title: Inferring circadian phases and quantifying biological desynchrony across single-cell transcriptomes

status: Public on Mar 30 2026

city: Lausanne

first: Felix

last: Naef

address: EPFL SV IBI

country: Switzerland

institute: EPFL

type: Expression profiling by high throughput sequencing

summary: Single-cell RNA sequencing (scRNA-seq) offers a unique opportunity to dissect circadian heterogeneity, yet accurately inferring circadian phase remains challenging due to natural biological fluctuations, technical noise, and low sequencing depth. Here, we introduce scRitmo, an unsupervised, model-based approach that jointly infers single-cell circadian phases and their posterior uncertainties. Using a simulation framework, we demonstrate that the non-uniform expression of core clock genes creates phase "attractor zones" that bias inference at low sequencing depths, and show that incorporating a broader set of rhythmically expressed genes allows scRitmo to overcome this limitation. We applied scRitmo to diverse datasets to unravel cell-type-specific circadian organization. Benchmarking across multiple murine scRNA-seq datasets demonstrates that scRitmo outperforms existing methods. We further validated our approach using a novel dataset composed of deeply sequenced unsynchronized fibroblasts, showing that transcriptomic phases accurately reconstruct temporal ordering of the corresponding protein levels. Beyond point estimates, scRitmo provides a measure of phase confidence through its posterior circular standard deviation. We show that this quantity is a robust predictor of inference quality: in both simulations and diverse murine datasets, cells with lower posterior uncertainty consistently exhibit significantly lower absolute errors relative to external time, allowing for principled quality control of single-cell predictions. Crucially, scRitmo enables the quantification of biological (de)synchrony by disentangling true phase dispersion from technical noise. We validated this capability using both synthetic datasets with known ground-truth desynchrony and time-series single-molecule RNA FISH (SABER-FISH) data. In the latter, scRitmo accurately recovered the progressive accumulation of phase heterogeneity following synchronization, confirming its ability to track dynamic changes in population synchrony. Applying this capability to Drosophila brains, scRitmo captured the expected increased desynchrony in constant darkness compared to light-dark cycles, effectively quantifying the biological loss of synchrony in the absence of Zeitgebers. Together, scRitmo provides a rigorous framework for high-precision phase inference, establishing a principled approach to decouple technical noise from biological variance and enabling the quantitative study of circadian (de)synchrony as a fundamental property of multi-scale temporal organization.

overall_design: Unsynchronized NIH3T3-Rev-VNP1-H2B-iRFP fibroblasts were FACS-sorted into 384-well plates using a binned strategy (4 bins along the Nr1d1-Venus axis, 2 bins along the H2B-iRFP axis) to capture the full range of circadian reporter expression. Full-length scRNA-seq libraries were generated using FLASH-seq (Alithea Genomics)

first: Andrea

last: Salati

first: Yves

last: Paychère

first: Vincent

last: Hahaut

first: Cédric

last: Gobet

first: Félix


title: Anticancer activity of RAS-GTP inhibition in cholangiocarcinoma [Mouse_iAKP_Abr24]

city: Pamplona

first: Silvestre

last: Vicent

address: Avenida Pío XII 55

department: Oncology

country: Spain

institute: Center for Applied Medical Research (CIMA)

summary: About 25% of cholangiocarcinoma (CCA) patients harbor KRAS mutations, being potentially amenable to direct KRAS inhibitors. Here, we investigated the activity of RAS(ON) multi-selective inhibitors, which target the active, GTP-bound state of both mutant and wild-type variants of canonical RAS isoforms, in CCA preclinical models and patients. RAS(ON) multi-selective inhibitors yield strong anticancer responses in cell- and patient-derived xenografts and in immunocompetent allograft models. Consistently, we describe responses to the RAS(ON) multi-selective inhibitor daraxonrasib in two KRASG12X patients with advanced CCA. RAS-GTP inhibition also potentiates the activity of the current standard-of-care CCA therapy to prolong survival in cell-derived human xenografts and mouse allografts. Furthermore, we demonstrate that both intrinsic and acquired resistance to RAS(ON) multi-selective inhibitors mainly rely on reconstitution of RAS signalling. Overall, our findings indicate that KRAS mutant CCA is addicted to RAS signalling for proliferation and support the potential clinical evaluation of RAS-GTP inhibition in CCA.

overall_design: KPCh cells and iAKP cells were treated or not with 100nM of RMC-7977 for 24hrs to assess alterations in biological pathways upon treatment.

first: Rodrigo Entrialgo

last: Cadierno

first: Konstantina

last: Morali

first: Silve


title: Behavioral, Physiological, and Transcriptional Mechanisms of Memory in a Synthetic Living Construct

city: Medford

first: Vaibhav

middle: P

last: Pai

state: MA

address: 200 Boston Ave suite 4600

department: Biology

country: USA

institute: Tufts University

summary: Synthetic living constructs, which lack the long histories of selection in ecological contexts that shape behaviors of conventional organisms, offer an important complement to traditional studies of learning. Could novel biobots exhibit sensing and memory of experiences? Here, we investigated the effects of chemical stimuli on basal Xenobots – autonomously motile entities derived from Xenopus embryonic ectodermal explants (with no additional sculpting or bioengineering). We quantified and characterized the coordinated ciliary activity that generates fluid flow fields guiding the trajectory of Xenobot motion. We also show distinct and specific changes in Xenobot behavior after brief exposure to Xenopus embryonic cell extract and to ATP. These two experiences produced distinct, long-term, stimulus-specific memories, detectable through both transcriptional and physiological signatures. Exposure to specific environmental stimuli induced alterations in the spatiotemporal patterns of calcium signaling across Xenobots. Together, these data lay a foundation for characterizing the capabilities of synthetic cellular collectives to sense and discriminate among stimuli, as well as store functional information in a non-neural context. Understanding behavioral competencies in novel, non-neural systems have broad implications across evolutionary biology, behavioral science, bioengineering, and bio/hybrid robotics.

overall_design: Comparative gene expression profiling analysis of RNA-seq data from Xenobots before stimulus (Control/Ctrl) and after simulus of either embryo extract stimulus (Ext) or ATP at two time points: immediately after stimulus (0 hours) and four hours after stimulus (4 hours)

first: James

middle: A

last: Traer

first: Megan

middle: M

last: Sperry

first: Yuxin

last: Zeng

first: Michael

last: Levin


title: Cohesin complex cooperates with PU.1 at super-enhancers to regulate the differentiation and identity of conventional dendritic cells

city: Bangalore

first: Kushagra

last: Bansal

state: Karnataka

address: Jakkur

department: MBGU

country: India

institute: JNCASR

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Dendritic cell (DC) network consists of two major subsets- conventional (cDC) and plasmacytoid (pDC). cDCs undergo chromatin reorganization during their differentiation from hematopoietic progenitors to establish cDC-specific gene expression program. However, the requirement of architectural protein complex Cohesin in the differentiation of cDCs is poorly understood. Herein we report that ablation of SMC3 or STAG2 subunits of Cohesin complex in DC-restricted progenitors leads to decrease in cDC, and increase in pDC compartment size. Cohesin complex controlled the functions of cDCs, such as antigen presentation and the expression of co-stimulatory molecules. The ablation of Cohesin complex repressed the expression of cDC identity genes and de-repressed the pDC-specific transcriptional signature in cDCs and pre-DCs. Mechanistically, Cohesin complex promoted the binding of PU.1, an ETS family transcription factor, at super-enhancers and supported chromatin loops favouring cDC differentiation. Notably, Cohesin-deficient DCs disturbed the steady-state haematopoiesis leading to myeloid hyperplasia. Our results reveal a transcriptional node involving Cohesin complex, PU.1 and super-enhancers in the regulation of lineage-specific gene expression program in cDCs.

overall_design: RNA-seq in splenic cDCs, splenic pDCs, and bone marrow pre-DCs from Smc3-WT and Smc3-cKO mice. SMC1A, PU.1 and H3K27Ac ChIP-seq in splenic cDCs from Smc3-WT and Smc3-cKO mice. Hi-C in  in splenic cDCs from Smc3-WT and Smc3-cKO mice. Single cell RNA-seq from RBC depleted splenocytes from Smc3-WT and Smc3-cKO mice.

first: Pallawi

last: Choubey

first: Amit

last: Kumar


title: Spatiomolecular mapping reveals anatomical organization of heterogeneous cell types in the human nucleus accumbens

city: Baltimore

first: Keri

last: Martinowich

state: Maryland

address: 855 N Wolfe St Suite 300

institute: Lieber Institute for Brain Development

summary: The primary goal of the Xenium experiments was to understand the cell type composition of D1-islands identified with Visium spatial transcriptomics, and to evaluate gradients of gene expression across the NAc.

overall_design: For Xenium experiments, 1 tissue section from 4 donors was used. Each tissue section was located within the intermediate Nac. Xenium was performed using a custom 100 gene panel with a 266 gene base panel. The custom gene panel was focused on identification of D1-islands and included genes with gradients of expression across the NAc.

first: Prashanti

last: Ravichandran

first: Svitlana

middle: V

last: Bach

first: Robert

last: Phillips III

first: Madeline

middle: R

last: Valentine

first: Nicholas

middle: J

last: Eagles

first: Yufeng

last: Du

first: Ishbel

last: del Rosario

first: Haya

last: AlGrain

first: Sarah

middle: E

last: Maguire

first: Ryan

last: Miller

first: Heena

last: Divecha

first: Madhavi

last: Tippani

first: Kelsey

middle: D

last: Montgomery

first: Joel

last: E Kleinman

first: Shizhong

last: Han

first: Stephanie

middle: C

last: Page

first: Thomas

last: Hyde

last: Hicks

first: Kristen

last: Maynard


title: Mitochondrial remodeling in skeletal muscle underlies exercise-induced reversal of age-associated functional decline in mice and humans

city: Madrid

first: Jose

middle: Luis

last: Cabrera

state: Madrid

address: Melchor Fernández Almagro, 3

institute: CNIC

summary: RNA-seq experiment to investigate the effect of training on muscle tissue in WT C57BL/6J and G6PD-tg mice. The goal of the study is to assess differential gene expression related to genotype and training status.

overall_design: A total of 40 samples were collected under trained or non-trained conditions. RNA-seq libraries were prepared from 50 ng total RNA using the NEBNext Ultra II Directional kit, including poly(A)+ selection, fragmentation, cDNA synthesis, adaptor ligation, and PCR amplification.  Sequencing was performed on a NextSeq 2000 (Illumina) platform.

first: Esther

last: García-Domínguez

first: José

last: Enríquez

first: María

last: Gómez-Cabrera


title: Anticancer activity of RAS-GTP inhibition in cholangiocarcinoma [Human_Abr24]

first: Silvestre

last: Vicent

overall_design: HUCCT-1 cells and KKU-213 cells were treated or not with 100nM of RMC-7977 for 24hrs to assess alterations in biological pathways upon treatment.

first: Rodrigo Entrialgo

last: Cadierno

first: Konstantina

last: Morali

first: Silve


title: Hypoxia-EPAS1 imprints immunoregulation and maintenance of type 2 tissue-resident CD4+ T cells I

city: Chiba

first: Atsushi

last: Sasaki

state: Chiba

address: Chuo ku Inohana 1-8-1

department: Immunology

country: Japan

institute: Chiba University

summary: CD4+ tissue-resident memory T cells (TRM) drive tissue-specific chronic inflammation by expressing characteristic functional molecules, but how tissue microenvironment-derived stimuli orchestrate their characteristics remains unclear. We found that intracellular hypoxia-dependent activation of EPAS1 programmed human and mouse type 2 CD4+ TRM to acquire immunoregulatory properties through the upregulation of CTLA-4 and IL-10. Genetic deletion of Epas1 in CD4+ T cells decreases the expression of immunoregulatory molecules in type 2 CD4+ TRM, consequently exacerbating lung inflammation. Spatial transcriptomic profiling of human asthmatic lungs identified FOXP3-negative CTLA4+GATA3+ TRM cells with activated hypoxic gene signatures in tertiary lymphoid structures. Our findings revealed that hypoxic pathway activation is a critical determinant of type 2 CD4+ TRM characteristics and highlight Epas1-dependent pathways as promising targets for treating chronic inflammatory disorders.

