiltration and activation—ultimately improving chemotherapeutic efficacy. These findings underscore that inflammatory pyroptosis is not inherently immunogenic, instead, it reshapes systemic immune landscape towards neutrophil-dominant inflammation in the chemotherapy context.

overall_design: Mouse hematopoetic progenitor cells were isolated by immunomagnetic separation from bone marrow preparations of different treatment groups

first: Fotis

last: Nikolos

first: Kazukuni

last: Hayashi

first: Keith

last: Chan


title: Correcting open chromatin bias in bulk and single-cell CUT&Tag data with PATTY [CUT&Tag]

city: Charlottesville

first: Chongzhi

last: Zang

state: VA

address: 1300 Jefferson Park Avenue

department: Genome Sciences

institute: University of Virginia

summary: Precise profiling of epigenomes, including histone modifications and transcription factor binding sites, is essential for better understanding gene regulatory mechanisms. Cleavage Under Targets & Tagmentation (CUT&Tag) is an easy and low-cost epigenomic profiling method that can be performed on a low number of cells and at the single-cell level. A large number of CUT&Tag datasets have been generated in various biological systems, providing a valuable resource. CUT&Tag experiments use the hyperactive transposase Tn5 for tagmentation. We found that the preference of Tn5 captured reads toward accessible chromatin regions can influence the distribution of CUT&Tag reads and cause open chromatin biases, further confounding the analysis of CUT&Tag data. The high sparsity of single-cell sequencing data makes the open chromatin biases more substantial than in bulk sequencing data. Here, we present a comprehensive computational method, PATTY (Propensity Analyzer for Tn5 Transposase Yielded bias), to mitigate the open chromatin bias inherent in CUT&Tag data at both bulk and single-cell levels. By integrating existing transcriptome and epigenome data using machine learning and comprehensive modeling, we demonstrate that PATTY yields more accurate and robust detection of occupancy sites for both active and repressive histone marks than existing methods, with experimental validation. We further designed a single-cell CUT&Tag analysis framework by utilizing this model and showing improved cell clustering from bias-corrected single-cell CUT&Tag data compared to using raw data. This model paved the way for further development of computational tools for improving bulk and single-cell CUT&Tag data analysis.

overall_design: To validate the performance of PATTY on H3K9me3 CUT&Tag data, we performed H3K9me3 CUT&Tag in wild-type HCT116 cells.

first: Shengen

last: Hu

first: Zhangli

last: Su

first: Anindya

last: Dutta


title: Protein kinase CK2α’ as a Dual Modulator of Immune Signaling and Synaptic Dysfunction in Tauopathy

city: Minneapolis

first: Ying

last: Zhang

state: MN

address: 117 Pleasant St S.E.

department: Minnesota Supercomputing Institute

institute: University of Minnesota

summary: Protein kinase CK2α’ is upregulated in patients with tau-associated dementias and in the PS19 mouse model of tauopathy. CK2α’ haploinsufficiency in the PS19 mouse model improved tau pathology, synaptic density, synaptic function, cognitive behavior and disease-associated microglia phenotypes. We hypothesized CK2α’  haploinsufficiency would impact dysregulated gene expression in the PS19 model.  We found that CK2α’  haploinsufficiency led to enhanced synaptic gene network expression and enhance immune gene network expression.

overall_design: RNA-Seq analysis of samples obtained from the hippocampus of ~7 month-old mice using: 4 Wildtype, 5 CK2α’(+/-), 3 high-pathology PS19, 4 low-pathology PS19 and 4 PS19;CK2α’(+/-) mice. Mice are on a mixed C57Bl/6 x B6C3H background. Animals were anesthetized with Avertin and intracardially perfused with Heparin in Tris-buffered Saline. Brains were dissected and hippocampus samples were flash frozen in liquid nitrogen prior to RNA extraction.

first: Angel

last: White

first: Rocio

last: Gomez-Pastor


title: Single-Cell Multimodal Profiling Highlights Persistent Aortic Smooth Muscle Cell Changes in Diabetic Mice Despite Glycemic Control

city: Duarte

first: Rama

last: Natarajan

address: 1500 E Duarte Rd

department: Department of Diabetes Complications and Metabolism

institute: Beckman Research Institute, City of Hope

summary: Type 2 diabetes (T2D) is associated with accelerated vascular complications like hypertension and atherosclerosis. “Phenotypic switching” of vascular smooth muscle cells (SMC), a major driver of these complications, is enhanced in diabetes. Despite adequate glycemic control, SMC dysfunction can persist due to “metabolic memory” of prior hyperglycemia. However, the mechanisms of hyperglycemic memory associated with persistent SMC dysfunction are unclear. Here, leveraging single-cell (sc) multi-omics, we examined the effect of glucose normalization on transcriptomic and epigenomic changes associated with SMC phenotypic transition in T2D mice. We treated T2D db/db mice with the antidiabetic drug dapagliflozin (DAPA) (db/dbDAPA) or vehicle (db/db), and non-diabetic control db/+ mice with vehicle for 6 weeks. Dissected aortas were subjected to scRNA-seq, scATAC-seq, and spatial transcriptomics (Xenium) to determine single-cell changes in gene expression and chromatin accessibility. DAPA treatment conferred effective glycemic control in db/db mice, with significant reduction in blood glucose/hemoglobin A1c. scRNA and scATAC-seq analysis of aortas identified major cell populations, including SMC, fibroblasts, endothelial and immune cells. SMC were further clustered into 9 subtypes, including contractile and fibromyocyte-like cells. Cell composition analysis revealed decreases in contractile SMC and increases in vascular remodeling associated fibromyocyte-like SMC in db/db versus db/+ mice. Interestingly, DAPA did not reverse diabetes-induced decreases in contractile markers but reversed changes in several fibromyocyte markers in db/db mice. Pseudotime trajectory analysis revealed increased activities of fibromyocyte enriched transcription factors (TFs) during contractile to fibromyocyte transition. Furthermore, increased expression of TFs regulating fibromyocyte phenotype (e.g. Atf4, Bach1, Hand2, Fosl2) in db/db were partially reversed by DAPA, whereas reduced contractile TF (Mef2c) expression was unchanged. Spatial transcriptomics analysis further mapped aortic cell types within intact aortas and confirmed that DAPA reversed alterations in key fibromyocyte but not contractile genes in db/db mouse aortas.   Persistent epigenetic changes may contribute to sustained vascular remodeling and dysfunction in T2D. T2D reduced contractile SMC gene expression and related chromatin accessibility and promoted phenotypic transition to fibromyocytes. These changes are only partially reversed by a widely used anti-diabetic drug like DAPA, underscoring the need for more effective therapies that target hyperglycemic memory.

overall_design: We performed Xenium spatial transcriptomics on aorta from db/db and db/db mice treated with DAPA for 6 weeks (db/dbDAPA).

first: Vinay

last: Tanwar

first: Vajir

last: Malek


title: Inflammatory epiCaspase-1 dampens immunogenic cell death by remotely skewing bone marrow hematopoiesis to drive systemic neutrophil-dominant inflammation [T24 RNAseq]

overall_design: T24 human bladder cancer cell lines were treated with LPS and Nigericin, and then subjected to RNA sequencing.

first: Ethan

last: Subel

first: Yung Hsing

last: Huang


title: PAN2 Maintains mRNA PolyA Tail Homeostasis and Regulates Translation during Spermiogenesis in Mouse

city: Hangzhou

first: Heng-yu

last: Fan

state: Zhejiang

address: Yuhangtang Road

country: China

institute: Life Sciences  Institute

summary: Elongation and shortening of the polyA tail are the most common type of post-transcriptional mRNA regulation in eukaryotic cells. Two protein complexes, PAN2-PAN3 and CCR4-NOT, are major cytoplasmic deadenylases that trim the mRNA polyA tails. While PAN2-PAN3 removes the distal portion of the long polyA tails, CCR4-NOT removes adenosine closer to the 3'-UTR. Some subunits in the CCR4-NOT complex have been shown to play synergistic biochemical functions during mouse germ cell development. However, the in vivo functions of the upstream deadenylase PAN2-PAN3 remain unclear, particularly in mammals. In this study, we found that germline-specific deletion of Pan2 leads to male infertility. Pan2 knockout did not affect the prophase of meiosis I, but led to abnormal spermiogenesis and apoptosis of male germ-cells. By Palso-seq and Ribo-lite, we found that the deletion of Pan2 increased the number of long-tail mRNAs in spermiogenesis and disrupted the translation efficiency of genes that should be highly translated at this stage. These findings highlight the crucial physiological functions of PAN2 in gametogenesis and expand our understanding of the posttranscriptional regulation of mRNAs in specific physiological processes.

