title: Bruceine A protects nuclear receptor 4A1 from ubiquitin-degradation to alleviate mesangial proliferative glomerulonephritis

city: Taipa

first: Huating

last: Hu

address: Avenida WaiLong

institute: Macau University of Science and Technology

summary: The nuclear receptor 4A1(NR4A1) plays a crucial role in maintaining cellular homeostasis and is involved in various disease processes; however, its functional role and pharmacological potential in mesangial proliferative glomerulonephritis (MsPGN) remain unexplored. In this study, we found that downregulation of NR4A1 promotes the pathogenesis of MsPGN by regulating inflammatory and proliferative responses in mesangial cells (MCs), whereas overexpression of NR4A1 reverses these processes. Bruceine A (BA) binds to NR4A1 at residues D481/Q568 and exhibits NR4A1-dependent anti-inflammatory and anti-proliferative effects both in vitro and in vivo. Notably, adeno-associated virus serotype 9 (AAV9)-mediated overexpression of NR4A1 alleviates glomerular injury and inflammatory cascades, while knockout of NR4A1 impairs the renoprotective effects of BA. BA binds to the ligand-binding domain (LBD) of NR4A1 and further sterically blocks K48-linked polyubiquitination at K558, thereby stabilizing NR4A1 protein levels. This stabilization enables NR4A1 to auto-activate its own promoter, amplifying the transcriptional repression of nuclear factor kappa-B (NF-κB) signaling phosphorylation, which ultimately attenuates inflammatory cascades and mesangial proliferation to confer renal protection. This study provides a promising therapeutic avenue for the development of next-generation therapies against MsPGN.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for healthy control group, anti-Thy1 nephritis model group  and its treatment groups (Bruceine A and Olmesartan).

first: Hudan

last: Pan

first: Runze

first: Kancheng

last: He

first: Ruimin

last: Tian

first: Chuanghai

first: Rongrong


title: T cell help transiently unlocks a high plasticity state in germinal center B cells during the humoral immune response [iPSC_RNA-seq]

status: Public on Oct 29 2025

city: New York

first: Prokopios Alexandros

last: Polyzos

address: 413 E 69th St

country: USA

institute: Weill Cornell Medicine

type: Expression profiling by high throughput sequencing

summary: During cellular differentiation, phenotypic plasticity is gradually lost due to the strengthening of epigenetic barriers, although it may be re-acquired upon disease or injury. Here, we report an unprecedented physiological gain of epigenetic plasticity among mature B cells during the humoral immune response. Acquisition of plasticity was strictly dependent on T follicular helper (TFH) cells and restricted to germinal center (GC) but not activated B cells, indicating both non- and cell-autonomous contributions to this phenotype. Importantly, GC plasticity was not dependent on proliferation and could not be solely explained by the activation of MYC programs. Instead, GC plasticity was associated with the reciprocal weakening of B cell identity and the de- repression of progenitor and stem cell-like programs induced by NFκB and other signals, downstream of TFH signaling. Therefore, physiological acquisition of GC B cell plasticity is tightly controlled by the affinity maturation positive selection checkpoint. Notably, Histone 1 loss of function, which results in aberrant stem cell gene reactivation through chromatin decompaction, allowed GC B cells to bypass this gatekeeping mechanism. Finally, patients with Diffuse LargeB Cell Lymphoma (DLBCL) that enriched for GC plasticity signatures had significantly worse outcomes, implicating a role for this mechanism in immune regulation and fitness and potential relevance to lymphomagenesis.

overall_design: Splenic B cells from mice (males) immunized with SRBC for 9 days were pre-enriched for B220pos cells by negative magnetic enrichment and stained for germinal center B cell markers (B220+ CD38- Fas+), including the Myc-GFP endogeneous reported for isolation of TFH-activated GC B cells by flow sorting. Bulk GC or TFH-activated GC B cells were plated on Mouse Embryonic Fibroblasts (MEF) for 6 days with cytokines for somatic cell reprogramming to induced Pluripotent Stem Cells (iPSC) upon treatment with Doxycyline (Dox). After 10 days of reprogramming, the iPSC colonies emerging were culture in abscence of Dox for 4 additional days to select for truly transgene-independent and pluripotent iPSCs. 3 individual colonies (clones) were manually picked and isolated from each Bulk GC or Myc-GFP GC-derived iPSCs. Cells were passaged once on gelatin only and pre-plated for an additional 45 min to remove the remaining MEF. Collected cells were processed for RNA extraction using the RNAeasy Qiagen kit according to the provider specifications. RNAs sent to Novogene Bioinformatics Technology Co. Ltd. for QC and Libraries preparation (non-stranded Poly A) and clustered on an Illumina NovaSeq X Plus Series (Pair End 150)