overall_design: Single-cell RNA sequencing was performed to characterize tissue-resident memory T cell–associated transcriptional states in a chronic allergic airway inflammation model. Mice were subjected to repeated house dust mite (HDM) exposure for five weeks or left untreated as controls. Prior to sacrifice, intravascular staining was performed by intravenous injection of anti-CD45 antibodies to distinguish intravascular from tissue-resident T cells. CD4⁺CD44hi T cells were isolated from lung tissue and stratified based on intravascular staining status (i.v. positive or negative). Cells from two control mice and two HDM-treated mice were pooled, and single-cell RNA-seq was performed across four groups: control i.v. negative, control i.v. positive, HDM i.v. negative, and HDM i.v. positive. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was performed to assess chromatin accessibility changes associated with hypoxia in CD4⁺ T cells during chronic allergic airway inflammation. Mice were exposed to house dust mite (HDM) for five weeks or left untreated as controls. Prior to sacrifice, mice were administered Hypoxyprobe to label hypoxic cells in vivo. CD4⁺CD44^hi T cells were isolated from lung tissue and sorted based on Hypoxyprobe signal intensity into hypoxic (Hypoxyprobe^hi) and non-hypoxic (Hypoxyprobe^lo) populations. ATAC-seq was performed on four control Hypoxyprobe^lo samples and eight HDM samples (four Hypoxyprobe^lo and four Hypoxyprobe^hi).

first: Masahiro

last: Kiuchi

first: Hiroyuki

last: Yagyu

first: Takahiro

last: Arano

first: Tsuguko

last: Tomita

first: Kanae

last: Ohishi

first: Shigenori

last: Baba

first: Kaori

last: Tsuji

first: Rui

last: Hirasawa

last: Nemoto

first: Takahisa

last: Hishiya

first: Chiaki

last: Iwamura

first: Takahide

last: Toyoda

first: Masayuki

last: Ota

last: Onodera

first: Motoko

middle: Y

last: Kimura

first: Shinichiro

last: Motohashi

first: Hidemi

last: Suzuki

first: Jun-ichiro

last: Ikeda

first: Damon

last: Tumes

first: Toshinori

last: Nakayama

first: Takuji

first: Kiyoshi

last: Hirahara


title: Epigenome-Wide DNA Methylation Patterns in Prostate Adenocarcinoma, Benign Hyperplasia, Metastatic Disease, and Related Lifestyle Factors among African American Men [Georgetown ]

city: Washington

first: Bernard

last: Kwabi-Addo

state: DC

address: 520 W. Street N.W.

department: Biochemistry and Molecular Biology

institute: Howard University College of Medicine

type: Methylation profiling by genome tiling array

summary: African American (AA) men have higher prostate cancer (PCa) incidence and mortality rates than any other racial/ethnic group in the USA. Biological differences including aberrant epigenetic DNA methylation changes likely contribute to the PCa disparities. Our study aims to identify differentially methylated CpG sites and genes among histologic localized adenocarcinoma PCa, metastatic PCa (mCRPC), and Benign Prostatic Hyperplasia (BPH) samples. We performed genome-wide DNA methylation profiling in 100 AA prostate tissues using Illumina Methylation EPIC array. Global hypermethylation indicating epigenetic silence was observed in localized adenocarcinoma PCa compared to paired normal and BPH samples. On the other hand, significant hypomethylation suggesting tumor associated activation of oncogenes and chromatin remodeling was observed in mCRPC. Using Shannon entropy as a metric for epigenetic heterogeneity, mCRPC samples exhibited significant elevated entropy, reflecting greater methylation variability than localized adenocarcinoma PCa and BPH. We also identified distinct smoking and alcohol-associated methylation signatures, with specific CpG sites showing significant differential methylation compared to non-exposed patients. Our analysis provides insights into methylation profiles as predictive markers for distinguishing between BPH, PCa and mCRPC that are also influenced by lifestyle factors. More importantly, aberrations in methylation favor PCa aggressiveness to metastasis and genomic instability that implies aging and environmental factors. Overall, the differential methylation patterns suggest potential for patient stratification, biomarker-driven treatment strategies including epigenetic therapeutic strategies for AA PCa.

overall_design: In the present study, we generated genome-wide DNAm data from AA prostate biospecimens including BPH, paired normal and adenocarcinomas, and metastatic samples. For each case, we obtained clinicopathological data, lifestyle factors (tobacco and alcohol use), and co-morbidities such as diabetes and hypertension. Our goals were to identify lifestyle-related and comorbidity-associated methylation signatures, and to quantify genome-wide methylation disorder using Shannon entropy as a measure of epigenetic heterogeneity.

first: H

last: Song

first: A

last: Duttargi

first: C

last: Albanese

first: V

last: Apprey

first: W

middle: F

last: Huang

first: B


title: Comparative and integrative analysis of transcriptomic and epigenomic-wide DNA methylation changes in African American prostate cancer [Baylor]

summary: African American (AA) men have the highest incidence and mortality rate from Prostate cancer (PCa) than any other racial/ethnic group. To date, PCa genomic studies have largely under- represented tumour samples from AA men. We measured genome-wide DNA methylation in benign and tumor prostate tissues from AA men using the Illumina Infunium 850 K EPIC array. mRNA expression database from a subset of the AA biospecimen were used to assess correlation of transcriptome and methylation datasets. Genome-wide methylation analysis identified 11,460 probes that were significant (p < 0.01) and differentially methylated in AA PCa compared to normal prostate tissues and showed significant (p < 0.01) inverse-correlation with mRNA expression. Ingenuity pathway analysis and Gene Ontology analysis in our AA dataset compared with TCGA dataset showed similarities in methylation patterns: top candidate genes with significant hypermethylation and corresponding down-regulated gene expression were associated with biological pathways in hemidesmosome assembly, mammary gland development, epidermis development, hormone biosynthesis, and cell communication. In addition, top candidate genes with significant hypomethylation and corresponding up-regulated gene expression were associated with biological pathways in macrophage differentiation, cAMP-dependent protein kinase activity, protein destabilization, transcription co-repression, and fatty acid biosynthesis. In contrast, differences in genome-wide methylation in our AA dataset compared with TCGA dataset were enriched for genes in steroid signalling, immune signalling, chromatin structure remodelling and RNA processing. Overall, differential methylation of AMIGO3, IER3, UPB1, GRM7, TFAP2C, TOX2, PLSCR2, ZNF292, ESR2, MIXL1, BOLL, and FGF6 were significant and uniquely associated with PCa progression in our AA cohort.

overall_design: The primary goal of the present study is to carry out Epigenome-wide DNA methylation changes in 113 prostate tissues from AA to identify biological pathways and genetic alterations associated with AA PCa, compare differential methylation changes with AA mRNA expression dataset to see whether differences corresponded with changes in mRNA expression and to comprehensively interrogate our AA methylation and mRNA expression (Howard/ BCM) dataset with the TCGA (EUR and AA) methylation and mRNA expression dataset for differences in the two datasets.

first: Chad

last: Creighton

first: Flora

last: Zhang

first: Yiqun

first: Patricia

last: Castro

first: Rong

last: Hu

first: Md

last: Islam

first: Somiranjan

last: Ghosh

first: Michael

last: Ittmann


title: Identification of host lncRNAs that impact Venezuelan equine encephalitis  virus replication

city: Albuquerque

first: Steven

last: Bradfute

state: NM

address: 1 University of New Mexico

institute: University of New Mexico Health Sciences Center

summary: Venezuelan equine encephalitis virus (VEEV) causes encephalitis in humans and equids, and there are no vaccines or therapeutics available for humans. In recent years, non-coding RNAs have emerged as critical regulatory factors affecting different cellular pathways. Specifically, long non-coding RNAs (lncRNAs) have been identified  as regulators of antiviral pathways during various viral infections; however, their role in  regulating VEEV infection has not been assessed. Here we show differential expression of several lncRNAs in primary mouse target cells infected with a vaccine strain of VEEV (TC-83) but not a pathogenic strain (TrD). Among the differentially expressed genes (DEGs), suppressing lncRNA small nucleolar RNA host gene 15 (Snhg15) resulted in about a 7-fold increase in VEEV TC-83 replication in primary mouse a strocytes. Knockdown of Snhg15 during VEEV TC-83 infection resulted in thesuppression of ten genes including Irf1, Junb, Atf3, Relb, Pim1, Hbegf, Ccl5, Ankrd33b, and H2-K2, all of which were also increased during TC-83 infection when the expression of Snhg15 increased in primary mouse astrocytes. Most of these genes are involved inantiviral responses. KEGG pathway analysis confirmed the suppression of both pattern recognition receptor and inflammatory pathways after in Snhg15 knockdown. These data are the first to identify lncRNA responses in encephalitic alphavirus infection and demonstrate important roles for these overlooked RNAs on VEEV infection.

overall_design: Total RNA sequencing was used to profile lncRNA gene expression in primary mouse  astrocytes and neurons in reponse to VEEV Trinidad donkey (TrD) and VEEV TC-83  infections. The gene expression profiles of VEEV TrD and VEEV TC-83 at 16hpi and 24hpi were compared to those of uninfected cells.

first: Mahgol

last: Behnia

first: Chunyan

last: Ye

first: Kim

last: Somfleth

first: Olufunmilola

last: Oyebamiji

first: Kathryn

last: Brayer

first: Yan

last: Guo

first: Scott

last: Ness

first: Ram

last: Savan

middle: B


title: Translatome profiling reveals opposing alterations in inhibitory and excitatory neurons of Fragile X mice and identifies EPAC2 as a therapeutic target

city: Los Angeles

first: Anand

last: Suresh

state: CA

address: 650 CHARLES E YOUNG DR S, MED RECEIVING CHS, RM 76-200C

department: Neurology

institute: University of California Los Angeles

summary: Symptoms of Fragile X Syndrome (FXS), the leading monogenic cause of intellectual disability and autism, are thought to arise from an excitation/inhibition (E/I) imbalance. Here, we leverage cell type specific mRNA sequencing to profile molecular alterations of cortical excitatory (Camk2) and inhibitory (Pvalb) neurons in Fmr1 knockout (KO) mice, integrating transcriptomic results with circuit and behavioral readouts to prioritize novel therapeutic targets. We uncovered significant genotype-by-cell interactions for differential gene expression in Camk2a and Pvalb translatomes, and, strikingly, the underlying signaling pathways were often altered in opposite directions. Among the 184 DEGs that were concordantly dysregulated across both cell types, only Rapgef4 (Epac2; upregulated in Fmr1 KO) was also an FMRP target, brain-enriched and associated with neurodevelopmental disorders. Treatment of Fmr1 KO mice with a specific EPAC2 antagonist restored cortical circuit function and ameliorated multiple behavioral phenotypes. Thus, EPAC2 should be considered as a potential target for therapy in FXS.

overall_design: Young adult (P70-100) PV-Cre-Rpl22HA/HA and Camk2a-Cre-Rpl22HA/HA, WT or Fmr1 KO mice were used for the Ribotag RNA-seq experiments.

first: Nazim

last: Kourdougli

first: Toshihiro

last: Nomura

first: Jessie E

last: Buth

first: Soledad

last: Miranda-Rottmann

first: Carlos A

last: Sanchez-Leon

first: Michelle W

last: Wu

first: Sofia M

last: Nelson

first: Lauren T

last: Wall

first: Anne T

last: Tran

first: Roberto

last: Araya

first: Anis

last: Contractor

first: Michael J

last: Gandal

first: Carlos

last: Portera-Cailliau


title: Anticancer activity of RAS-GTP inhibition in cholangiocarcinoma [Mouse_hKPR.KPCHR_Sep25]

first: Silvestre

last: Vicent

overall_design: In vivo generated resistant cell lines (hKP117-RR, KPCh972-RR, KPCh993-RR) and parental (hKP and KPCh) were assessed for altered biological pathways.