overall_design: MII stage spermatocytes and round spermatids form Pan2flox/-;Stra8-Cre mice were subjected to RNA-seq using the Smart-seq2 method with minor modifications.

first: Xuan

last: Wu


title: SETD2 methyltransferase activity promotes correct transcription initiation and termination

city: Fukuoka

first: Takayuki

last: Nojima

state: Japan

address: Maidashi

department: Medical Sciences

country: Japan

institute: Kyushu University

summary:
SETD2 is a methyltransferase responsible for depositing histone H3 lysine 36 trimethylation (H3K36me3). Loss of its enzymatic activity occurs in some cancers, including renal cell carcinoma. SETD2 mutations have been linked to delayed transcription termination, but have not been explored in depth. Here, using nascent transcriptomics in SETD2 knockout and patient-derived cells, we reveal a dichotomy in SETD2 functions depending on the affected protein-coding gene. The majority of genes, named class I, are dependent on SETD2 function for transcription initiation, yet terminate transcription in the usual locations. In contrast, for class II genes, corresponding to 15-25% of active protein-coding genes, transcription initiation is robust in absence of SETD2 activity; however, widespread transcriptional readthrough occurs. Defective termination following SETD2 loss/mutation is associated with increased cryptic transcription initiation and impaired 3′ pre-mRNA cleavage. Additionally, alternative polyadenylation upon SETD2 activity loss is highly cell type specific, and no relationship with transcription readthrough was observed. We demonstrate that methyltransferase activity of SETD2 stimulates proper initiation, prevents cryptic initiation and promotes efficient 3′ end processing, however it does so indirectly.

This SuperSeries is composed of the SubSeries listed below.

first: Magda

last: Kopczyńska

first: Chihiro

last: Nakayama

first: Agata

last: Stępień

first: Shoko

last: Ito

first: Koshi

last: Imami

first: Michal

last: Gdula

first: Kinga

last: Kamieniarz-Gdula


title: Single-Cell Multimodal Profiling Highlights Persistent Aortic Smooth Muscle Cell Changes in Diabetic Mice Despite Glycemic Control [scATAC-seq]

summary: Type 2 diabetes (T2D) is associated with accelerated vascular complications like hypertension and atherosclerosis. “Phenotypic switching” of vascular smooth muscle cells (SMC), a major driver of these complications, is enhanced in diabetes. Despite adequate glycemic control, SMC dysfunction can persist due to “metabolic memory” of prior hyperglycemia. However, the mechanisms of hyperglycemic memory associated with persistent SMC dysfunction are unclear. Here, leveraging single-cell (sc) multi-omics, we examined the effect of glucose normalization on transcriptomic and epigenomic changes associated with SMC phenotypic transition in T2D mice. We treated T2D db/db mice with the antidiabetic drug dapagliflozin (DAPA) (db/dbDAPA) or vehicle (db/db), and non-diabetic control db/+ mice with vehicle for 6 weeks. Dissected aortas were subjected to scRNA-seq, scATAC-seq, and spatial transcriptomics (Xenium) to determine single-cell changes in gene expression and chromatin accessibility. DAPA treatment conferred effective glycemic control in db/db mice, with significant reduction in blood glucose/hemoglobin A1c. scRNA and scATAC-seq analysis of aortas identified major cell populations, including SMC, fibroblasts, endothelial and immune cells. SMC were further clustered into 9 subtypes, including contractile and fibromyocyte-like cells. Cell composition analysis revealed decreases in contractile SMC and increases in vascular remodeling associated fibromyocyte-like SMC in db/db versus db/+ mice. Interestingly, DAPA did not reverse diabetes-induced decreases in contractile markers but reversed changes in several fibromyocyte markers in db/db mice. Pseudotime trajectory analysis revealed increased activities of fibromyocyte enriched transcription factors (TFs) during contractile to fibromyocyte transition. Furthermore, increased expression of TFs regulating fibromyocyte phenotype (e.g. Atf4, Bach1, Hand2, Fosl2) in db/db were partially reversed by DAPA, whereas reduced contractile TF (Mef2c) expression was unchanged. Spatial transcriptomics analysis further mapped aortic cell types within intact aortas and confirmed that DAPA reversed alterations in key fibromyocyte but not contractile genes in db/db mouse aortas. Persistent epigenetic changes may contribute to sustained vascular remodeling and dysfunction in T2D. T2D reduced contractile SMC gene expression and related chromatin accessibility and promoted phenotypic transition to fibromyocytes. These changes are only partially reversed by a widely used anti-diabetic drug like DAPA, underscoring the need for more effective therapies that target hyperglycemic memory.

overall_design: We performed scATAC-seq on whole aorta from db/+, db/db and db/db mice tretated with DAPA for 6 weeks (db/dbDAPA).

first: Jingyi

last: Wang

first: Clint

last: Miller


title: Single-Cell Multimodal Profiling Highlights Persistent Aortic Smooth Muscle Cell Changes in Diabetic Mice Despite Glycemic Control [scRNA-seq]

first: Rama

last: Natarajan

overall_design: We performed scRNA-seq on whole aorta from db/+, db/db and db/db mice tretated with DAPA for 6 weeks (db/dbDAPA).


title: Transcriptome analysis of HCT116 cells subjected to low glucose or depleted of MAPKAPK2 expression.

city: Omaha

first: Brian

last: North

state: NE

address: 2500 California Plaza

department: Biomedical Science

institute: Creighton University School of Medicine

summary: MAPKAPK2, a component of p38 signaling pathway, is upregulated in colorectal cancer (CRC) and is known to regulate glucose metabolism. Utilizing HCT116, a CRC cell line, we identified that cancer cells can withstand low glucose conditions via upregulation of stress-induced survival pathways and downregulation of DNA metabolism and cell cycle related genes. As p38/MAPKAPK2 signaling is a well-known stress activated pathway, understanding the role played by this pathway in glucose-deprivation conditions was of particular interest. Interestingly, MAPKAPK2 levels decline in low glucose. We observed that MAPKAPK2 depletion results in upregulation or downregulation of pathways similar to glucose deprivation. Our findings suggest that MAPKAPK2 abundance plays critical role in proliferation versus survival depending on glucose availability.

overall_design: HCT116 cells expressing control shRNA were cultured in DMEM with or without glucose (20 mM vs 0 mM glucose) for 24 hours, followed by RNA extraction and RNA-sequencing.  HCT116 cells expressing control and MAPKAPK2 targeting shRNAs were also subjected to RNA-sequecing.