first: Laurianne

last: Scourzic

first: Alexander

first: Effie

last: Apostolou


title: ATACSeq of TET2-mutant human HSCs

city: London

first: Hector

last: Huerga Encabo

address: The Francis Crick Institute, 1 Midland Rd

country: United Kingdom

institute: The Francis Crick Institute

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The expansion of hematopoietic stem cell clones and their immune progeny carrying somatic mutations is defined as clonal hematopoiesis (CH) and its prevalence increases with age. Individuals with CH are at higher risk of developing hematological malignancies as well as other pathologies, such as cardiovascular or inflammatory pulmonary diseases. Understanding the environmental conditions that favor mutant clones and the CH-immune system response to such environments is key to designing therapeutic strategies to stall the expansion of mutant clones and the development of CH-associated pathologies. In this work, we characterized how mutations in TET2, increase the competitive advantage of human HSCs upon environmental stress conditions and simultaneously shape the immune response of their myeloid progeny. Employing a humanized mouse model, we unravel a cell-specific impact of TET2 mutations on HSCs and their myeloid progeny to inflammatory stress. In such environments, TET2Mut HSCs have a mitigated transcriptional upregulation of inflammatory pathways, and by contrast, TET2Mut myeloid cells have an exacerbated inflammatory response. A distinct monocyte-macrophage trajectory derived from TET2Mut HSCs contributes to pathological inflammation. These findings provide evidence at the human cell level and reconcile the notion that TET2-derived CH is associated with both increased stemness in the HSC compartment and inflammation elicited by the myeloid progeny.

overall_design: Using CRISPR-edited human HSCs and transplantation in immunodeficient mice, we have sorted CD34+CD38-CD45RA- cells and analyzed the chromatin conformation of TET2 mutant (TET2Mut) HSCs.

first: Huerga Encabo

last: Hector

first: Garcia-Albornoz

last: Manuel

first: Llorian

last: Miriam

first: Bonnet

last: Dominique


title: Circadian regulator REV-ERBα is a master regulator of tumor lineage plasticity and an effective therapeutic target [ChIP-Seq]

city: sacramento

first: Xiong

last: Zhang

state: CA

address: 4645 2nd Ave

institute: University of California, Davis

summary: Epigenetic and transcriptional dysregulation plays a fundamental role in tumor lineage plasticity (LP). However, the underlying mechanisms, especially for the initial events of LP development, are still poorly understood. Here, we report that in progression of prostate cancer from adenocarcinoma to treatment-induced neuroendocrine prostate cancer (t-NEPC), anti-AR signaling inhibitors (ARSIs) reprogram the function of a circadian regulator/nuclear receptor REV-ERBα by switching its target gene programs from kinase signaling and metabolic programs to programs of LP, which includes neurogenesis, stem cell and epithelia to mesenchymal transition (EMT) as well as over fifteen LP drivers including POU3F2/BRN2, ASCL1, FOXA2, ONECUT2 and MYCN. Unexpectedly, REV-ERBα facilitates the chromatin occupancy of BRN2, ASCL1 and FOXA1 in their activation of LP programs, thus functioning as a master regulator of ARSI-induced LP driver network. Mechanistically, REV-ERBα induces chromatin accessibility and H3K27ac modification at promoters of LP genes through its recruitment of BRD4 and p300. Overexpression of REV-ERBα alone is sufficient to induce LP and neuroendocrine phenotype and confers cell resistance to ARSI in adenocarcinoma cells. Loss of REV-ERBα potently inhibits NEPC cell growth and abolishes the expression of LP drivers and gene programs. Pharmacological inhibition of REV-ERBα exhibits high potency in blocking the growth of NEPC tumors including patient-derived xenografts.  Our findings reveal that therapy-induced LP development entails a coordinated induction of a network of LP drivers and that REV-ERBα is a novel master regulator of the network and a promising therapeutic target for treatment of advanced prostate cancer such as NEPC.