first: Rodrigo Entrialgo

last: Cadierno

first: Konstantina

last: Morali

first: Silve


title: Lactate-driven H3K18 lactylation promotes cisplatin resistance in bladder cancer via HNRNPF-Parkin mediated mitophagy

city: Guangzhou

first: Dongqing

last: Li

address: South Shatai road

department: Department of Urology

country: China

institute: Southern medical university

summary: Cisplatin resistance in bladder cancer (BCa) is driven by metabolic reprogramming that enhances glycolysis and lactate production. Here, we report that lactate-induced histone H3K18 lactylation (H3K18la) drives chemoresistance by activating a novel signaling axis that couples epigenetic regulation with mitochondrial quality control. Through integrative multi-omics analysis, we identified HNRNPF as a key effector downstream of H3K18la, with its promoter being directly enriched by H3K18la in cisplatin-resistant BCa cells. Unexpectedly, HNRNPF, primarily known as an RNA-binding protein, promotes chemoresistance through a non-canonical mechanism: it directly interacts with the core mitophagy protein Parkin. Mechanistically, the RRM2 domain of HNRNPF binds the R0 domain of Parkin, facilitating Parkin’s recruitment to damaged mitochondria. This interaction potentiates Parkin’s E3 ubiquitin ligase activity, leading to enhanced ubiquitination of VDAC1 and robust activation of mitophagy. Collectively, our findings establish the H3K18la-HNRNPF-Parkin axis as a previously unrecognized signaling cascade that bridges epigenetic reprogramming with to mitochondrial quality control in chemoresistance. Targeting this axis, particularly the HNRNPF-Parkin interaction or mitophagy activation, may represent a novel therapeutic strategy to overcome cisplatin resistance in BCa, pending further validation in preclinical models.

overall_design: Cells were fixed with 1% formaldehyde, sonicated for 8 min on ice, then diluted the fragments and added Protein G agarose. After shaking, the supernatant was collected, followed by adding Anti-H3K18la antibody (PTM-BIO, PTM-1406RM), and incubated overnight. Protein A and Protein G beads were used to precipitate protein. Then, the releasing DNA from chromatin, were precipitated and diluted. Purified DNA fragments were added to the ChIP-seq library and sequenced on the HiSeq 2 platform according to the protocol from company (LC-BIO, China).

first: Henghui

last: Zhang

first: Mingqiang

last: Su

first: Zaosong

last: Zheng

first: Wei

last: Chen

first: Haiyong


title: Single-cell CRISPRi perturbation of POU5F1 in human and cynomolgus macaque iPSCs

city: Planegg

first: Ines

last: Hellmann

address: Großhaderner Straße 2

country: Germany

institute: Ludwig-Maximilians-Universität München

summary: To understand phenotypic evolution, it is essential to investigate the underlying gene regulatory networks (GRNs). However, most comparative GRN analyses remain descriptive due to the low signal-to-noise ratio inherent in single-cell transcriptomics data. To address this, we introduced CroCoNet (Cross-species Comparison of Networks), an R-package for quantitative GRN comparison across species. CroCoNet builds comparable network modules centered on putative regulators and compares module topologies within and between species, distinguishing true evolutionary divergence from technical and biological confounders. We demonstrate its utility by comparing early neural differentiation across primates and validating results with a CRISPRi analysis of the diverged POU5F1 module.

sample_organism:
Macaca fascicularis
Homo sapiens

platform_organism:
Macaca fascicularis
Homo sapiens

overall_design: Using a single-cell CRISPRi screening approach, we perturbed 94 transcription factors in human (Homo sapiens) and cynomolgus macaque (Macaca fascicularis) induced pluripotent stem cells (iPSCs). We performed the experiments in two human and two cynomolgus cell lines harboring an inducible KRAB–dCas9 construct stably integrated into their AAVS1 locus. The cells were transduced with custom-designed, species-specific lentiviral gRNA libraries targeting the 94 transcription factors of interest with 4 gRNAs each and containing ~12% non-targeting control gRNAs in addition. After selecting for the successfully transduced cells, KRAB-dCas9 expression was induced by adding doxycycline to the medium, enabling the downregulation of the target transcription factors. 5 days after doxycycline induction, we performed scRNA-seq using the 10x Genomics 5’ platform, profiling 1) the transcriptome of each cells by poly(A) capture (GEX libraries) and 2) the gRNA(s) in each cell by capture of the gRNA constant region (CRISPR libraries). This repository is a subset of the full dataset (GEO accession: GSE298717), containing only the POU5F1-perturbed and control cells.

first: Anita

last: Térmeg

first: Fiona

last: Edenhofer

first: Jessica

last: Jocher

first: Zane

last: Kliesmete

first: Philipp

last: Janssen

first: Dana

last: Lopez-Parra

first: Wolfgang

last: Enard


title: Divergent Macrophage-Regulated T cell States Determine Response to Bacillus Calmette-Guérin (BCG) in High-Risk Bladder Cancer

city: Chicago

first: Joshua

last: Meeks

state: IL

address: 303 East Superior Street

institute: Northwestern University

summary: The primary therapy for high-risk bladder cancer (BCa) is repeated instillations of the tuberculosis vaccine, Bacillus Calmette-Guerin (BCG). While BCG decreases the risk of recurrence by more than half, the concerted mechanisms of immune activation from BCG are unknown. Our objective was to investigate how the immune response differs between responders and non-responders to BCG therapy. We performed single-cell RNA-sequencing of isolated immune cells adjacent to high-risk bladders before and after BCG in BCG responders and non-responders. We identify an increase in Th17-like Th1 cells in BCG responders, characterized by greater expression of pro-inflammatory cytokines. Alternatively, non-responders had increased CD8+ T-cell exhaustion and T-regulatory cells. We identify that the primary mechanism of divergent T cell activity is driven by altered polarization and immunosuppressive signaling with myeloid cells. Through a machine-learning-based approach, we identified a Th17-like Th1 cytokines, such as IL17, IL21, and IL26, were predictive of a response, which were then validated in a separate BCG-treated BCa cohort. Together, this suggests that dynamic regulation of myeloid-T cell interactions can be targeted to improve BCG activity.

overall_design: We performed single-cell RNA sequencing (scRNA-seq) of isolated immune cells adjacent to high-risk bladders in BCG responders and non-responders before and after BCG. We also compared concurrent scRNA-seq profiles of circulating immune cell populations with those of bladder immune cells.

first: Andrew

last: Houston

first: Mairah

last: Khan

first: Ryan

last: Brown


title: High-resolution spatial transcriptomics of human adrenal neoplasms

city: Shinjuku

first: Tomohiro

last: Tamura

state: Tokyo

address: Shinanomachi 35

department: Department of Extended Intelligence for Medicine, The Ishii-Ishibashi Laboratory

institute: Keio University School of Medicine

summary: To characterize the tumor microenvironment and elucidate mechanisms of immune evasion in malignant adrenal tumors, we performed spatial transcriptomics (Visium HD) on pathological specimens from adrenocortical adenoma (ACA) and pheochromocytoma mixed tumor. Using each cell’s spatial coordinates and gene expression profiles, we inferred tumor cell–immune cell interactions.

overall_design: Using an FFPE specimen from patients with ACA and pheochromocytoma mixed tumor for whom clinical data such as clinical course had been accumulated, we performed high-resolution spatial transcriptomics analysis using Visium HD. (Deposited files were binned outputs)

first: Masaki

last: Uchihara

first: Kotaro

last: Umamoto

first: Akiyo

last: Tanabe


title: FACT maintains nucleosomes around promoters and opposes to the spreading of chromatin factors, thereby playing a major role in chromatin architecture [ATAC-seq]

city: Oxford

first: Catherine

last: Chahrour

address: MRC Weatherall Institute of Molecular Medicine

country: United Kingdom

institute: University of Oxford

summary: Amounting evidence suggests nucleosome organization is key for higher levels of chromatin packaging. However, studying it in higher eukaryotes is challenging due to the difficulty to manipulate nucleosomes in vivo and reconstitute their large higher-order chromatin domains in vitro. Facilitates chromatin transcription (FACT) is a H2A/B histone chaperone that displaces and re-assembles histones, being important for processes such as transcription. Recent studies report a minor role of FACT in chromatin architecture. Here, we studied chromatin organization upon rapid FACT degradation employing a multidimensional 3C technique with base-pair resolution (MCC-u) and genomic approaches. FACT loss leads to nucleosome depletion around active promoters, nano-scale domain collapse and emergence of larger nucleosome-free regions with increased protein binding. At a larger scale, we detect increased interactions between promoters across topologically associated domains. Thus, we demonstrate FACT plays a major role in chromatin organization and gain insights into the role of nucleosomes in genome architecture.

overall_design: Accessible chromatin sequencing via ATAC-seq for SSRP1-FKBP E14 cells treated for 6h with DMSO and 3h with dTAG-13

first: Ana

last: Dopico-Fernandez

first: Robert

last: Beagrie

first: Thomas

last: Milne


title: FACT maintains nucleosomes around promoters and opposes to the spreading of chromatin factors, thereby playing a major role in chromatin architecture [CUT&TAG]

overall_design: CUT&TAG for CBP, P300, TBP, RING1B, KDM2B, and no primary control (IgG) in SSRP1-FKBP E14 cells treated for 6h with DMSO and 3h with dTAG-13. Drosophila spike-in was added for reference normalization.


title: Identification of host lncRNAs that impact Venezuelan equine encephalitis virus replication

first: Steven

last: Bradfute

summary: Venezuelan equine encephalitis virus (VEEV) causes encephalitis in humans and equids, and there are no vaccines or therapeutics available for humans. In recent years, non-coding RNAs have emerged as critical regulatory factors affecting different cellular pathways. Specifically, long non-coding RNAs (lncRNAs) have been identified as regulators of antiviral pathways during various viral infections; however, their role in regulating VEEV infection has not been assessed. Here we show differential expression of several lncRNAs in primary mouse target cells infected with a vaccine strain of VEEV (TC-83) but not a pathogenic strain (TrD). Among the differentially expressed genes (DEGs), suppressing lncRNA small nucleolar RNA host gene 15 (Snhg15) resulted in about a 7-fold increase in VEEV TC-83 replication in primary mouse astrocytes. Knockdown of Snhg15 during VEEV TC-83 infection resulted in the suppression of ten genes including Irf1, Junb, Atf3, Relb, Pim1, Hbegf, Ccl5, Ankrd33b, and H2-K2, all of which were also increased during TC-83 infection when the expression of Snhg15 increased in primary mouse astrocytes. Most of these genes are involved in antiviral responses. KEGG pathway analysis confirmed the suppression of both pattern recognition receptor and inflammatory pathways after in Snhg15 knockdown. These data are the first to identify lncRNA responses in encephalitic alphavirus infection and demonstrate important roles for these overlooked RNAs on VEEV infection.

overall_design: Total RNA sequencing was used to profile lncRNA gene expression in primary mouse astrocytes and neurons in reponse to VEEV Trinidad donkey (TrD) and VEEV TC-83 infections. The gene expression profiles of VEEV TrD and VEEV TC-83  at 16hpi and 24hpi were compared to those of uninfected cells.