first: Niti

last: Kumari


title: Transcriptomic profiling of co-cultured cancer-host cells identifies hypoxia as a driver of the skeletal muscle cell’s antiproliferative effect on cancer cells

city: Oslo

first: Jerome

last: Ruzzin

address: Sognsvannveien 9

department: Dept. of Molecular Medicine, Section of Biochemistry

country: Norway

institute: University of Oslo

summary:
Cancer metastasis is the leading cause of cancer-related death. While organs such as the lung are hotspots for metastases, others
-like skeletal muscle- remain rarely colonized, a phenomenon that remains poorly understood. In this study, we show that EO771
breast cancer cells proliferated robustly when co-cultured with MLg lung stromal cells, whereas their proliferation was restrained
when maintained in direct contact with differentiated C2C12 skeletal muscle myotubes. Notably, these effects were not cell-typespecific, as similar results were obtained with 4T1 breast cancer cells and Sol8 myotubes. After two days of co-culture, both cancer and host cells (MLg and C2C12) exhibited distinct niche-specific transcriptional remodeling. Strikingly, the poorly proliferative
EO771 cells co-cultured with C2C12 myotubes acquired a hypoxia-associated gene-expression signature despite normoxic
conditions (~20% O₂), showing that muscle cells reprogram cancer cells into a hypoxic, anti-proliferative state. Under hypoxic
conditions, we confirmed that the depletion of oxygen allows C2C12 cells to nearly abolish EO771 proliferation. Neither exogenous
lactate, culture acidosis, their combination, altered glucose levels, nor conditioned medium could reproduce the suppressive
environment created by C2C12 myotubes. In contrast, MLg cells induced minimal transcriptional changes in EO771 cells and were
themselves broadly reprogrammed by the cancer cells. Moreover, hypoxia enhanced EO771 proliferation in MLg co-cultures,
emphasizing the permissive nature of the MLg environment. Collectively, these findings uncover a unique, paradoxical, muscleinduced
pseudo-hypoxic program that restricts cancer cell proliferation. They also highlight the need for caution in targeting
hypoxia signaling in anti-metastatic therapies, as such interventions could weaken skeletal muscle’s natural defense against tumor
colonization.

overall_design: RNAseq profie of a co-culture model in which EO771 breast cancer cells  are maintained directly on either MLg cells or C2C12 myotubes,

first: Anine

last: Aunan

first: Charlotte

last: Claeyssen

first: Mohamed

last: Abelhalim

first: Jérôme


title: RNA-Seq analysis of ATOH8 knockdown in human podocytes

city: Maebashi

first: Hiroko

last: Hamatani

state: Gunma

department: Nephrology and Rheumatology

institute: Gunma University

summary: This study investigates the effect of ATOH8 knockdown on human podocytes using RNA-Seq analysis

overall_design: This study investigates the effects of ATOH8 knockdown on human podocytes (AB8/13). ATOH8 knockdown was achieved using shRNA, and control samples were transfected with a non-targeting shRNA. Cells were cultured under differentiation conditions at 37°C for 5 days before RNA extraction. Total RNA was extracted from four biological replicates per experimental group, and RNA sequencing was performed. The study aims to compare gene expression profiles between ATOH8 knockdown and control groups.


title: Integrated small and long RNA sequencing in single oocytes reveals piRNA-mediated transposon repression during human oogenesis

city: Shanghai

first: Fengjuan

last: Zhang

state: Shanghai

address: 320 Yueyang Road

department: Institute of Biochemistry and Cell Biology

institute: Shanghai Institutes for Biological Sciences, CAS

summary: In this study, we simultaneously profiled small and long RNA transcriptomes in individual human oocytes across four developmental stages. piRNAs, especially PIWIL3-associated short piRNAs (short-piRNAs), are the predominant small non-coding RNAs during human oogenesis. A marked increase in short-piRNAs after the primordial follicle stage coincided with a global downregulation of TE expression, particularly LINE-1 (L1) and endogenous retroviruses (ERVs). On the other hand, PIWIL1- and PIWIL2-associated long piRNAs (long-piRNAs) were correlated with the silencing of certain specific ERV subfamilies. Genomic-context analyses revealed that highly productive piRNA clusters have evolved asymmetric antisense insertion bias toward L1 and ERVs, contributing to TE families-specific regulation.

overall_design: A total of 30 human oocytes spanning four developmental stages, primordial follicle (PrF), primary follicle (PF), germinal vesicle (GV), and metaphase II (MII), were collected. Small and long RNA transcriptomes were simultaneously profiled at the single-oocyte level.

first: Hongdao

first: Yali

last: Xiao

first: Miao

last: Liu

first: Suying

first: Aimin

last: Ren

first: Huijuan

last: Shi

first: Ligang

last: Wu


title: Tailoring a CRISPR/Cas-based Epigenome Editor for Programmable Chromatin Acylation and Decreased Cytotoxicity [RNA-Seq,2]

city: Houston

first: Isaac

last: Hilton

address: 6500 Main St., BRC Rm 660

institute: Rice University

summary: Precisely engineering histone acylation states can inform mechanistic epigenetics and catalyze new therapeutic epigenome editing opportunities. Here, we developed engineered lysine acyltransferases that enable the deposition of acetylation and longer-chain acylations in a programmable manner using a dead Cas9 (dCas9) fusion protein. We show that targeting an engineered lysine crotonyltransferase, developed by mutagenizing the native human p300 protein, results in relatively weak levels of endogenous enhancer activation yet retains strong potency when targeted to promoters. We further identify a single mutation within the catalytic core of human p300 that preserves enzymatic activity while promoting more target-specific acetylation and substantially reducing downstream transcriptomic perturbations. Further, this novel fusion protein exhibits low cytotoxicity but maintains high levels of H3K27ac deposition, enabling markedly improved lentiviral delivery. We leveraged this enhanced delivery and improved cytotoxicity profile to perform single-cell CRISPR activation screening. Using proteomics and a panel of engineered p300 variants, we also discover acylation-specific interactions and link the cytotoxicity of the wild-type p300 core domain to altered activities among DNA repair machinery components. These new programable epigenome editing tools and insights expand our ability to perform functional genomic screens, multiplexed cell engineering, and, more broadly, understand the mechanistic role of lysine acylation in epigenetic and cellular processes.

overall_design: mRNA sequencing for dCas9-p300 variant specificity in HEK293T cells

first: Jacob

last: Goell


title: Tailoring a CRISPR/Cas-based Epigenome Editor for Programmable Chromatin Acylation and Decreased Cytotoxicity [Targeting Amplicon Sequencing II]

overall_design: Deep mutational scanning for rTetR-p300 variant specificity in HEK293T reporter cell line


title: Mapping convergent regulators of melanoma drug resistance by PerturbFate

city: New York

first: Zihan

last: Xu

state: NY

address: 1230 York Ave

institute: The Rockefeller University

summary: High-throughput genomic studies have uncovered associations between diverse genetic alterations and disease phenotypes; however, elucidating how perturbations in functionally disparate genes give rise to convergent cellular states remains challenging. Here, we present PerturbFate, a high-throughput, cost-effective, combinatorial-indexing single-cell platform that enables systematic interrogation of massively parallel CRISPR interference (CRISPRi) perturbations across the full spectrum of gene regulation, from chromatin remodeling and nascent transcription to steady-state transcriptomic phenotypes. Using PerturbFate, we profiled over 300,000 cultured melanoma cells to characterize multi-modal phenotypic and gene regulatory responses to perturbations in more than 140 Vemurafenib resistance-associated genes. We uncovered a shared dedifferentiated cell state marked by convergent cooperative transcription factor (TF) activities across diverse genetic perturbations. We further dissected phenotypic responses to perturbations in Mediator Complex components, linking module-specific biochemical properties to convergent transcriptional activations. Together, we reveal common regulatory nodes that drive similar phenotypic outcomes across distinct genetic perturbations. We also delineate how perturbations in functionally unrelated genes reshape cell state. PerturbFate thus establishes a versatile platform for identifying key molecular regulators by anchoring multi-modal regulatory dynamics to disease-relevant phenotypes.

overall_design: Single-cell and parallel bulk CRISPR screen including 143 gRNA pairs targeting 143 genes and no-target with either DMSO or Vemurafenib treatment

first: Ziyu

last: Lu

first: Aileen

last: Ugurbil

first: Abdulraouf

last: Abdulraouf

first: Andrew

last: Liao

first: Jianxiang

first: Wei

last: Zhou

first: Junyue

last: Cao


title: Deciphering the Transcriptomic Signatures of Aging Across Organs in Mice

city: Bonn

first: Dan

last: Ehninger

state: NRW

address: Venusberg-Campus 1/99

department: AG Ehninger

country: Germany

institute: DZNE

summary: Aging is a key risk factor for disease in mammals, yet its molecular basis across organs remains unclear. Here, we performed bulk RNA sequencing on eight organs (brain, heart, kidney, liver, lung, skeletal muscle, spleen, testis) from male C57BL/6J mice at distinct life stages. Our analysis revealed that age-related transcriptomic shifts vary in timing and magnitude: early in lung, spleen, testis; mid-life in heart, kidney, skeletal muscle; and late in brain and liver. Magnitude ranged from very low (testis), low (brain, heart), moderate (lungs, skeletal muscle) to high (kidneys, liver, spleen). We uncovered organ-specific aging signatures, for instance, mitochondrial and epigenetic regulation in the kidney, metabolic/detoxification in the lung, and angiogenesis as well as ribosome biogenesis in the spleen). We also identified shared transcriptomic signatures, such as cellular senescence in the kidney and skeletal muscle, ECM remodeling in the heart, skeletal muscle and spleen), or inflammation  in the heart, kidney, liver and lungs. These findings highlight unique and overlapping transcriptomic aging signatures, informing future therapeutic strategies to improve healthspan.