overall_design: ChIP-seq profiling of circadian regulator REV-ERBα, BRN2, ASCL1, FOXA1, BRD4,p300, H3K27ac  in ARSI-resistant prostate cancer cells and PDX tumors.

first: Yatian

last: Yang

first: Ronald M

last: Evans

first: Hongwu

last: Chen


title: Transcriptome-wide off target examining of AIM systems.

status: Public on Oct 30 2025

city: Beijing

first: Wu

last: Hao

address: No.5 Yiheyuan Road Haidian District, Beijing

department: School of Life Science

country: China

institute: Pecking University

summary:
Purpose: Development of specific and precise RNA base editing toolkit.
Methods: The transcriptome-wide off-target sites identification, annotation, and transcriptome-wide genes regulation of each editor were analyzed by RNA-seq data. The raw reads (.fastq) were first processed using Trim Glore (v0.6.7) to remove adapter sequences with default parameters. Then the trimmed data were used to remove rRNA data by Bowtie2 (v2.5.0) with key parameters –no-unal -I 1 -X 1000. The cleaned reads data were then aligned to the indexed human GRCh38 reference genome by STAR (v2.7.1a) with key parameters --outSAMstrandField intronMotif  --outFilterIntronMotifs RemoveNoncanonical. The aligned BAMs were added RG tag of sample information, sorted, indexed, stated by Samtools (v1.15.1) with default parameters. Then the processed BAMs were removed duplications by the Picard Markduplicates tool (v2.23.9) with key parameters ASO=coordinate REMOVE_DUPLICATES=true. Then remove duplicate BAMs were used to filter reads containing clips by Samtools with default parameters. All the cleaned BAMs were downsampled to an equivalent level for subsequent analysis to ensure a fair comparison of transcriptome-wide off-targets. The uniquely mapped reads in final BAMs were used to calculate gene expression level by Cuffdiff (v2.2.1) with default parameters. Finally, we filtered the differential expression genes using cutoff |Log2FoldChange| > 1 and P-value < 0.05, based on the Cuffdiff quantification results.
Results: Our new programmable RNA base editing toolkits have high editing efficiency, flexible editing scope and high transcriptome-wide specificity.
Conclusions: We deleveped verstaile RNA  editing toolkits enabling presice, specific and versatile RNA base editing.

overall_design: We developed new RNA base editing toolkits using single strand RNA deaminase TadA. The RNA-seq data were used to evaluate  the transcriptome-wide off-targets of the newly developded RNA base editing toolkits.

first: Hao

last: Wu


title: To investigate role of bile acids in modulating regulatory T cell phenotype under cholestatic conditions

city: Cincinnati

last: Miethke

state: OH

address: 3333 Burnet Ave, MLC 2010

institute: Cincinnati Childrens Hospital Medical Ce

summary: Regulatory T cells (Tregs) were shown to protect from cholestatic liver injury. Here we investigate whether amphiregulin (AREG), a ligand for epithelial growth factor receptor (EGFR) which promotes epithelial repair, mediates innate Treg functions in cholangiopathies.

overall_design:
CD25+CD127-/lowCD4+CD3+CD56-CD45+eflour506-cells FACS sorted from peripheral blood of healthy donors. FACs sorted Tregs were cultured with  α-CD3, α-CD28 (Beads 1:1ratio), hIL-2(100U), with either H2O(Vehicle) or with TCDCA (100 μM), 48hrs.
contributor: Zhang, Xiang and his Laboratory for Statistical Genomics and Systems Biology at the University of Cincinnati.