first: Mahgol

last: Behnia

first: Chunyan

last: Ye

first: Kim

last: Somfleth

first: Olufunmilola

last: Oyebamiji

first: Kathryn

last: Brayer

first: Yan

last: Guo

first: Scott

last: Ness

first: Ram

last: Savan


title: Hypoxia-EPAS1 imprints immunoregulation and maintenance of type 2 tissue-resident CD4+ T cells II

first: Atsushi

last: Sasaki

overall_design: Naive CD4⁺ T cells were isolated from mouse spleens and differentiated into T helper 2 (Th2) cells in vitro. During the differentiation process, cells were cultured under either normoxic or hypoxic conditions. Bulk RNA sequencing was performed to compare transcriptional profiles between normoxia-induced Th2 cells and hypoxia-induced Th2 cells. Three independent biological replicates were included for each condition.

first: Masahiro

last: Kiuchi

first: Hiroyuki

last: Yagyu

first: Takahiro

last: Arano

first: Tsuguko

last: Tomita

first: Kanae

last: Ohishi

first: Shigenori

last: Baba

first: Kaori

last: Tsuji

first: Rui

last: Hirasawa

last: Nemoto

first: Takahisa

last: Hishiya

first: Chiaki

last: Iwamura

first: Takahide

last: Toyoda

first: Masayuki

last: Ota

last: Onodera

first: Motoko

last: Kimura

first: Shinichiro

last: Motohashi

first: Hidemi

last: Suzuki

first: Jun-ichiro

last: Ikeda

first: Damon

last: Tumes

first: Toshinori

last: Nakayama

first: Takuji

first: Kiyoshi

last: Hirahara


title: Identification of  Therapeutic Targets for Low-Grade Serous Ovarian Carcinoma

city: Melbourne

first: Kathleen

last: Pishas

state: Victoria

address: 305 Grattan Street

department: Cancer Research

country: Australia

institute: Peter MacCallum Cancer Centre

summary: Low-grade serous ovarian carcinoma (LGSOC) is a rare, indolent ovarian cancer subtype with limited effective therapies. Approximately 40% of cases lack canonical MAPK/ERK or PI3K/AKT/mTOR pathway alterations and are classified as having no specific molecular profile (NSMP). These patients have poor responses to chemotherapy, MEK inhibitors, and hormonal therapies, highlighting the need for alternative strategies. This study aimed to identify novel therapeutic targets in NSMP LGSOC. A high-throughput drug screen of over 3,500 compounds (including FDA-approved, clinically tested, and investigational agents) was conducted across 12 LGSOC and one control ovarian epithelial cell line. EGFR inhibitors emerged as selective hits in NSMP cell lines and were further tested in two NSMP and two MAPK-mutant lines in combination with standard-of-care chemotherapy agents, carboplatin and paclitaxel. EGFR expression was assessed using RNA sequencing, DNA methylation profiling, and immunohistochemistry in primary tumours, followed by survival analysis based on expression levels.

overall_design: RNA-sequencing of 12 LGSOC cell lines and two normal controls (no treatment)

middle: I

first: Hannah

last: Kim

first: Marta

last: Llaurado-Ferandez

first: Mark

middle: S

last: Carey

first: Timothy

last: Semple

first: Ian

middle: G

last: Campbell

first: Dane

last: Cheasley


title: HyperTRIBE uncovers MEX3A RNA-binding activity in colorectal cancer (CRC) cells

city: Porto

first: Bruno

last: Pereira

address: Rua Alfredo Allen, 208

country: Portugal

summary: A comprehensive list of RNA targets of the RNA-binding protein MEX3A remains elusive. To address this gap, we applied the high-throughput HyperTRIBE (Targets of RNA Identified By Editing) methodology in the SW480 cell line in order to map the MEX3A RNA interactome in CRC. An hyperactive version of the catalytic domain (cd) of the fruit fly RNA-editing enzyme Adenosine Deaminase Acting on RNA (ADAR) was fused either to the human MEX3A coding sequence (MEX3A-hyperADARcd) or its RNA-binding mutant version (MEX3ADKH-hyperADARcd) as a control. Once expressed, the MEX3A-hyperADARcd fusion protein creates irreversible adenosine-to-inosine (A-to-I) RNA editing events that are identified as adenosine-to-guanosine (A-to-G) mismatches near MEX3A-binding sites by RNA-sequencing. Editing specificity is guaranteed by the lack of RNA-binding of ADARcd. To prevent analytical bias by ADAR continuous activity, the MEX3A-hyperADARcd and MEX3ADKH-hyperADARcd sequences were placed under the control of a doxycycline (DOX)-inducible promoter for transient expression, together with a TurboRFP reporter for sorting of positive cells expressing the constructs.

overall_design: We generated an hyperactive version of Drosophila’s ADARcd by introducing the human ADAR2 E488Q matching mutation, previously described to increase ADAR2cd editing efficiency and reduce sequence recognition bias (Kuttan et al., 2012; Xu et al., 2018). The MEX3AΔKH mutant was generated as a control for background editing from a MEX3A expression vector (Pereira et al., 2013) by introducing a double amino acid change in both KH domains, previously predicted to impair nucleic acid binding (Hollingworth et al., 2012). Fragments were assembled and cloned into the pCW57-RFP-P2A-MCS lentiviral vector (Addgene #78933) to generate stable SW480 isogenic lines integrating MEX3A-hyperADARcd and MEX3ADKH-hyperADARcd constructs. Fusion proteins overexpression was induced by doxycycline (DOX) addition for 48h. RFP+ induced cells and the corresponding non-induced controls (NIC) were collected by fluorescence-activated cell sorting (FACS) and used for RNA-sequencing. Two independent induction experiments were used as biological replicates (R1 and R2).

first: Ana R

last: Silva

last: Cavadas

first: Raquel

last: Almeida


title: Bulk RNA Sequencing of Chimera-Derived Monocyte-Derived Macrophages Targeted for Mitochondrial ROS During Peak Experimental Autoimmune Encephalomyelitis

city: Zürich

first: Juan

last: Villar Vesga

address: Winterthurerstrasse 190

institute: University of Zurich

summary: Reactive oxygen species (ROS) produced by mononuclear phagocytes (MPs) are widely believed to drive tissue damage in multiple sclerosis (MS), yet the distinct roles of central nervous system (CNS)-resident versus CNS-invading MPs remain unclear. Here, we combined single-cell profiling and conditional gene targeting to map and modulate ROS production across CNS MPs in a preclinical mouse model of MS. We show that monocyte-derived macrophages (MdMs) exhibit a higher oxidative stress gene signature and produce more ROS than Mglia. Challenging previous assumptions, our findings reveal that phagocytic NADPH oxidase 2 is dispensable for neuroinflammation. In contrast, quenching mitochondrial ROS (mtROS) through mitochondria-targeted catalase (mCAT) expression in MdMs, but not in Mglia, ameliorated disease severity in acute neuroinflammation. While core phagocyte functions were unaltered in mCAT-expressing MdMs, our results demonstrate a direct neurotoxic role of mtROS. Together, we identify MdMs as the primary driver of ROS-mediated oxidative neurological tissue damage.

overall_design: Bulk RNA sequencing of CNS-infiltrating monocyte-derived macrophages (MdMs) isolated from central nervous system with targeted modulation of mitochondrial ROS pathways during peak experimental autoimmune encephalomyelitis (EAE). The dataset contains sorted MdMs from donor wt (CD45.1) and donor mitochondrial catalase(CD45.2) targeted mice.

last: Villar-Vesga

first: Donatella

last: De Feo

first: Pauline

last: Clément

first: Viola

last: Bugada

first: Elèni

last: Meuffels

first: Sarah

last: Mundt


title: Cell size-dependent mRNA transcription drives proteome remodeling [SLAM-seq]

city: Stanford

first: Dong Shin

last: You

address: 327 Campus Drive

institute: Stanford University

summary: Increasing cell size drives proteomic changes that impact cell physiology. However, the molecular basis of size-dependent proteome remodeling has remained unclear. Here, we develop an inducible Cyclin D1 expression system in human cells to generate proliferating cells spanning over a two-fold size range. We use this system to make comprehensive genome-wide measurements of mRNA and protein concentrations and stability. We find that protein and mRNA turnover rates are weakly related to cell size, but that mRNA concentrations are strongly size-dependent. This establishes that transcriptional regulation is the basis of proteome remodeling. Live-cell imaging of nascent mRNAs using the MS2 system is used to measure how transcriptional dynamics change with cell size. Larger cells prolong transcriptional bursts and shorten inactive periods between bursts but maintain similar burst amplitudes to achieve transcriptional scaling. Together, our results show how transcription is modulated by cell size to remodel the proteome and alter cell physiology.

overall_design: SLAM-seq profiling of CCND1 overexpression or knockdown hTERT-RPE-1 lines with  induction by doxycycline at day 4. Cells are pulsed with 10uM thiouridine at day 4 for 24 hours, with the media refreshed every 3 hours. Chase is started by washing twice with PBS and applying growth media with 1mM uridine. Negative control of unlabelled samples are also provided. Note that time points are not uniform across replicates and samples as only high quality datasets were selected for downstream analysis and upload

first: Christopher

middle: H

last: Bohrer

first: Purva

last: Rumde

first: Ioannis

last: Sanidas

first: Matthew

last: Swaffer

first: Daniel

last: Larson

first: Josh

last: Elias

first: Michael

last: Lanz

first: Jan

last: Skotheim


title: In vivo RNA metabolic labeling identifies the sentinel cells of the regenerating zebrafish heart [scslam-seq]

city: Berlin

first: Janita

last: Mintcheva

address: Robert-Rössle-Str. 10

institute: Max-Delbrück Center for Molecular Medicine

summary: The adult zebrafish heart has high regenerative capacity.  Heart injury triggers a cascade of events including inflammation, fibrosis, revascularization, and proliferation of cardiomyocytes, culminating in full functional recovery of the heart. However, the first events that are induced upon injury and trigger the regenerative cascade remain elusive. Here, we established in vivo single-cell RNA metabolic labeling in the adult heart to systematically identify the cells that first respond to injury, as well as the processes that are induced in these sentinel cells. We found that activation of damage response pathways including Toll-like receptor signaling and expression of pro-inflammatory genes in macrophages are the first response to heart injury. In conjunction with detailed dissection of single-cell transcriptomics atlases, this finding led us to postulate that macrophage-specific inhibition of Toll-like receptor signaling would improve hallmarks of regeneration, a hypothesis we validated in functional experiments. In summary, our work establishes RNA metabolic labeling as a powerful approach for measuring perturbation response in vivo, identifies macrophages as the sentinel cells of the heart, and highlights the importance of fine-tuning the early pro-inflammatory phase for optimal regenerative outcomes.

overall_design: scSLAM-seq data of sham or cryoinjured adult zebrafish hearts

first: Tzu-Lun

last: Tseng

first: Pinelopi

last: Goumenaki

first: Anika

last: Neuschulz

first: Anis

last: Senoussi

first: Ronny

last: Schäfer

first: Sara

last: Lelek

first: Daniela

last: Panakova

first: Didier

last: Stainier

first: Jan Philipp

last: Junker


title: Hypoxia-EPAS1 imprints immunoregulation and maintenance of type 2 tissue-resident CD4+ T cells III

first: Atsushi

last: Sasaki

overall_design: Naive CD4⁺ T cells were isolated from mouse spleens and differentiated into Th2 cells in vitro. During Th2 differentiation, cells were transduced with an empty vector (Empty), an EPAS1 wild-type (Epas1WT) vector, or a constitutively stabilized EPAS1 mutant (Epas1TM) vector. Bulk RNA sequencing was performed to compare transcriptional profiles among the three groups, with two independent biological replicates per condition. Chromatin immunoprecipitation sequencing (ChIP-seq) was performed to map EPAS1TM-associated chromatin binding in Th2 cells. Naive CD4⁺ T cells were differentiated into Th2 cells and transduced with either an EPAS1TM vector carrying a Myc tag or an empty vector (Empty). ChIP was performed using an anti-Myc antibody to immunoprecipitate EPAS1TM-Myc–bound chromatin. Two independent biological replicates were analyzed per condition, and matched input DNA controls were included for each replicate.