overall_design: Towards addressing some of the shortcomings from previous studies, we employed an experimental design with a relatively large sample size (n = 45), 6 time points- 3, 5 (both young adult), 8 (adult), 14 (mid-life), 20 and 26 (late life) months and profiled overall age-associated transcriptomic changes in 8 organs harvested from male C57BL/6J mice, using the likelihood ratio test (LRT).

first: Enzo

last: Scifo

first: Sarah

last: Morsy


title: Epi-transcriptome analysis reveals the absence of m6A modifications in two watermelon-infected RNA viruses

city: Beijing

first: Jia-xian

last: Jing

address: Beijing Academy of Agriculture and Forestry Science

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

overall_design: Refer to individual Series


title: G1-APOL1 bone marrow chimeric B6 mice and rejection of fully MHC mismatched heterotopic Heart Transplants (BALBc-to-B6 model)

first: Zeguo

last: Sun

address: 1468 Madison Avenue

institute: Ichan School of Medicine at Mount Sinai

summary: G1-mice demonstrated greater CD8+T-cell graft infiltration and reduced survival in a mismatched heart transplant model following sensitization. Single-cell transcriptomics of graft infiltrating TCMs showed enrichment of T-cell receptor (TCR) response pathways including calcium signaling.

overall_design: To evaluate APOL1-RVs in an allotransplantation model, we generated bone marrow chimeric mice from G0- and G1-BAC-Tg (in a B6/45.2 background) in B6/45.1 hosts. B6/45.2 chimerism was confirmed at 4 weeks in B6/45.1 hosts (both H-2b). Bacterial artificial chromosome transgenic mice (BAC-Tg) show physiologic expression of APOL1 gene and each mouse line expresses only that respective variant, Co-stimulation blockade with CTLA4-Ig (methods) was provided and the mice were monitored for rejection till 28 days. To study G1-CD8+T-cell subsets, we evaluated the single-cell transcriptome of graft infiltrating T-cells from the BALBc/J cardiac allografts harvested from chimeric G1- or -G0 recipients at 4-weeks (N=3 each). We flow-sorted live CD3+ cells from allografts and performed single-cell RNA sequencing using a hashtagging approach to multiplex the 6 samples during sequencing (n~17000 total cells).

first: Madhav

last: Menon


title: Characterization of human senescent cell biomarkers for clinical trials

city: Rochester

first: David

last: Monroe

address: 200 1st St SW

institute: Mayo Clinic

summary: There is an increasing need for biomarkers of senescent cell burden to facilitate selection of participants for clinical trials. p16Ink4a is encoded by the CDKN2A locus, which produces five variant transcripts in humans, two of which encode homologous p16 proteins: p16Inka4a, encoded by p16_variant 1, and p16ɣ, encoded by p16_variant 5. While distinct quantitative polymerase chain reaction primers can be designed for p16_variant 5, primers for p16_variant 1 also measure p16_variant 5 (p16_variant 1+5). In a recent clinical trial evaluating effects of the senolytic combination, dasatinib + quercetin (D+Q), on bone metabolism in postmenopausal women, we found that women in the highest tertile for T-cell expression of p16_variant 5 had the most robust skeletal responses to D+Q. Importantly, assessment of p16_variant 5 was more predictive of these responses than p16_variant 1+5. Here, we demonstrate that in vitro, p16_variant 1+5 increased rapidly (week 1) following the induction of DNA damage, whereas p16_variant 5 increased later (week 4), suggesting that p16_variant 5 becomes detectable only when the abundance of senescent cells reached some threshold.  Further analysis identified a SASP panel in plasma that performed as well in identifying postmenopausal women with a positive skeletal response to D+Q. Collectively, our findings provide further support for the T-cell p16_variant 5 assay as a biomarker for selecting participants in clinical trials of senolytic interventions. In addition, our data indicate that correlated plasma SASP markers could be used in lieu of the more technically challenging T-cell p16 assay.

overall_design: Bulk RNA-seq profiling of human IMR-90 cells, comparing non-senescent (Week 0) and senescent cells (Weeks 1 and 4) following induction of senescence using etoposide treatment in vitro.

first: Joshua

middle: N

last: Farr

first: Mitchell

last: Froemming

first: Sundeep

last: Khosla


title: Multi-omic landscape of squamous cell lung cancer

city: TAmpa

first: Yonghong

last: Zhang

state: FL

address: 12902 USF Magnolia Drive

department: Biomedical Informatics

institute: Moffitt Cancer Center

summary: We identified 116 patients consented to the Total Cancer Care (TM) protocol that had donated snap frozen lung squamous cell carcinoma tumor tissues with linked molecular data as well as clinical history, tumor pathology, and patient outcomes. Samples were collected under the Moffitt’s Total Cancer Care protocol (Liberty IRB # 12.11.0023) and Moffitt’s General Banking protocol (IRB #: USF 101642). Described experiments were considered non-human subjects research and performed under protocol MCC #50083.

overall_design: Qiagen’s AllPrep DNA/RNA Mini kit was used for the simultaneous purification of genomic DNA and total RNA from the homogenized tumor tissue aliquot. RNAseq was performed using the NuGen Ovation Encore Complete RNAseq kit, which generates strand-specific total RNAseq libraries (Nugen, Inc., San Carlos, CA). A custom Agilent SureSelect panel covering 154 total genes (151 genes from the ClearSeq Comprehensive Cancer Panel plus KMT2D, KEAP1, and NFE2L2) was designed and used for the enrichment of DNA libraries generated using the Agilent SureSelect XT Library Kit (Agilent Technologies, Santa Clara, CA).

first: Paul

last: Stewart

first: Eric

last: Haura

first: Elsa

middle: R

last: Flores


title: Wnt-dependent spatiotemporal reprogramming of bone marrow niches drives fibrosis

city: Aachen

first: Ivan

middle: Gesteira

last: Costa

state: Northrhine-Westphalia

address: Pauwelsstr. 19

department: Joint Research Center for Computational Biomedicine

institute: UKA RWTH Aachen

summary: Bone marrow fibrosis is the most extensive matrix remodeling of the microenvironment, and can include de novo formation of bone (osteosclerosis). Spatiotemporal information on the contribution of distinct bone marrow niche populations to this process is incomplete. We demonstrate that fibrosis-inducing hematopoietic cells cause profibrotic reprogramming of perivascular CXCL12 abundant reticular (CAR) progenitor cells resulting in loss of their hematopoiesis-support and upregulation of osteogenic and pro-apoptotic programs. In turn, peritrabecular osteolineage cells (OLCs) are activated in an injury-specific, Wnt-dependent manner, comparable to skeletal repair. OLCs fuel bone marrow fibrosis through their expansion and skewed differentiation, resulting in osteosclerosis and expansion of Ly6a+ fibroblasts. Ncam1 expression marks peritrabecular OLCs and their expansion into the central marrow is specific for fibrosis in mice and patients. Peritrabecular stromal b-catenin expression is linked to fibrosis in patients and inhibition of Wnt signaling reduces bone marrow fibrosis and osteosclerosis, possibly being a clinically relevant therapeutic target.