first: Ramesh

last: Kudira

first: Zhang

last: Xiang


title: ERRγ impedes neuroendocrine prostate cancer development [ChIP-Seq]

city: Montréal

first: Vincent

last: Giguère

state: QC

address: 1160 Pine Ave West, McIntyre Bldg Room 710

department: Goodman Cancer Centre

country: Canada

institute: McGill University

summary: Neuroendocrine prostate cancer (NEPC) is a lethal subtype of castration-resistant prostate cancer (CRPC). The molecular mechanisms underlying the progression of CRPC toward NEPC remain incompletely understood and effective treatments remain to be discovered. Herein, we report that loss of the nuclear receptor ERRγ promotes neuroendocrine differentiation in a Pten-deficient mouse model of prostate adenocarcinoma. These findings were recapitulated in advanced cellular and xenograft models of human prostate cancer (PCa). Critically, we show that ERRγ gain-of-function can reverse instilled NEPC features accompanied by suppression of growth and oncogenic metabolic reprogramming. Activation of a neuroendocrine transcriptional program enabled by ERRγ deficiency unveiled a targetable vulnerability exploited by combined pharmacological inhibition of EZH2 and RET kinase that effectively inhibited the growth of ERRγ-deficient tumor organoids and cells. Collectively, our findings demonstrate that ERRγ downregulation facilitates PCa adeno-to-neuroendocrine transformation and offer potential therapeutic strategies to prevent/treat the development of poor outcome NEPC.

overall_design: ERRγ ChIP-seq experiments were performed in engineered PTEN-ERRγ+ (sgPTEN+ERRγ) and PTEN-ERRγlow (sgPTEN) DU145 cells, the latter used as a control for ERRγ peak calling in PTEN-ERRγ+ cells. DU145 cells have high PTEN and low ERRγ status to which we generated PTEN-ERRγlow cells by stably knocking out PTEN by CRISPR/Cas9 followed by lentivirus-mediated ERRγ overexpression in PTEN-ERRγlow cells to generate PTEN-ERRγ+ cells. H3K27ac and H3K27me3 ChIP-seq experiments were performed in PTEN-ERRγ+ (sgPTEN+ERRγ) and PTEN-ERRγlow (sgPTEN) DU145 cells and peaks were called using respective input controls.

first: Ting

last: Li

first: Catherine

middle: R

last: Dufour

first: Lingwei

last: Han


title: Transcriptional dysregulation in the spinal cord of SCA3 provides insights into disease mechanisms

city: Ann Arbor

first: Hayley

middle: S

last: McLoughlin

state: MI

address: 109 Zina Pitcher Pl

department: Neurology, Human Genetics

institute: University of Michigan

summary: Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disease caused by polyglutamine repeat expansion in the ATXN3 gene. Despite the ubiquitous expression of ATXN3 throughout the body, SCA3 pathology is pronounced in select, vulnerable regions within the central nervous system (CNS). Notably, imaging studies of SCA3 patients have revealed spinal cord atrophy prior to ataxia symptom onset and progressing with disease severity. However, the molecular mechanisms underlying SCA3 pathology in the spinal cord remain largely unexplored. Here, we present, for the first time, a comprehensive analysis of the spinal cord transcriptome using SCA3 tissue from humans and mouse models. Our data demonstrates both early and progressive transcriptional dysregulation in the spinal cord, impacting key biological processes such as lipid metabolism, inflammation, cellular structure, and nucleic acid processing. Transcriptomic profiling of Atxn3 knockout mice revealed only weak transcriptional changes in the spinal cord, with minimal overlap compared to the robust alterations observed in SCA3 knock-in mice, indicating that the molecular signature of the disease in the spinal cord is likely not driven by ATXN3 loss of function. In addition, we identified aberrant RNA splicing, particularly affecting genes involved in cytoskeletal organization. Collectively, our findings indicate that SCA3-associated transcriptional dysregulation in the spinal cord contributes to canonical pathological mechanisms of SCA3 both early and progressively. These results underscore the central role of the spinal cord in SCA3 pathogenesis and advocate for its inclusion as a key therapeutic target.

overall_design: RNAseq of RNA collected from the spinal cord of SCA3 KIQ300 and WT mice at 24 and 56 weeks of age.