first: Masahiro

last: Kiuchi

first: Hiroyuki

last: Yagyu

first: Takahiro

last: Arano

first: Tsuguko

last: Tomita

first: Kanae

last: Ohishi

first: Shigenori

last: Baba

first: Kaori

last: Tsuji

first: Rui

last: Hirasawa

last: Nemoto

first: Takahisa

last: Hishiya

first: Chiaki

last: Iwamura

first: Takahide

last: Toyoda

first: Masayuki

last: Ota

last: Onodera

first: Motoko

last: Kimura

first: Shinichiro

last: Motohashi

first: Hidemi

last: Suzuki

first: Jun-ichiro

last: Ikeda

first: Damon

last: Tumes

first: Toshinori

last: Nakayama

first: Takuji

first: Kiyoshi

last: Hirahara


title: Transcriptomic profiling of AC16 cardiomyocytes under TPEN-induced zinc deficiency

city: Hefei

first: Zhaoyuan

last: Wu

state: Anhui

address: Luyang District

department: Central Laboratory

institute: Wanbei Coal Electricity Group General Hospital

summary: Zinc deficiency is an important risk factor for myocardial injury, yet the underlying cytoprotective mechanisms remain incompletely understood. In this study, bulk RNA sequencing was performed in human AC16 cardiomyocytes treated with the zinc chelator TPEN or vehicle control to characterize transcriptional changes induced by zinc deficiency. Differential expression and pathway enrichment analyses revealed activation of apoptosis- and stress-related pathways, providing a transcriptomic framework for subsequent mechanistic investigations of ATF3-mediated mitochondrial and survival signaling.

overall_design: Bulk RNA-seq was performed on AC16 cardiomyocytes treated with TPEN (6.25 μM, 24 h) or vehicle control. Each group contained three independent biological replicates. Differential gene expression analysis was conducted to identify transcriptional responses to zinc deficiency.


title: The shelterin subunit TRF-1 mediates PRC2 function at ectopic telomere repeats in Neurospora crassa [ChIP-seq]

city: Sapporo

first: Hideki

last: Tanizawa

state: Hokkaido

address: Kita 15, Nishi 7, Kita-ku

department: Institute for Genetic Medicine

institute: Hokkaido University

summary: Telomeres are crucial for maintaining chromosomal integrity and are characterized by repetitive sequences, which may be stabilized by the shelterin protein complex and/or by formation of secondary structures, such as G-quadruplexes (G4 DNA). Frequently, subtelomeric regions are decorated with di- and tri-methylated lysine 27 on histone H3 (H3K27me), repressive marks associated with facultative heterochromatin in many eukaryotes.  Our previous work with the filamentous fungus Neurospora crassa demonstrated that native telomere repeats induce H3K27me at ectopic loci.  Here we report investigations into the mechanism of this and demonstrate that nonnative telomere repeats can also induce H3K27me.  Chromatin immunoprecipitation (ChIP) experiments with an anti-G4-DNA antibody showed that establishment of H3K27me does not depend on the formation of G4 DNA.  Other ChIP experiments demonstrated binding of the shelterin member TRF-1 to interstitial telomere repeats that induce H3K27me but not to similar sequences that do not.  Tethering experiments revealed that TRF-1, but not other proteins tested, is sufficient to induce H3K27me.  Hi-C analyses demonstrated that ectopic telomeric repeats can interact with native telomeres.  Together these results suggest that the shelterin complex plays a crucial role in establishment of H3K27me and repression at telomere sequences.

overall_design: ChIP-seq experiments were conducted to assess the genome-wide distribution of H3K27me2/3 and to evaluate the binding of shelterin component TRF-1 in Neurospora crassa. Strains containing various telomere or telomere-like repeat sequences inserted at the csr-1 locus were analyzed. For TRF-1 binding studies, a TRF-1-3xFLAG knock-in strain was used. ChIP was performed using antibodies against H3K27me2/3 or FLAG.

first: Colleen

last: Mumford

first: Peregrine

last: Painter

first: Kevin

last: McNaught

first: Nolan

last: Smith

first: Shinji

last: Honda

first: Osamu

last: Iwasaki

first: Sanki

last: Tashiro

first: Ken-ichi

last: Noma

first: Eric

middle: U

last: Selker


title: Effect of HNF1A-MODY on stem cell islets [scRNA-Seq]

city: Bergen

first: Lucas

last: Unger

address: Haukelansbakken 15

country: Norway

institute: University of bergen

summary: This project investigates the impact of the hotspot mutation P291fsinsC in HNF1A-MODY (Maturity-Onset Diabetes of the Young) on stem cell-derived islets. RNA sequencing (RNA-seq) was performed on islets differentiated from mutant and control HNF1A-MODY stem cells to study the mutation's effect on gene expression. By comparing the transcriptomic profiles of these islets, the study aims to uncover molecular mechanisms underlying the dysfunction caused by the P291fsinsC mutation during islet development and maturation.

overall_design: HNF1A-MODY stem cells with the P291fsinsC hotspot mutation and wild-type controls were differentiated into the posterior foregut stage.

first: Simona

last: Chera


title: The Chromatin Remodeler, Mit1, Enables the Propagation of the Histone Modification, H3K9me

city: Waltham

first: Kaushik

last: Ragunathan

address: 415 South St

institute: Brandeis University

summary: Chromatin structure dynamically regulates the balance between DNA accessibility and compaction, which is essential for controlling gene expression in eukaryotic cells. ATP-dependent chromatin remodelers are key effectors in this process, modulating nucleosome positioning to establish either open or repressive chromatin states. The CHD family of remodelers exhibits diverse roles in transcriptional regulation, yet the mechanisms by which they contribute to transcriptional silencing remain incompletely understood. In Schizosaccharomyces pombe, the CHD remodeler Mit1 functions within the SHREC complex to enforce heterochromatin formation, but its precise mode of action is unclear. Here, we directly tether Mit1 to an ectopic chromatin locus and demonstrate that Mit1 recruitment induces transcriptional gene silencing and nucleates broad domains of H3K9 methylation in a manner dependent on the Clr4 methyltransferase. This silencing requires Mit1’s remodeling activity but does not involve direct physical interaction with Clr4, indicating that Mit1 remodels chromatin to create a substrate favorable for Clr4 function. Furthermore, we find that enables the formation of de novo heterochromatin domains genome-wide. Our findings uncover a novel feedback mechanism whereby CHD-family remodelers initiate stable heterochromatin formation through chromatin remodeling that promotes methylation-based silencing, revealing new insights into the interplay between nucleosome dynamics and epigenetic gene regulation.

overall_design: Chromatin immunoprecipitation sequencing (ChIP-seq) for H3K9me2 and H3K9me3

first: Melissa

last: Seman

first: Alexandria

last: Latuda

first: Fenting

last: Huang

first: Agnisrota

last: Mazumder

first: Luca

last: Wolfstadter

first: Sigurd

last: Braun


title: CRISPR/Cas9 screening reveals key role of STK11/LKB1 in vasopressin-mediated regulation of Aqp2 transcription

city: Bethesda

first: Lihe

last: Chen

state: MD

address: 10 center drive MSC 1603, Building 10, Room 6N228

institute: NHLBI

summary: Identification of signaling networks is an essential goal in systems biology.  Here, we use CRISPR knockout screening (employing a whole kinome sgRNA library) to identify functionally critical protein kinases in a well-studied Gs-dependent G-protein coupled receptor (GPCR)-signaling model, namely the vasopressin V2 receptor (V2R). Screening was done using a specially-designed fluorescence-based reporter cell line with green-fluorescent protein (GFP) co-transcribed with Aqp2, a gene whose transcription is dependent on vasopressin-mediated activation of protein kinase A (PKA). The reporter line endogenously expresses V2R. Positive regulators (n=16) included PKA-catalytic subunit α (but not β). Mark2 (Par1b) and Dyrk1a (minibrain homolog).  Negative regulators (n=13) included PKA-regulatory subunit type Iα, Stk11 (liver kinase B1 [LKB1]), and three TGF-β receptor subunits (Tgfbr1, Tgfbr2, Tgfbr3) (see https://esbl.nhlbi.nih.gov/Databases/Kinome-CRISPR-screen/ for full list). Dyrk1a and Stk11/LKB1 knockout cell lines were created for further study.  Dyrk1a knockout cell lines failed to express AQP2 protein and exhibited a profound decrease in AQP2 mRNA. RNA-sequencing demonstrated widespread increases in cell-cycle transcripts, with a general defect in cell differentiation, accounting for AQP2 loss. Stk11/LKB1 knockout lines displayed marked increases in AQP2 protein and mRNA.  RNA-sequencing and phospho-proteomic findings point to a signaling model in which Stk11/LKB1- and PKA-mediated phosphorylation events exert counteracting effects on the activities of cAMP-responsive transcriptional coactivator (CRTC) proteins.  Finally, additional studies confirmed that TGF-β exposure to un-transformed cells results in a profound decrease in AQP2 mRNA abundance along with most recognized differentiation markers, consistent with our prior conclusion that TGF-β represses Aqp2 gene expression by inducing epithelial-mesenchymal transition.

overall_design:
mpkCCDc11-38 cells (n= 4 per group) were treated under four experimental condition: vehicle-treated, dDAVP (0.1 nM), 1 ng/mL TGF-b + dDAVP (0.1 nM), and 10 ng/mL TGF-b + dDAVP (0.1 nM) for 48 h. dDAVP was applied to the basolateral side, whereas TGF-b was applied to both apical and basolateral side of polarized cells, followed by RNA sequencing.
CRISPR/Cas9-mediated Stk11 KO mpkCCDc11-38 cells (n=5) were compared with wild-type mpkCCDc11-38 cells (n=5) under vehicle or vasopressin analog dDAVP (0.1 nM) treatment for 48 h, followed by sample collection for RNA sequencing.
CRISPR/Cas9-mediated Dyrk1a KO mpkCCDc11-38 cells (n=4) were compared with wild-type mpkCCDc11-38 cells (n=4). Cells were treated with vasopressin analog dDAVP (0.1 nM) for 48 h, followed by sample collection for RNA sequencing.

first: Euijung

last: Park

first: Mark

middle: A

last: Knepper


title: Ceramide Disrupts TM9SF2-PGK1 Axis to Redirect PD-L1 Trafficking and Enhance Antitumor Immunity

city: jinan

first: Mengmeng

last: Wang

address: wenhuaxilu

institute: shandong university of pharm

summary: The programmed cell death protein 1 (PD-1) / programmed death-ligand 1 (PD-L1) axis represents a cornerstone of cancer immunotherapy, yet the dynamic shuttling of PD-L1 between endosomal recycling and lysosomal degradation routes limits durable responses. Using a CRISPR screen targeting glycosphingolipid metabolism, we identify transmembrane 9 superfamily member 2 (TM9SF2) as a key regulator of PD-L1 levels. TM9SF2 orchestrates a dual mechanism: it recruits phosphoglycerate kinase 1 (PGK1) to promote PD-L1 recycling to the plasma membrane while dismantling the huntingtin-interacting protein 1-related protein (HIP1R)-mediated lysosomal degradation pathway. Genetic or pharmacological disruption of the TM9SF2-PGK1 complex depletes PD-L1 levels and boosts antitumor immunity. Further, the endogenous ceramide species Cer(d18:1/26:0) destabilizes this complex, triggering PD-L1 lysosomal destruction and potentiating antitumor immunity. These findings delineate a ceramide-gated sorting mechanism within the endosomal network, revealing a druggable metabolic switch to disrupt immune evasion and amplify checkpoint blockade efficacy.