overall_design: Murine bone marrow from long bones and spine was crushed, flushed, RBC lysed,  and MACS-depleted for CD11b, Gr1, Ter119, CD4, CD8, B220, CD45, and CD71, and subsequently combined with the digested bone chip fraction, after which the samples were FACS-sorted for lineage-, tdTom+ viable cells for sequencing

first: Bella

last: Banjamin

first: James Shiniti

last: Nagai

first: YeVin

last: Mun

first: Rafael

last: Krammann

first: Hélène F.E

last: Gleitz

first: César

last: Nombela-Arrieta

last: G. Costa

first: Rebekka K.

last: Schneider


title: Macrophage Smad1 contributes to post-infarction dysfunction, suppressing angiogenic chemokine expression, and stimulating extracellular matrix remodeling

city: Bronx

first: Silvia

last: Hernandez

state: New York

address: 1300 Morris Park Avenue

department: Medicine-Cardiology

institute: Albert Einstein College of Medicine

summary: In healing infarcts, macrophages undergo dramatic phenotypic changes in response to cytokines and growth factors and serve as central effectors in cardiac repair and remodeling. TGF-β superfamily members are key regulators of macrophages and signal predominantly through activation of Smad-dependent cascades. Although the involvement of TGF-β/Smad3 in regulation of macrophage phenotype has been documented, there is no information on the in vivo effects of macrophage Smad1 signaling. To study the role of macrophage Smad1 in myocardial infarction, we performed in vitro studies and in vivo experiments using mice with myeloid cell-specific Smad1 loss. Macrophage Smad1 activation peaked 7 days after infarction. In vitro, TGF-β1, TGF-β3 and BMP6, but not by BMP2, BMP4 and BMP7, stimulated Smad1 activation. In vivo, myeloid cell-specific Smad1 loss attenuated post-infarction adverse remodeling and ventricular dysfunction, increasing angiogenesis and decreasing extracellular matrix denaturation. Acquisition of an angiogenic profile by macrophages in the absence of Smad1, involved upregulation of the angiogenic chemokine CXCL12. In conclusion, our findings demonstrate for the first time that macrophage Smad1 inhibits release of angiogenic chemokines and enhances expression of genes involved in extracellular matrix remodeling.

overall_design:
This study is designed to compare gene expression profiles between Smad1fl/fl and MyS1KO (Smad1fl/fl + LyzMCre) bone marrow derived macrophages in the presence or absence of 10ng/ml TGF- β1, or 50ng/ml BMP4. The loss of Smad1 in MyS1KO macrophages has been documented by western blot.

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Submitter states that missing raw files are due to file loss.
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first: Bijun

last: Chen

first: Shuaibo

last: Huang

first: Anis

last: Hanna

first: Nikolaos

last: Frangogiannis


title: Spatial Transcriptomics, Histopathology, and Magnetic Resonance Subfield Segmentation of Hippocampal Sclerosis Compared to Normal Hippocampus: A Proof-of-Concept Study

city: Sheridan

first: Kenneth

last: Jones

state: Wyoming

address: 1309 Coffeen Avenue STE 10090

institute: Bioinformatic Solutions LLC

summary: Despite recent major advances in work-up and treatment of Drug Resistant Mesial Temporal Lobe Epilepsy (MTLE) with Hippocampal Sclerosis (HS), the underlying pathophysiology of this disease remains elusive. Depending on the type of HS, as defined by the histopathologic International League Against Epilepsy (ILAE), studies report variable post-surgical seizure freedom durations, with diverse underlying pathophysiology and gene expression. We here describe our initial experience with incorporating spatial transcriptomics along with Magnetic Resonance Imaging (MRI) and histopathology in a patient with Drug Resistant MTLE and HS compared to the hippocampus of an age-sex matched control case. Cases were assessed by MRI hippocampal subfields segmentation, neuropathology, and spatial transcriptomics to identify candidate genes that play a role in the biology of HS.  A significant finding was a partial inhibition of the methyl-CpG-binding protein 2 (MeCP2) gene in all subfields of the hippocampus, which is important for neuronal survival and differentiation. Together, our results have shown the feasibility of hippocampal subfield segmentation on MRI and direct correlation with histology and spatial transcriptomics to identify key areas of gene dysregulation in epilepsy. These novel methods can be used to implement personalized medicine and discovery of new drug targets.

overall_design: Compare one pathologically diagnosed case of hippocampal sclerosis (ILAE type 1) to a age-sex matched control.

first: Samuel

last: Guzman

first: Ken

first: Saman

last: Hazany


title: Transcriptome of BAC-transgenic G1- and G0-APOL1 murine CD8+T-cells after ex-vivo stimulation.

first: Zeguo

last: Sun

summary: BAC-transgenic G1-APOL1 mice ex-vivo stimulated  CD8+T-cells show  showed enrichment of canonical T-cell receptor (TCR) response pathways including calcium signaling.

overall_design: Bacterial artificial chromosome transgenic mice (BAC-Tg) show physiologic expression of APOL1 transgenes and each mouse line expresses only that respective variant. G0, G1 BAC-Tg mice were thus generated. Isolated naïve CD8+T-cells were stimulated ex vivo with using anti-CD3/CD28 (n=4 G0 vs 3 G1 adult animals). RNA was extracted and RNA-sequencing was performed at 72 hrs stimulation.

first: Madhav

last: Menon


title: Dedifferentiation-Driven Oncogenic Stemness Promotes Tumor-Sustaining Adaptability in the Intestinal Epithelium

city: Hoboken

first: ansu

last: perekatt

state: NJ

address: 507 River street

department: CCB

institute: Stevens Institute of Technology

summary: Intestinal tumors can arise either from mutations sustained in Lgr5⁺ stem cells (bottom-up) or through dedifferentiation of lineage-committed epithelial cells (top-down). Using a Smad4 loss-of-function and β-catenin gain-of-function mutant mouse model, we show that dedifferentiation-derived stem cells sustain tumorigenesis more effectively than mutant Lgr5⁺ cells in the endogenous crypts. Bulk and single-cell RNA sequencing revealed transcriptional reprogramming and the emergence of oncogenic villus-derived stem-like cells. Complementary in vivo and organoid studies suggest that dedifferentiation-derived oncogenic stem cells possess superior tumor-initiating and sustaining capacity

overall_design: Intestinal epithelial cells expressing red fluorescent protein, or the the stem cell marker CD44, was FACS isolated from wild type crypt, and the  Smad4 loss-of-function and β-catenin gain-of-function mutant crypt and villus epithelium and analyzed by single cell RNA-sequencing. Unlabeled cells from the wild type villus epithelium was also subjected to sc-RNA-sequencing.

first: Kylee

last: Zgeib

first: Thompson

last: Hui

first: Atharva

last: Inamdar

first: Ansu

last: Perekatt

first: Simon

last: Garcia

first: Binfeng

last: Lu

first: Crysta

last: Lim

first: Shima

last: Nejati

first: Amartya

last: Singh

first: Zahra

last: Hashemi

first: Dahlia

last: Matouba

first: Christina

last: Li


title: Differentially expressed genes in the heart tissues between wildtype control and the ttna+/- heterozygous knockout zebrafish

city: Baltimore

first: Yuji

last: Zhang

state: MD

address: 660 W. Redwood Street

institute: University of Maryland

summary: To seek the potential molecular mechanisms altered in ttna+/- heterozygous knockout zebrafish, we performed whole transcriptome RNA-sequencing experiments using heart tissues isolated from ttna+/- and WT control zebrafish at sx months of age.

overall_design: Total RNA was extracted from the heart tissues of six-month-old ttna+/- heterozygous mutant hearts and WT sibling controls. Genes were considered to be differentially expressed between the two groups if they exhibited a greater than 2-fold change and an False Discovery Rate (FDR) of less than 0.05 according to the DESeq approach.