overall_design: A CRISPR-Cas9 knockout (KO) screen targeting 50 genes involved in the sphingolipid synthesis/metabolic pathway

first: Z

last: Yi


title: R-loops shape the H2A.Z landscape and promote balanced lineage allocation during differentiation [ZX-1 CUT&Tag]

city: Worcester

first: Chun-Hao

last: Chao

address: 364 Plantation St. LRB560D

department: MCCB

institute: UMass Chan Medical School

summary: R-loops—RNA:DNA hybrids that often form co-transcriptionally—are emerging as key regulators of genome function, yet their roles in shaping chromatin architecture and developmental potential remain incompletely defined. Here, we use inducible Rnaseh1 expression in mouse embryonic stem cells (mESCs) to achieve acute, global R-loop depletion and systematically interrogate their impact on chromatin structure and lineage specification. We find that R-loop loss has minimal effect on steady-state gene expression or self-renewal. Instead, it leads to a striking reduction in H2A.Z occupancy at both active and bivalent promoters, accompanied by increased nucleosome density—revealing a previously unrecognized role for R-loops in maintaining promoter architecture. During gastruloid differentiation, R-loop-depleted mESCs exhibit accelerated ectodermal differentiation, along with dysregulation of lineage-specific transcription factors and impaired cell-cell signaling. Consistent with these alterations, R-loop-depleted cells show widespread perturbations in gene regulatory networks across several early cell types. These findings uncover a critical role for R-loops in shaping the H2A.Z chromatin landscape and preserving balanced lineage trajectories during early development, offering new insights into the epigenomic regulation of stem cell fate.

overall_design: Rnaseh1-inducible cells or ZX-1 with or without Dox treatment were subjected to CUT&Tag with antibodies/proteins including H2A.Z, H3K4me3, H3K9me3, H3K27me3, H3K27ac, H3K36me3, RNAPIISer2P, H3.3, H4ac and IgG

first: Thomas

last: Fazzio


title: FACT maintains nucleosomes around promoters and opposes to the spreading of chromatin factors, thereby playing a major role in chromatin architecture

first: Catherine

last: Chahrour

summary:
Amounting evidence suggests nucleosome organization is key for higher levels of chromatin packaging. However, studying it in higher eukaryotes is challenging due to the difficulty to manipulate nucleosomes in vivo and reconstitute their large higher-order chromatin domains in vitro.
Facilitates chromatin transcription (FACT) is a H2A/B histone chaperone that displaces and re-assembles histones, being important for processes such as transcription. Recent studies report a minor role of FACT in chromatin architecture. Here, we studied chromatin organization upon rapid FACT degradation employing a multidimensional 3C technique with base-pair resolution (MCC-u) and genomic approaches.
FACT loss leads to nucleosome depletion around active promoters, nano-scale domain collapse and emergence of larger nucleosome-free regions with increased protein binding. At a larger scale, we detect increased interactions between promoters across topologically associated domains. Thus, we demonstrate FACT plays a major role in chromatin organization and gain insights into the role of nucleosomes in genome architecture.

overall_design: Refer to individual Series


title: The retinoic acid receptor transcriptional coregulator RIP140 predicts poor outcome and restrains ATRA response in acute myeloid leukemia

city: Montpellier

last: Teyssier

address: 208, rue des apothicaires

country: France

summary: Despite advances in acute myeloid leukemia (AML) therapy, overall survival remains poor except in acute promyelocytic leukemia (APL), where all-trans retinoic acid (ATRA) dramatically improved outcomes. However, ATRA showed limited efficacy in non-APL AML, highlighting the need for biomarkers to identify differentiation-responsive patients. Here, we demonstrated that the transcriptional coregulator RIP140 was upregulated in hematopoietic stem cells, immature AML blasts, and at relapse, and acted as a poor prognostic factor across most AML subtypes except APL, where high expression was associated with a better predicts ATRA response. RNA-seq analysis in RIP140-silenced MOLM14 cells revealed that RIP140 did not block ATRA-induced myeloid differentiation but redirected it: in RIP140-expressing cells, ATRA enhanced immune cell activation and mitochondrial metabolism while it repressed cell-cycle and DNA replication/repair pathways. RIP140 silencing enhanced ATRA effect on chromatin, transcription and macromolecules biosynthesis, whereas RIP140 shifted ATRA inhibitory effects toward translational control and ubiquitin-proteasome-mediated protein degradation. Consistent with this transcriptional reprogramming, RIP140 knockdown enhanced ATRA-induced differentiation, apoptosis, and proliferation arrest. These findings position RIP140 as a novel biomarker and potential therapeutic target to optimize ATRA responsiveness in non-APL AML patients.

overall_design: RNAseq profiling of MOLM14 AML cells under four treatment conditions: untreated control, doxycyclinn (DOX), all-trans retinoic acid (ATRA), and combined ATRA+DOX treatment for 48h, with three biological replicates per condition (n=3)

first: Valentin

last: Jacquier

first: Vincent

last: Cavaillès


title: Hypoxia-EPAS1 imprints immunoregulation and maintenance of type 2 tissue-resident CD4+ T cells IV

city: Chiba

first: Atsushi

last: Sasaki

address: Chuo ku Inohana 1-8-1

institute: Chiba University

summary: CD4+ tissue-resident memory T cells (TRM) drive tissue-specific chronic inflammation by expressing characteristic functional molecules, but how tissue microenvironment-derived stimuli orchestrate their characteristics remains unclear. We found that intracellular hypoxia-dependent activation of EPAS1 programmed human and mouse type 2 CD4+ TRM to acquire immunoregulatory properties through the upregulation of CTLA-4 and IL-10. Genetic deletion of Epas1 in CD4+ T cells decreases the expression of immunoregulatory molecules in type 2 CD4+ TRM, consequently exacerbating lung inflammation. Spatial transcriptomic profiling of human asthmatic lungs identified FOXP3-negative CTLA4+GATA3+ TRM cells with activated hypoxic gene signatures in tertiary lymphoid structures. Our findings revealed that hypoxic pathway activation is a critical determinant of type 2 CD4+ TRM characteristics and highlight Epas1-dependent pathways as promising targets for treating chronic inflammatory disorders.

overall_design: Single-cell RNA sequencing was performed to assess the role of Epas1 in tissue-localized CD4⁺ T cells during chronic allergic airway inflammation. Mice carrying floxed Epas1 alleles (Epas1 fl/fl) or T cell–specific Epas1 deletion (Epas1 fl/fl, CD4-Cre) were subjected to repeated house dust mite (HDM) exposure for five weeks. Prior to sacrifice, intravascular staining was performed by intravenous injection of anti-CD45 antibodies to distinguish intravascular from tissue-localized T cells. CD44hi CD4⁺ T cells were isolated from lung tissue and stratified based on intravascular staining status (i.v. positive or negative). Cells from two mice per genotype were pooled, and single-cell RNA-seq was performed across four groups defined by genotype and intravascular staining status.

first: Masahiro

last: Kiuchi

first: Hiroyuki

last: Yagyu

first: Takahiro

last: Arano

first: Tsuguko

last: Tomita

first: Kanae

last: Ohishi

first: Shigenori

last: Baba

first: Kaori

last: Tsuji

first: Rui

last: Hirasawa

last: Nemoto

first: Takahisa

last: Hishiya

first: Chiaki

last: Iwamura

first: Takahide

last: Toyoda

first: Masayuki

last: Ota

last: Onodera

first: Motoko

last: Kimura

first: Shinichiro

last: Motohashi

first: Hidemi

last: Suzuki

first: Jun-ichiro

last: Ikeda

first: Damon

last: Tumes

first: Toshinori

last: Nakayama

first: Takuji

first: Kiyoshi

last: Hirahara


title: R-loops shape the H2A.Z landscape and promote balanced lineage allocation during differentiation [RNA-Seq]

first: Chun-Hao

last: Chao

overall_design: Rnaseh1-inducible cells were differentiated into gastruloids with and without Dox treatment. Samples were collected at Days 0 (0 hr), 3 (72 hrs), and 5 (120 hrs) for bulk RNA-seq

first: Thomas

last: Fazzio


title: Anticancer activity of RAS-GTP inhibition in cholangiocarcinoma

city: Pamplona

first: Silvestre

last: Vicent

address: Avenida Pío XII 55

institute: Center for Applied Medical Research (CIMA)

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: R-loops shape the H2A.Z landscape and promote balanced lineage allocation during differentiation [MNase-Seq]

overall_design: Rnaseh1-inducible cells with or without Dox treatment were subjected to MNase-seq with different amount of MNase for the digestion.


title: FACT maintains nucleosomes around promoters and opposes to the spreading of chromatin factors, thereby playing a major role in chromatin architecture [ChIP-seq]

first: Catherine

last: Chahrour

summary: Amounting evidence suggests nucleosome organization is key for higher levels of chromatin packaging. However, studying it in higher eukaryotes is challenging due to the difficulty to manipulate nucleosomes in vivo and reconstitute their large higher-order chromatin domains in vitro. Facilitates chromatin transcription (FACT) is a H2A/B histone chaperone that displaces and re-assembles histones, being important for processes such as transcription. Recent studies report a minor role of FACT in chromatin architecture. Here, we studied chromatin organization upon rapid FACT degradation employing a multidimensional 3C technique with base-pair resolution (MCC-u) and genomic approaches. FACT loss leads to nucleosome depletion around active promoters, nano-scale domain collapse and emergence of larger nucleosome-free regions with increased protein binding. At a larger scale, we detect increased interactions between promoters across topologically associated domains. Thus, we demonstrate FACT plays a major role in chromatin organization and gain insights into the role of nucleosomes in genome architecture.

overall_design:
ChIP-seq for H3, H2A, H2B, H3K27ac in SSRP1-FKBP E14 cells treated for 6h with DMSO and 3h with dTAG-13. Drosophila spike-in was added for reference normalization.
ChIP-seq for TBP in SSRP1-FKBP E14 cells treated for 3h with DMSO and 3h with dTAG-13. Drosophila spike-in was added for reference normalization.

first: Ana

middle: M

last: Dopico-Fernandez

first: Robert

last: Beagrie

last: Milne


title: FACT maintains nucleosomes around promoters and opposes to the spreading of chromatin factors, thereby playing a major role in chromatin architecture [TT-seq]

overall_design: TT-seq in SSRP1-FKBP E14 cells treated for 6h with DMSO and 3h with dTAG-13. Drosophila cells were used as spike-in for reference normalization.


title: FACT maintains nucleosomes around promoters and opposes to the spreading of chromatin factors, thereby playing a major role in chromatin architecture [MCC]

overall_design: MCC-ultra for SSRP1-FKBP E14 cells treated with DMSO 6h and dTAG-13 3h

first: Hangpeng

last: Li

first: James

last: Dalgleish


title: In situ fate mapping identifies the neonatal origin of platelet-biased hematopoietic stem cells [Native Developmental Atlas]

city: BARCELONA

first: Elvira

last: Gonzalez-Saiz

state: BARCELONA

address: CARRER BALDIRI REIXAC 10

institute: IRB BARCELONA

summary: Hematopoietic stem cells (HSCs) display heritable and deterministic gene regulatory states associated with functional heterogeneity. However how these biased fate programs change through development remains poorly understood. Here, using single-cell lineage tracing, we map the function and state of thousands of individual mouse HSCs spanning from the earliest mature fetal stages until young adults. We identify a burst of platelet- and myeloid-biased HSCs during the first postnatal week. Fetal HSCs retain a fetal-like program even after 4 months in the recipient bone marrow, suggesting that the adult HSC program is epigenetically configured early after birth. Creating a single-cell atlas of mouse perinatal HSCs, we identify a transient HSC state with hallmarks of Epithelial-Mesenchymal Transition (EMT), mTOR complex 1 activity, and cholesterol synthesis (SREBP targets). Tracing the rare cells that traverse this transient state, we reveal the neonatal origin of long-term platelet/myeloid-biased HSCs in both native and transplantation hematopoiesis. Transient treatment with mTORC1 inhibitor rapamycin during the first postnatal week suffices to reduce the platelet-biased adult HSC programming, without affecting total HSC numbers. In sum, we reveal a late developmental origin for platelet-biased HSCs, with important implications for hematopoietic aging.