first: Xiaolei

last: Xu

first: Feixiang

last: Yan


title: Effect of CAR-M2 hydrogel treatment on the expression of related genes such as fibrosis and endothelial angiogenesis in mice with renal fibrosis (bulk RNA-seq)

city: Chongqing

first: Wen

last: Zeng

address: Gaotanyan street

department: Department of Cell Biology

institute: Army Medical University

summary: Currently there is no effective treatment for renal fibrosis, which is the common outcome of chronic kidney disease (CKD). The renal fibrosis was aggravated by the unfavorable microenvironment and cell-to-cell interactions, accompanied by fibroblast activation and vascular rarefaction. Here, M2 macrophage engineered with chimeric antigen receptor (CAR) was created to promote renal revascularization for the synergistic treatment of phagocytosis of activated fibroblast. To achieve efficient delivery of parenchymal organs, a hydrogel was used to deliver CAR-M2 by renal subcapsule injection. CAR-M2 significantly reduced renal fibrosis and promoted revascularization. The single-cell RNA sequencing (scRNA-seq) revealed the heterogenicity and interaction of stroma and endothelial cells (ECs). A fibrosis-related Cxcr2+ EC subset was defined, which induced renal fibrosis by upregulating lipocalin 2 (Lcn2). Combined analysis of CKD patients demonstrated that CAR-M2 could release Matrix Metalloproteinase 2 (MMP2) in close proximity to activate the Cxcr2+ EC retinoid X receptor alpha (Rxra) to regulate fibrosis. Our study provides an effective strategy to develop an advanced cell therapy for organ fibrosis.

overall_design: The study includes two groups: the CAR-M group and the CAR-M macrophage-fibroblast group, with three replicates in each group.

first: Wenyan

last: Zhao

first: Xin

last: Zhou


title: Radiotherapy synergizes with AAV-based immunotherapy to program local and systemic antitumor immunity

city: Pamplona

first: Juan

last: Dubrot

state: (None)

address: Avda. Pío XII 55

country: Spain

institute: Cima-Universidad de Navarra; Cancer Center Clinica Universidad de Navarra (CCUN)


title: Diverse Roles of Arabidopsis ROS1 in Regulating Chromatin Accessibility and DNA Methylation [ChIP-seq]

city: Wuhan

first: Wenying

last: Zhong

address: Lion Rock Street

institute: Huazhong Agricultural University

summary: Over two decades after the discovery of ROS1 as the first eukaryotic DNA demethylase, its genome-wide binding sites and functions beyond active DNA demethylation remain unknown. Here, using advanced ChIP-seq, we reveal that ROS1 specifically occupies nearly all accessible chromatin and dynamically correlates with changes in chromatin accessibility across tissues, establishing it as a marker of accessible chromatin. Furthermore, we demonstrate that ROS1 maintains DNA hypomethylation through an occupancy-based mechanism that prevents the recruitment of RNA-directed DNA methylation, distinct from its active DNA demethylation. Additionally, ROS1 plays a regulatory role in chromatin accessibility, both independently and in cooperation with other epigenetic regulators. This regulation occurs in both DNA methylation-dependent and independent contexts, with ROS1 functioning as a potential or actual protector of accessible chromatin, depending on the presence and targeting of DNA methylation systems. Our results provide a comprehensive understanding of the regulatory roles of ROS1 in chromatin accessibility and DNA methylation, highlighting the intricate crosstalk between these mechanisms.

sample_organism:
Arabidopsis thaliana
Brassica napus

platform_organism:
Arabidopsis thaliana
Brassica napus

overall_design: We take advantage of our recently developed aChIP method to map the genome-wide binding landscape of ROS1. We reveal that ROS1 specifically occupies nearly all accessible chromatin regions and dynamically correlates with changes in chromatin accessibility across distinct tissues and in the hda6 histone deacetylase mutant, thereby marking accessible chromatin. By integrating ROS1 and RNA-directed DNA methylation (RdDM) occupancy data with chromatin accessibility and DNA methylation profiles from various mutants and wild-type plants, we demonstrate the regulatory role of ROS1 in chromatin accessibility. Additionally, we propose a previously unreported mechanism by which ROS1 occupancy physically prevents the recruitment of RdDM to maintain DNA hypomethylation. Our findings elucidate the intricate crosstalk among these mechanisms and provide a framework for a deeper understanding of chromatin protein functions.

first: Li

last: Deng

first: Guangfeng

last: Zhu

first: Lun


title: Weaning-Driven Gut Microbiome Shapes Intestinal Stem Cell Epigenetics to Train Immunological Memory (RNA-seq)

first: Lanlan

last: Shen

state: Texas

address: 1100 Bates Ave

department: Pediatrics

institute: Baylor College of Medicine

summary: During weaning, the transition to solid food diversifies the gut microbiome, triggering a programmed immune response critical for long-lasting mucosal immunity. Previous work showed that the gut microbiome mediates epigenetic development in intestinal stem cells (ISCs) during suckling, but what happens during weaning is unclear. Here, using genome-wide methylation profiling revealed that weaning-driven microbiome changes shape the DNA methylome and transcriptome of murine ISCs in an IFN-g dependent manner. Specifically, we observe demethylation of enhancer elements essential for MHC class II genes, which result in a transcriptional memory that persists through differentiation into adulthood. IFN-g blockade, or low-dose penicillin to target Gram-positive bacteria, in early life impaired microbiome-mediated epigenetic control and mucosal immunity, and exacerbated colitis. Murine organoids primed with IFN-g showed rapid, amplified transcriptional responses upon secondary stimulations. These findings reveal that early-life events alter the gut microbiome and these changes reprogram ISC epigenetic memory to shape mucosal immunity.

overall_design: Comparative transcriptome profiles of colonic stem cells and differentiated cells from Lgr5 mice under specific-pathogen-free (SPF) and germ-free (GF) conditions at 15 weeks of age.

last: Yang


title: ROS1: Key Marker and Regulator of Accessible Chromatin in Arabidopsis [ATAC-Seq]


title: MYC binding to nascent RNA suppresses innate immune signaling by R-loop-derived RNA-DNA hybrids

city: Wuerzburg

first: Martin

last: Eilers

address: Am Hubland

department: Chair for Biochemistry and Molecular Biology

institute: University of Wuerzburg

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: sc-RNA-seq study of dedifferentiating intestinal epithelial cells reveals oncogenic reprogramming to heterogeneous stem cell states [dataset 1]

first: ansu

last: perekatt

overall_design: Intestinal epithelial cells expressing red fluorescent protein, or the the stem cell marker CD44, was FACS isolated from wild type crypt, and the Smad4 loss-of-function and β-catenin gain-of-function mutant crypt and villus epithelium and analyzed by single cell RNA-sequencing. Unlabeled cells from the wild type villus epithelium was also subjected to sc-RNA-sequencing

first: Kylee

last: Zgeib

first: Thompson

last: Hui

first: Atharva

last: Inamdar

first: Ansu

last: Perekatt

first: Crystal

last: Lim

first: Shima

last: Nejati

first: Binfeng

last: Lu

first: Simon

last: Garcia

first: Amartya

last: Sigh


title: Radiotherapy synergizes with AAV-based immunotherapy to program local and systemic antitumor immunity [RNA-Seq]

first: Juan

last: Dubrot

summary: Radiotherapy (RT) primes the immune system against cancer antigens but fails to trigger effective antitumor responses due to concomitant induction of immunosuppressive factors. To expand the benefits of RT, we combine local radiation with immuno-gene therapy by harnessing the ability of ionizing radiation to enhance AAV-mediated tumor transduction. To successfully deliver AAV-based local immunotherapy and restrict transgene expression within tumors, we designed a recombinant AAV that expresses IL-12 through an interferon (IFN)-inducible promoter (AAV-iIL12). Local administration of AAV-iIL12 upon RT results in marked and durable elevation of intratumor IL-12 with minor and transient systemic release. This strategy generates a highly immunostimulatory tumor microenvironment (TME) leading to strong antitumor responses in an IFNg-dependent manner. Moreover, local treatment with RT and AAV-iIL12 elicits systemic antitumor immunity with marked CD8+ T cell infiltration and regression of distant tumors. Our work shows that radiation coupled with AAV-based immuno-gene delivery is a novel, efficient and safe approach that can be exploited as cancer therapy.

overall_design: Comparative gene expression profiling analysis after RT treatment between control AAV mice (n=4) and mice receiving AAV-iIL12 (n=3)

first: Jesús

last: Prieto

first: Elizabeth

last: Guruceaga

first: Sonia

last: Marco


title: ROS1: Key Marker and Regulator of Accessible Chromatin in Arabidopsis [RNA-Seq]

first: Wenying

last: Zhong

first: Li

last: Deng

first: Guangfeng

last: Zhu

first: Lun

last: Zhao


title: Weaning-Driven Gut Microbiome Shapes Intestinal Stem Cell Epigenetics to Train Immunological Memory (WGBS)

first: Lanlan

last: Shen

type: Methylation profiling by high throughput sequencing

summary: During weaning, the transition from milk to solid food rapidly expands and diversifies the gut microbiome, triggering the weaning reaction, a programmed immune response critical for long-lasting mucosal immunity. We characterized how these microbiome changes from weaning through young adulthood in mice shape the DNA methylome and transcriptome of intestinal stem cells (ISCs). We identified a weaning-induced IFN-g–TET3 axis as a key driver of MHC class II enhancer-linked epigenetic reprogramming, creating a transcriptional memory that persists through differentiation into adulthood. Early-life disruption of this axis, via IFN-g blockade or low-dose penicillin, impaired microbiota-mediated epigenetic control and mucosal immunity. Weaning-associated microbiome shifts enriched metabolites such as α-ketoglutarate, Fe2+, and methionine g-lyase, promoting TET-dependent DNA demethylation. These findings reveal how early-life events imprint lifelong epigenetic regulation of mucosal immunity, highlighting epithelial cells as innate immune sentinels that acquire trained transcriptional memory in response to microbial cues.

overall_design: Genome-wide DNA methylation profiling by WGBS was performed in mice colonic stem cells and differentiated cells at 3 weeks and 15 weeks of age.

last: Yang


title: Diverse Roles of Arabidopsis ROS1 in Regulating Chromatin Accessibility and DNA Methylation [Bisulfite-Seq]


title: HR3/RORα-mediated cholesterol sensing regulates TOR signaling

city: København Ø

first: Kim

last: Rewitz

state: Capital region

address: Universitetsparken 15, Building 3.3.401

country: Denmark

institute: University of Copenhagen

summary: Cells and organisms adjust their growth based on the availability of cholesterol, which is essential for cellular functions. However, the mechanisms by which cells sense cholesterol levels and translate these into growth signals are not fully understood. We report that cholesterol rapidly activates the master growth-regulatory TOR pathway in Drosophila tissues. We identify the nuclear receptor HR3, an ortholog of mammalian RORα, as an essential factor in cholesterol-induced TOR activation. We demonstrate that HR3 binds cholesterol and promotes TOR pathway activation through a non-genomic mechanism acting upstream of the Rag GTPases. Similarly, we find that RORα is necessary for cholesterol-mediated TOR activation in human cells, suggesting that HR3/RORα represents a conserved mechanism for cholesterol sensing that couples cholesterol availability to TOR-pathway activity. These findings advance our understanding of how cholesterol influences cell growth, with implications for cholesterol-related diseases and cancer.

overall_design: To determine the biological effects of dietary cholesterol replenishment, and to assess which effects require HR3 or TOR, we performed RNAseq on whole larvae expressing no RNAi, Hr3-RNAi, or Tor-RNAi throughout the body (using ubiquitous driver Tub-GAL80TS; Tub-GAL4), after the animals had been stimulated with dietary cholesterol or not. Thus there are three genotypes x two feeding conditions, with five replicates each. Each replicate contains five late-third-instar larvae.

first: Mette

last: Lassen

first: Michael

last: Texada


title: miR-203 controls developmental timing and early fate restriction during preimplantation embryogenesis (RNA-Seq)

city: Barcelona

first: Agustín

middle: Sánchez

last: Belmonte

state: Barcelona

address: Nazaret 115 -117

department: Systems Oncology Program

institute: Vall d ́Hebron Institute of Oncology (VHIO)

summary: Commonly expressed at developmental transitions, microRNAs operate as fine tuners of gene expression to facilitate cell fate acquisition and lineage segregation. Nevertheless, how they might regulate the earliest developmental transitions in early mammalian embryogenesis remains obscure. Here, in a strictly in vivo approach based on novel genetically-engineered mouse models and single-cell RNA sequencing, we identify miR-203 as a critical regulator of timing and cell fate restriction within the totipotency to pluripotency transition in mouse embryos. Genetically engineered mouse models show that loss of miR-203 slows down developmental timing during preimplantation leading to the accumulation of embryos with high expression of totipotency-associated markers, including MERVL endogenous retroviral elements. A new embryonic reporter (eE-Reporter) transgenic mouse carrying MERVL-Tomato and Sox2-GFP transgenes showed that lack of miR-203 leads to sustained expression of MERVL and reduced Sox2 expression in preimplantation developmental stages. A combination of single-cell transcriptional studies and epigenetic analyses identified the central coactivator and histone acetyltransferase P300 as a major miR-203 target at the totipotency to pluripotency transition in vivo. By fine tuning P300 levels, miR-203 carves the epigenetic rewiring process needed for this developmental transition, allowing a timely and correctly paced development.

overall_design: embryps of the indicated developmental stages were microinjected with siRNAs or treated with inhibitors when indicated. At least three biological replicates per experimental group were used, with 15-20 embryos approximately in each one of them.

first: José

last: González-Martínez

first: Marcos

last: Malumbres

last: Sánchez-Belmonte


title: Radiotherapy synergizes with AAV-based immunotherapy to program local and systemic antitumor immunity [scRNA-Seq]

last: Dubrot

overall_design: Transcriptomic analysis by scRNA seq of CD45+ tumor infiltrating cells obtained 6 days after treatment with RT + control AAV (n=4) or RT +  AAV-iIL12 (n=4).

first: Seth

last: Anderson


title: miR-203 controls developmental timing and early fate restriction during preimplantation embryogenesis (CUT&RUN)

overall_design: Embryos were harvested from female donors at E2.75. Zona pellucida was removed and embryos were dissagregated in accutase. Blastomeres were processed for CUT&RUN analysis as stated in the material and methods section. Each sample contains approximately 1000 blastomeres.


title: miR-203 controls developmental timing and early fate restriction during preimplantation embryogenesis (scRNA-Seq)

overall_design: Embryos were harvested directly from the female donors, dissagregated and analysed by scRNA-seq.


title: TGF-β controls epigenetic re-programing of IFN-antiviral trained immunity and impairs virologic control in SIV-infected rhesus macaques [bulk RNA-seq]

city: Atlanta

first: Felipe

last: ten Caten

state: Georgia

address: 1750 Haygood Road

department: Department of Pathology and Laboratory Medicine

institute: Emory University

summary: Current immunotherapeutic approaches to eliminate latently HIV-infected cells have focused on targeting the adaptive arm of the immune system using vaccines and passive transfer of broadly neutralizing antibodies (bNAbs). Epigenetic reprogramming of innate myeloid cells triggered by adjuvants was recently shown to promote a state of interferon (IFN)-induced antiviral immunity and resistance to heterologous viral infections. Herein, flow cytometry, bulk and single-cell RNA-Seq/ATAC-Seq were performed on samples from a cohort of SIV-infected, virologically suppressed rhesus macaques (RMs) treated with anti-IL-10 and anti-PD-1 monoclonal antibodies (combo treatment) prior to analytical treatment interruption (ATI). These experiments revealed that combo treatment led to selective changes in chromatin accessibility, expression of IRF7/3 and STAT1, and expression of IFN-stimulated antiviral genes in myeloid cells and CD4+ T cells. These RMs showed a stringent and prolonged control of SIV viral rebound following ATI and a decay in levels of cell-associated viral DNA (CA-vDNA) in lymph nodes. These RMs showed reduced chromatin accessibility for the SMAD2/SMAD3, AP-1, and NFKB1 loci and lower expression of TGF-b and SMADs target genes. This signature is consistent with that observed in HIV elite controllers, who maintain undetectable levels of virus in the absence of ART and whose HIV reservoir size is small. These results highlight the critical role of the interplay between TGF-b and IFN in epigenetically regulated innate antiviral immune responses that can impact the viral reservoir.