overall_design: P2: HSCs were isolated from 7 2-days-old WT mice to get this scRNAseq dataset to build the naive developmental atlas. 2 more pups Tppp3-CreERT2 Rosa26-lox-STOP-lox-TdTomato were sequenced at P2 as replicates of this timepoint after labeling them with Tamoxifen at P0. E19 dataset: HSCs were isolated at day ~19 of gestation (embryos from E19) to get another timepoint dataset for the atlas. These datasets were used to build an integrated developmental HSC naive atlas together with already published GSE128761 series (Y. Li et al. Cell Stem Cell 2021)

first: Marta

last: López-Osias

last: González-Saiz

first: Daniel

last: Fernández-Pérez

first: Pedro

last: Sánchez

first: Indranil

last: Singh

first: Anna

last: Adrover

first: Alejo

last: Rodríguez-Fraticelli


title: FACT maintains nucleosomes around promoters and opposes to the spreading of chromatin factors, thereby playing a major role in chromatin architecture [RNA-Seq]

city: Oxford

address: MRC Weatherall Institute of Molecular Medicine

overall_design: RNA-seq in WT cells treated with DMSO for 6h and SSRP1-FKBP E14 cells treated for 6h with DMSO and dTAG-13. Drosophila cells were used as spike-in for reference normalization.


title: Transcriptomic responses to acute or chronic ethanol exposure observed across five brain regions in male mice

city: St. Louis

first: Gabor

last: Egervari

email: gabor@wustl.edu, egervarilab@gmail.com

state: MO

address: 4515 McKinley Ave, Couch Building

department: Genetics, Biochemistry and Molecular Biophysics

institute: Washington University School of Medicine

summary: Alcohol use disorder (AUD) has been consistently linked to transcriptomic dysregulation in the brain. Alcohol itself produces transcriptomic changes in the brain, and resulting molecular changes may contribute to the progression or maintenance of AUD. However, little is known about how ethanol-induced transcriptomic outcomes differ between acute and chronic exposure. Here, we systematically investigated the transcriptomic effects of ethanol exposure across key brain regions relevant to AUD and how these outcomes differ between acute and chronic exposure. To this end, RNA sequencing was done for the cortex, dorsal hippocampus, ventral hippocampus, dorsal striatum, and ventral striatum of adult male mice that were given a daily i.p. dose of ethanol or saline for 1 day (acute) or 10 days (chronic). Both ethanol exposure paradigms resulted in dysregulated expression (gene and transcript) and splicing in the investigated brain regions. Transcriptomic dysregulation varied across regions and was more robust following acute than chronic ethanol exposure. Paradigm-dependent transcriptional effects of ethanol were limited in most regions but were striking in the ventral hippocampus, suggesting alcohol's transcriptomic effects in this region might play a relevant role in the transition from limited to chronic alcohol consumption. Overall, this systematic study illuminates alcohol's effects on transcriptional regulation in the brain and how these effects differ across brain regions and by extent of exposure, ultimately providing valuable insight for developing novel therapeutics for AUD.

overall_design: RNA-seq samples are from adult male C57BL/6J mice 8-10 weeks of age that were given a daily i.p. dose of 2 g/kg ethanol or saline (control) for 1 day (acute) or 10 days (chronic) and sacrificed 4 hours after the final injection. Five brain regions were investigated: cortex (CTX), dorsal hippocampus (dHPC), ventral hippocampus (vHPC), dorsal striatum (DS), ventral striatum (VS). Experimental conditions are defined by paradigm (acute or chronic) and treatment (saline or ethanol): acute saline (AP), acute ethanol (AE), chronic saline (CP), chronic ethanol (CE). Each sample corresponds to a biological replicate (mouse); there are n=3-4 biological replicates (mice) per group.

first: Erica

last: Periandri

first: Kala

last: Dodson

first: Mariana

last: Lopes

first: Francisca

middle: N

last: de Luna Vitorino

first: Anjola

last: Ola

first: Joanna

last: Gongora

first: Benjamin

last: Garcia

first: Karl

last: Glastad


title: Identification of  Therapeutic Targets for Low-Grade Serous Ovarian Carcinoma [EM-seq]

city: Melbourne

first: Kathleen

last: Pishas

address: 305 Grattan Street

type: Methylation profiling by high throughput sequencing

summary: Low-grade serous ovarian carcinoma (LGSOC) is a rare, indolent ovarian cancer subtype with limited effective therapies. Approximately 40% of cases lack canonical MAPK/ERK or PI3K/AKT/mTOR pathway alterations and are classified as having no specific molecular profile (NSMP). These patients have poor responses to chemotherapy, MEK inhibitors, and hormonal therapies, highlighting the need for alternative strategies. This study aimed to identify novel therapeutic targets in NSMP LGSOC. A high-throughput drug screen of over 3,500 compounds (including FDA-approved, clinically tested, and investigational agents) was conducted across 12 LGSOC and one control ovarian epithelial cell line. EGFR inhibitors emerged as selective hits in NSMP cell lines and were further tested in two NSMP and two MAPK-mutant lines in combination with standard-of-care chemotherapy agents, carboplatin and paclitaxel. EGFR expression was assessed using RNA sequencing, DNA methylation profiling, and immunohistochemistry in primary tumours, followed by survival analysis based on expression levels.

overall_design: We aimed to profile the whole genome methylation landscape of 12 Low Grade Serous Ovarian Carcinoma patient derived cell lines and compared this to two normal cell lines

first: Hannah

last: Kim

last: Llaurado-Ferandez

first: Mark

last: Carey

first: Ian

last: Campbell

first: Dane

last: Cheasley

first: Raunak

last: Shrestha


title: R-loops shape the H2A.Z landscape and promote balanced lineage allocation during differentiation

first: Chun-Hao

last: Chao

overall_design: Rnaseh1-inducible cells with or without Dox treatment were subjected to DRIPc-seq.

first: Thomas

last: Fazzio


title: A Compendium of Cancer Organoid Models for Diverse Cancer Type

city: New York

first: Luca

last: Zanella

state: New York

address: 1130 St Nicholas Ave

department: Systems Biology

institute: Columbia University

summary: Development of new cancer therapeutics and validation of pathogenetic mechanisms in cancer require representative laboratory models. However, existing model collections do not yet represent the full spectrum of diversity observed in human cancer. Recent technologies enable more efficient model derivation (e.g., tumor organoids), yet whether such models maintain the molecular states of the originating tumor in long term ex vivo expansion has not been systematically investigated. Here, we present the results of a large-scale international program—the Human Cancer Models Initiative (HCMI)—including 665 patient-derived models from tumors provided by 2,594 donors. The collection enables tumor/model comparisons by whole genome, exome, methylome and transcriptome analyses. It includes 522 models with comprehensive clinical data, 153 models of rare cancers, and 71 models derived from non-European populations. Analysis of 421 matched tumor/model pairs revealed a high degree of genetic (97.8%) and epigenetic (95%) concordance. In glioblastoma, single-nucleus RNA sequencing of 16 tumor/model pairs revealed cell differentiation state shifts induced by specific culture medium formulations. We characterize extrachromosomal DNA-based gene amplification and post-treatment mutational signatures, which provide opportunities to study mechanisms of resistance to therapy. This publicly available collection of models, with accompanying molecular and clinical characterization and integrative software tools, provides the cancer research community with a powerful resource for preclinical investigation of cancer pathogenesis and treatment response.

overall_design: We performed single-nucleus RNA sequencing (snRNA-seq) of a set models and their parental tumors from three cohorts of the HCMI cancer model collection (colorectal, glioblastoma, pancreatic cancer). Multiple tumors or models (2 or 3) were multiplexed together in each run (see "title" under the "SAMPLES" section for the case ID of the multiplexed tumors/models, and "tumor or model" for the description of the sample type, either model or tumor). Frozen samples were obtained from the HCMI project collaborators at BPC Nationwide Children's Hospital, DFCI and ATCC.

first: Adina

last: Grunn

first: Mikko

last: Turunen

first: Patrick

last: Kerwin

first: Gaetano

last: Viscido

first: Iuliana-Cristina

last: Caescu

first: Andrea

last: Califano


title: Inhalable Nanomedicine Targeting Macrophages for Modulation of the STING Pathway in Pulmonary Fibrosis Therapy

city: Guangzhou

first: Yangeng

last: Wang

state: Guangdong

address: No. 777 East Xingye Avenue, Panyu District, Guangzhou, Guangdong, China

institute: South China University of Technology

summary: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease characterized by excessive immune activation and irreversible scarring of lung tissue. Recent evidence suggests that aberrant activation of the stimulator of interferon genes (STING) pathway in macrophages plays a critical role in driving inflammatory and fibrotic responses. To investigate this, we developed an inhalable lipid nanoparticle (LNP) platform encapsulating Cas9 mRNA and a sgRNA targeting Sting (mCas9/gSting@DOPS LNPs), enabling selective disruption of STING signaling in lung macrophages. Using a bleomycin-induced mouse model of pulmonary fibrosis, we performed bulk RNA sequencing on whole-lung tissues from healthy controls, bleomycin-challenged mice, and mCas9/gSting@DOPS LNP–treated mice. Transcriptomic profiling revealed extensive remodeling of the fibrotic lung environment, including suppression of type I interferon and chemokine signaling, restoration of immune homeostasis, and enrichment of pathways associated with tissue repair. Functionally, inhaled mCas9/gSting@DOPS LNPs mitigated alveolar collapse, reduced collagen deposition, and prolonged survival without eliciting systemic toxicity. These findings establish STING as a key pathogenic driver in pulmonary fibrosis and highlight the potential of inhalable gene-editing nanomedicine as a therapeutic strategy for chronic lung diseases.

overall_design: Whole-lung tissues were collected from three groups of C57BL/6 mice: healthy controls, bleomycin-challenged mice, and bleomycin-challenged mice treated with inhalable mCas9/gSting@DOPS LNPs. Total RNA was extracted, libraries were prepared with the Illumina TruSeq Stranded mRNA Kit, and sequencing was performed on an Illumina NovaSeq X Plus.

first: Yang

last: Liu


title: Adaptive cellular evolution in the intestinal tracts of hyperdiverse African cichlid fishes [scRNA-Seq]

city: Basel

first: Antoine

last: Fages

address: Vesalgasse 1

institute: University of Basel

summary: Adaptations relating to how nutrients are acquired and processed play a central role in the colonization of novel ecological niches and, therefore, in organismal diversification. While the evolution of feeding structures has been studied extensively in this context, the nature of dietary adaptations in the digestive tract remains largely unexplored. Here, we investigate the cellular and molecular basis of dietary adaptations in the massive radiation of cichlid fishes in Lake Tanganyika using comprehensive single-cell transcriptomic data derived from the intestines of 25 African cichlid species with distinct habitats and diets. We show that, at the cellular level, dietary adaptations are primarily driven by anterior enterocytes, and that both the abundance and gene expression profiles of these cells have evolved in response to dietary specializations. These dietary adaptations in enterocytes are driven by rapidly evolving cell population-specific genes, suggesting that alterations in gene regulatory programmes and cell-state specification promote ecological diversification.