overall_design: Twenty-eight rhesus macaques were infected intravenously with SIVmac239. After 14 months on antiretroviral therapy (ART), RMs were randomized into 3 groups: 8 RMs received vehicle and served as controls; 10 RMs received deimmunized aIL-10, and 10 RMs received deimmunized aIL-10 and deimmunized aPD-1 (combo group). Bulk RNA-seq analyses were performed on lymph node mononuclear cells (LNMCs) and peripheral blood mononuclear cells (PBMCs) collected at week 0 and 24-weeks post-ATI from all animals. Combo-treated animals were dichotomized as CA-vDNAlo (n = 4) and CA-vDNAhi (n = 6) based on the size of the SIV reservoir 24 weeks post-ATI (<2 log10 and >2 log10 CA-vDNA copies per 106 CD4+ T cells, respectively)

last: ten-Caten

first: Khader

last: Ghneim

first: Mirko

last: Paiardini

first: Susan

middle: P

last: Ribeiro

first: Rafick

last: Sekaly


title: miR-203 controls developmental timing and early fate restriction during preimplantation embryogenesis

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

overall_design: Refer to individual Series

first: Jose

last: González Martínez

first: Agustin

last: Sanchez Belmonte


title: Tailoring a CRISPR/Cas-based Epigenome Editor for Programmable Chromatin Acylation and Decreased Cytotoxicity [Targeted Amplicon Sequencing]

city: Houston

first: Isaac

last: Hilton

address: 6500 Main St., BRC Rm 660

institute: Rice University

summary: Precisely engineering histone acylation states can inform mechanistic epigenetics and catalyze new therapeutic epigenome editing opportunities. Here, we developed engineered lysine acyltransferases that enable the deposition of acetylation and longer-chain acylations in a programmable manner using a dead Cas9 (dCas9) fusion protein. We show that targeting an engineered lysine crotonyltransferase, developed by mutagenizing the native human p300 protein, results in relatively weak levels of endogenous enhancer activation yet retains strong potency when targeted to promoters. We further identify a single mutation within the catalytic core of human p300 that preserves enzymatic activity while promoting more target-specific acetylation and substantially reducing downstream transcriptomic perturbations. Further, this novel fusion protein exhibits low cytotoxicity but maintains high levels of H3K27ac deposition, enabling markedly improved lentiviral delivery. We leveraged this enhanced delivery and improved cytotoxicity profile to perform single-cell CRISPR activation screening. Using proteomics and a panel of engineered p300 variants, we also discover acylation-specific interactions and link the cytotoxicity of the wild-type p300 core domain to altered activities among DNA repair machinery components. These new programable epigenome editing tools and insights expand our ability to perform functional genomic screens, multiplexed cell engineering, and, more broadly, understand the mechanistic role of lysine acylation in epigenetic and cellular processes.

overall_design: Deep mutational scanning for rTetR-p300 variant specificity in HEK293T reporter cell line

first: Jacob

last: Goell


title: Tailoring a CRISPR/Cas-based Epigenome Editor for Programmable Chromatin Acylation and Decreased Cytotoxicity [CUT&RUN]

overall_design: CUT&RUN sequencing for H3K27ac in HEK293T cells expressing dCas9-p300 variants


title: Effect of CAR-M2 hydrogel treatment on the expression of related genes such as fibrosis and endothelial angiogenesis in mice with renal fibrosis

first: Wen

last: Zeng

summary: Kidney fibrosis involves the dynamic interactions of various cells, including immune cells, stromal cells, and ECs. Macrophages regulate regeneration and fibrosis through the interaction with other cell types, inducing some effects including the clearance of apoptotic myofibroblasts. Moreover, M2 macrophages promote tissue angiogenesis and participate in the reparative processes of soft tissue remodeling. Therefore, in addition to the direct effect of macrophage on renal fibrosis, its indirect regulatory role is also worthy to be explored. Its interaction with activated fibroblasts and ECs in renal fibrosis is unknown. Here our data discovered the heterogenicity and interaction of stroma and EC. Moreover, the infiltration of Cxcr2+ EC and expression of Lcn2 were indentfied highly related renal fibrosis. Taken together, our data provide a deeper understanding of the expression profile of renal fibrosis in mice, provide novel molecular targets for the treatment of renal fibrosis.

overall_design: To investigate the effect of CAR-M2 on renal fibrosis expression genes, we set the Sham group, fibrosis model UUO group, and CAR-M2 treatment group for sequencing. We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 different groups at 14 days. Comparative gene expression profiling analysis of RNA-seq data for 3 groups.

first: Wenyan

last: Zhao


title: Establishing the Oocyte Proteome at the Primordial Follicle Stage and Cisplatin-induced Shifts through in situ APEX2-driven Biotinylation

city: Hangzhou

first: Heng-yu

last: Fan

state: Zhejiang

address: Yuhangtang Road

institute: Life Sciences  Institute

summary: The number and quality of primordial follicles play a decisive role in female fertility. The high sensitivity of oocytes in primordial follicles to chemotherapeutics makes antitumor therapy extremely prone to premature ovarian insufficiency and infertility; however, the specific mechanism remains unclear. A systematic proteomic profile of oocytes in primordial follicles is urgently needed to support basic and clinical research, which has been hindered by the rarity of oocyte samples and the technical challenges associated with isolating oocytes from primordial follicles. In this study, utilizing in vivo protein labeling by engineered ascorbate peroxidase (APEX) fluorescent mice, we identified 641 proteins and 2020 gene transcripts in primordial follicular oocytes, from which the abundance of several proteins involved in processes associated with DNA damage repair and histone modification changed after ovary exposure to the chemotherapeutic cisplatin. Notably, a histone modifier, EZH2, was induced by cisplatin in oocytes, and the simultaneous application of its inhibitor, GSK126, partially relieved cisplatin-induced oocyte developmental defects. Our study provides the first description of the primordial follicular oocyte proteome and further illustrates the dynamic shifts in protein abundance associated with chemotherapeutic agents, laying the foundation for further development of effective and targeted drugs to protect and prolong female fertility.

overall_design: The biotinylated RNAs from APEXflox/+;Gdf9-Cre mice were subjected to RNA-seq using the Smart-seq2 method with minor modifications.

first: Zhiyan

last: Jiang

first: Xuan

last: Wu

first: MO

last: Li

first: Hengyu


title: Tailoring a CRISPR/Cas-based Epigenome Editor for Programmable Chromatin Acylation and Decreased Cytotoxicity [RNA-Seq]

overall_design: mRNA sequencing for dCas9-p300 variant specificity in HEK293T cells


title: Ago2 protects against diabetic cardiomyopathy via activating mitochondrial gene translation [miRNA-seq]

first: zhan

last: jiabing

address: Jiefang Avenue

institute: Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology

summary: Diabetes is associated with cardiovascular complications. microRNAs translocate into subcellular organelles to modify genes involved in diabetic cardiomyopathy. However, functional properties of subcellular Ago2, a core microRNA, remain elusive. We elucidated the function and mechanism of subcellular localized Ago2 on mouse models for diabetes mellitus and diabetic cardiomyopathy. Ago2 decreased in cardiomyocyte mitochondria in both models. Overexpression of mitochondrial Ago2 attenuated diabetes-induced cardiac dysfunction. Ago2 recruited TUFM, a mitochondria translation elongation factor, to activate translation of electron transport chain (ETC) subunits and decrease reactive oxygen species. Malonylation, a post-translational modification of Ago2, reduced the importing of Ago2 into mitochondria in diabetic cardiomyopathy. Ago2 malonylation was regulated by a cytoplasmic-localized short isoform of SIRT3 through a previously unknown demalonylase function. Our results reveal that the SIRT3–Ago2–CYTB axis links glucotoxicity to cardiac ETC imbalance, providing new mechanistic insights and the basis to develop mitochondria targeting therapies for diabetic cardiomyopathy.

overall_design: Comparative microRNA profiling analysis of RNA-seq data for mitochondria extracted from db/db mouse heart and its control.

first: Jiabing

last: Zhan

first: Kunying

last: Jin

first: Jiahui

first: Chen

last: Chen

first: Huaping

first: Dao

last: Wang