sample_organism:
Oreochromis niloticus
Tropheus moorii
Haplochromis burtoni
Bathybates ferox
Callochromis pleurospilus
Cyphotilapia frontosa
Petrochromis orthognathus
Boulengerochromis microlepis
Lepidiolamprologus elongatus
Neolamprologus brichardi
Perissodus microlepis
Neolamprologus brevis
Telmatochromis vittatus
Triglachromis otostigma
Simochromis diagramma
Eretmodus cyanostictus
Haplotaxodon microlepis
Xenotilapia spiloptera
Limnotilapia dardennii
Lepidiolamprologus attenuatus
Julidochromis ornatus
Benthochromis horii
Cyprichromis coloratus
Trematocara zebra
Shuja horei

overall_design: single-cell RNAseq data from intestinal tracts of African cichlid fishes


title: Integrated Multiomic Profiling Enhances Risk Stratification and Prognostication in Canine Osteosarcoma

first: Anjali

last: Garg

address: Building 10, Room 1B53

department: Comparative Oncology Program,NCI

institute: National Institute of Health

summary: Osteosarcoma is a heterogeneous and aggressive primary bone malignancy that affects both canines and humans. Standardized treatment regimens prescribed to both species do not address the complexity of the disease and thus have resulted in stagnant patient outcomes for more than 30 years. Here we present the first multiomic dataset created from a large outcome-linked biobank of canine OS treatment-naïve primary tumors utilizing a computational framework designed to interrogate each dataset individually, and to compare and integrate findings. This work demonstrates the presence of MYC amplification as a poor prognostic indicator and as well as alterations in DNA damage repair, metabolism, and cell cycle genes that are shared with humans. Further, we show relationships between the local tumor immune microenvironment, TP53 mutations, MYC status and global gene methylation patterns that further highlight the complexity of the disease and provide new insight into the utility of prognostic biomarkers and potential druggable targets for future study.

overall_design: Whole genome bisulfite sequencing of primary OS (n=57) tumor tissue and normal bone samples from ribs and vertebrae (n=4) from NCI-COTC021/022 samples.Samples were sequenced on 8 NovaSeq 6000 S4 runs using EZ DNA Methylation-Gold Kit/Accel NGS Methyl-Seq DNA library preparation and paired-end sequencing.

first: Joshua

last: Mannheimer

first: Heather

last: Gardner

first: Cheryl

last: London

first: William

last: Hendricks

first: Guannan

first: Kenneth

last: Day

first: Sharadha

last: Sakthikumar

first: Manisha

last: Warrier

first: Christina

last: Mazcko

first: Jessica

last: Beck

first: Amy

middle: K

last: LeBlanc


title: Targeting amyloid-β pathology by chimeric antigen receptor astrocyte (CARA) therapy

city: Saint Louis

first: Khai

last: Nguyen

address: 425 South Euclid Ave, 8107, 8th floor BJCIH,

department: Pathology & Immunology

institute: Washington University in St. Louis

summary: Alzheimer’s disease (AD), the most common cause of dementia, is characterized by a complex pathological cascade initiated by amyloid buildup and progressing to tau-mediated neurodegeneration. While recent advances in anti-amyloid immunotherapies have shown promise, their inherent limitations underscore the need for innovative therapeutic paradigms. Here, we propose novel anti-amyloid chimeric antigen receptors expressed in astrocytes (CAR-A) and confirm their functionality in vitro. We further demonstrate the in vivo effectiveness of two designs in reducing amyloid plaque accumulation and related pathologies in a mouse model of amyloidosis. We also demonstrated the potential of CARA therapy in preventing amyloid burden and related pathologies prior to the plaque formation in the 5xFAD model. Our single nuclei analysis on pooled hippocampi showed that CAR-A treatment induced a unique glial cell response towards the amyloid pathology, reflecting that both astrocytes and microglia share the burden of amyloid aggregates. Upon the amyloid pathology, each CAR design induced additional astrocytic or microglial responses due to the distinct intracellular domains. These findings provide in vivo evidence and insights supporting the potential of CAR-A based cell or gene therapies for treating AD.

overall_design: 5xFAD mice were transfected with AAV containing Dectin1-CARA, Megf10-CARA, or GFP-CARA at 2 months old and 6 months old. Three months following transfection, brains from perfused mice were harvested and flash frozen. Nuclei were isolated from 4-5 randomly selected brain tissues. FACS was used to isolate NeuN- nuclei from cortical tissues, and NeuN- Olig2- nuclei from hippocampal tissues. Library was prepared using 10X Chromium Single-Cell 3' v3 and v4 kit (NeuN- samples) or 10X Chromium Single-Cell 5' v2 kit (NeuN- Olig2- samples) according to manufacturer instructions.

first: Yun

last: Chen

first: Yizhou

first: Marco

last: Colonna


title: Effect of HNF1A-MODY on stem cell islets

first: Lucas

last: Unger

address: Haukelansbakken 15

summary: This project investigates the impact of the hotspot mutation P291fsinsC in HNF1A-MODY (Maturity-Onset Diabetes of the Young) on stem cell-derived islets. RNA sequencing (RNA-seq) was performed on islets differentiated from mutant and control HNF1A-MODY stem cells to study the mutation's effect on gene expression. By comparing the transcriptomic profiles of these islets, the study aims to uncover molecular mechanisms underlying the dysfunction caused by the P291fsinsC mutation during islet development and maturation.

overall_design: HNF1A-MODY stem cells with the P291fsinsC hotspot mutation and wild-type controls were differentiated into pancreatic islets. RNA sequencing was performed on these islets to compare gene expression profiles and assess the impact of the mutation on islet development and function.

first: Simona

last: Chera


title: In vivo RNA metabolic labeling identifies the sentinel cells of the regenerating zebrafish heart [bulk_slamseq]

city: Berlin

first: Janita

last: Mintcheva

address: Robert-Rössle-Str. 10

institute: Max-Delbrück Center for Molecular Medicine

summary: The adult zebrafish heart has high regenerative capacity.  Heart injury triggers a cascade of events including inflammation, fibrosis, revascularization, and proliferation of cardiomyocytes, culminating in full functional recovery of the heart. However, the first events that are induced upon injury and trigger the regenerative cascade remain elusive. Here, we established in vivo single-cell RNA metabolic labeling in the adult heart to systematically identify the cells that first respond to injury, as well as the processes that are induced in these sentinel cells. We found that activation of damage response pathways including Toll-like receptor signaling and expression of pro-inflammatory genes in macrophages are the first response to heart injury. In conjunction with detailed dissection of single-cell transcriptomics atlases, this finding led us to postulate that macrophage-specific inhibition of Toll-like receptor signaling would improve hallmarks of regeneration, a hypothesis we validated in functional experiments. In summary, our work establishes RNA metabolic labeling as a powerful approach for measuring perturbation response in vivo, identifies macrophages as the sentinel cells of the heart, and highlights the importance of fine-tuning the early pro-inflammatory phase for optimal regenerative outcomes.

overall_design: bulk SLAM-seq data of zebrafish hearts injected with 4sU (different concentrations and injection methods), a bulk SLAM-seq time course and healthy hearts as a control

first: Tzu-Lun

last: Tseng

first: Pinelopi

last: Goumenaki

first: Anika

last: Neuschulz

first: Anis

last: Senoussi

first: Ronny

last: Schäfer

first: Sara

last: Lelek

first: Daniela

last: Panakova

first: Didier

last: Stainier

first: Jan Philipp

last: Junker


title: In vivo RNA metabolic labeling identifies the sentinel cells of the regenerating zebrafish heart [scrna-seq]

overall_design: scRNA-seq data of cryoinjured and healthy adult zebrafish hearts. Some data contain CRISPR/Cas9 based lineage information (not used in the publication)


title: A sympathetic-eosinophil neuroimmune axis orchestrates psychological stress to exacerbate skin inflammation

city: Shanghai

first: Shenbin

last: Liu

address: 131 Dongan road

department: Fudan University

institute: MOE Frontiers Center for Brain Science

summary: Psychological stress is hypothesized to trigger exacerbations of atopic dermatitis (AD). However, the neurobiological mechanisms linking stress to immune processes involved in atopy remain poorly understood. Here, we identified a subtype of skin-specific Prodynorphin (Pdyn)+ noradrenergic sympathetic neurons that closely interact with eosinophils, mediating the stress-induced aggravation of AD. Among AD patients, psychological stress was positively correlated with eosinophilic infiltration and the severity of inflammation. In AD-like murine models, genetic ablation of eosinophils or Pdyn+ sympathetic neurons mitigated stress-induced exacerbation of skin inflammation. Furthermore, optogenetic or chemogenetic activation of Pdyn+ sympathetic neurons precipitated AD inflammation through eosinophils. These neurons promote eosinophil recruitment via the CCL11-CCR3 axis and activate them through the noradrenaline-Adr2 pathway within the inflamed skin. Our findings uncover a neuroimmunological mechanism underlying psychological stress-induced exacerbation of AD, highlighting the Pdyn+ sympathetic-eosinophil axis as a crucial interface between the brain and skin inflammation, with potential therapeutic implications.

overall_design: For scRNA-seq of skin CD45+ cells, the tissues from EtOH- and MC903-treated mice were surgically removed and digested as described above (see the procedure of “flow cytometry”). After digestion, the samples were sieved through a 40 µM cell strainer and centrifuged at 300 g for 10 minutes. The obtained cell pellet was resuspended in 90 μL MACS buffer (supplemented with 0.5% bovine serum albumin (9048-46-8, Vetec) and 2 mM EDTA (60-00-4, Sigma-Aldrich) and 10 μL CD45 Microbeads (130-052-301, Miltenyi Biotec) per 107 cells, and the cells were incubated for 15 minutes at 4°C in the refrigerator. CD45+ cells were subsequently sorted as described above. Single cells were stained for viability assessment using Calcein-AM (Thermo Fisher Scientific) and Draq7 (BD Biosciences). The BD Rhapsody system was used to capture transcriptomic information of the single cells. Single-cell capture was achieved by randomly distributing a single-cell suspension across >200,000 microwells through a limited dilution approach.For sNucRNA-seq of sympathetic neurons, sympathetic ganglia (SCG, STG, T2-T4, CG) were dissected from thirty MC903-challenged mice and placed in the cold (4°C) artificial cerebrospinal fluid (ACSF) solution (in mM: 125 NaCl, 3 KCl, 20 NaHCO3, 1.25 NaH2PO4, 2 MgCl2, 20 D-glucose, 2 CaCl2, 0.0001 tetrodotoxin, 0.02 6,7-dinitroquinoxaline-2,3-dione, 0.05 DL-2-amino-5-phosphonovaleric acid), continually bubbled with 95%O2 and 5%CO2. Harvested tissue was homogenized at 4°C in a buffer containing 0.3% IGEPAL CA-630 and a protease inhibitor cocktail (ab65621, Abcam), and the following components (in mM): 250 sucrose, 25 KCl, 20 NaHCO3, 1.25 NaH2PO4, 5 MgCl2, 20 Tricine-KOH, 0.15 spermine, 0.5 spermidine, and 1 dithiothreitol. Samples were filtered through 70 mM strainers, diluted with iodixanol to a final concentration of 25%, and layered on an iodixanol gradient (30% and 40% iodixanol containing 0.04% BSA and RNasin (Promega N2611, 64 units/mL). The samples were centrifuged at 10,000 g for 20 minutes at 4°C, and the nuclei were collected from the 30-40% iodixanol interface. Sorted nuclei were immediately prepared as the 10x Genomics Cell Preparation Guide describes. The cell suspension was loaded into Chromium microfluidic chips using 3’ v3.1 chemistry and barcoded using a 10×Chromium Controller (10x Genomics). According to the manufacturer's instructions, RNA from the barcoded cells was reverse transcribed, and sequencing libraries were constructed with reagents from a Chromium Single Cell 3’ v 3.1 reagent kit (10x Genomics). Sequencing was performed using the Illumina NovaSeq 6000, following the manufacturer’s instructions (Illumina).

first: Jiahe

last: Tian

first: Yudian

last: Cao

first: Yilei

last: Li

first: Junlong

last: Sun


title: Methylome analysis of HGG_F samples

first: Leonille

last: Schweizer

address: Charitépl. 1

institute: Charité - Universitätsmedizin

summary: Methylome analysis of High Grade Glioma

overall_design: Genome-wide DNA methylation profiles were obtained using Infinium HumanMethylation450 (450 k) BeadChip or Infinium MethylationEPIC (850 k) BeadChip array (Illumina).

first: Amos

last: Münch