first: Jacen

last: Emerson

first: Brianna

last: Nelthrope

first: Emma

last: Walker

first: Grace

last: Mao

first: Hannah

last: Shorrock


title: Mitochondrial RNA cytosolic leakage drives the SASP

city: Rochester

first: Dominik

last: Saul

state: Minnesota

address: 200 First Street SW Rochester

institute: Mayo Clinic

summary: Senescent cells secrete proinflammatory factors known as the senescence-associated secretory phenotype (SASP), contributing to tissue dysfunction and aging. Mitochondrial dysfunction is a key feature of senescence, influencing SASP via mitochondrial DNA (mtDNA) release and cGAS/STING pathway activation. Here, we demonstrate that mitochondrial RNA (mtRNA) also accumulates in the cytosol of senescent cells, activating RNA sensors RIG-I and MDA5, leading to MAVS aggregation and SASP induction. Inhibition of these RNA sensors significantly reduces SASP factors. Furthermore, BAX and BAK play a key role in mtRNA leakage during senescence, and their deletion diminishes SASP expression in vitro and in a mouse model of Metabolic Dysfunction Associated Steatohepatitis (MASH). These findings highlight mtRNA's role in SASP regulation and its potential as a therapeutic target for mitigating age-related inflammation.

overall_design: RNA-seq profiling of proliferating and senescent fibroblasts with and without mitochondria, which were subsequently transfected with isolated mitochondrial RNA (mtRNA).

first: Victorelli

last: Stella

first: Han

last: Yeaeun

first: Saul

last: Dominik

first: Passos

last: Joao


title: Distinct miRNA profiles in human amniotic tissue and its vesicular and non-vesicular secretome

city: Wien

first: Andreas

middle: B

last: Diendorfer

address: Leberstraße 20

country: Austria

institute: TAmiRNA GmbH

type: Non-coding RNA profiling by high throughput sequencing

summary: The human amniotic membrane (hAM) is a valuable tissue in regenerative medicine due to its capacity to release cell-derived bioactive factors. Among these, microRNAs (miRNAs) play a critical role in modulating gene expression. This study aimed to comprehensively characterize the miRNA landscape of native viable hAM tissue and its secretome, with a focus on distinguishing between extracellular vesicles (EV)-associated and protein-bound miRNAs. We analyzed two anatomically and functionally distinct regions of the hAM, the reflected and placental amnion. After incubation of tissue biopsies ex vivo for 72 hours, vesicular and non-vesicular components in the conditioned medium were separated by size exclusion chromatography, and small RNA sequencing was performed on tissue and secretome fractions. Our analysis identified three main clusters of miRNA expression corresponding to tissue, EV-associated, and protein-bound fractions. We observed regional differences in miRNA expression between the reflected and placental amnion and identified miRNAs selectively released into EVs or protein-bound fractions. Data of gene ontology analysis suggest distinct biological roles for miRNA depending on the sample type. This dataset provides novel insights into the spatial and functional miRNA release profile of viable hAM and contributes to a better understanding of its regenerative potential.

overall_design: This study investigates small RNA expression profiles derived from different regions of the human amniotic membrane. Placental amnion (PA) and reflected amnion (RA) were separated from human placentae obtained after cesarean sections. Two biopsy punches (26 mm diameter) from each region were cultured for 72 hours (Dulbecco Modified Eagle’s medium/high glucose, 1 % v/v L-glutamine, 1 % v/v penicillin/streptomycin, 37 °C, 5 % CO2, humidified atmosphere), and extracellular vesicle and protein fractions were isolated from the conditioned media. Small RNAs from EV fractions, protein fractions, and tissue samples were analyzed using next-generation sequencing. Four biological replicates were included for each tissue type and sample fraction to assess differences in small RNA content.

first: Nefertiti

last: Chaves-Solano

first: Silvio

last: Kau-Strebinger

first: Johannes

last: Oesterreicher

first: Marianne

last: Pultar

first: Wolfgang

last: Holnthoner

last: Grillari

first: Simone

last: Hennerbichler

last: Brandstetter

last: Spittler

first: Matthias

last: Hackl

first: Susanne

last: Wolbank

first: Asmita

last: Banerjee

first: Adelheid

last: Weidinger


title: PolyA RNA seq of human glioblastoma cells treated with a pool of 11 miRNAs in vitro and in vivo (orthotopic tumors in mice)

city: Genova

first: Kiril

last: Tuntevski

address: Via Morego 30

country: Italy

institute: Istituto Italiano di Tecnologia

summary: Glioblastoma (GBM) has a poor prognosis due to infiltrative and therapy-resistant glioma stem-like cells. MicroRNAs (miRNAs) synergistically modulate biological pathways by repressing tumor-related genes, a mechanism lost in cancer. This study aimed to test a combination of different miRNAs exploiting their synergism as a multimodal therapy for GBM.

overall_design: In vitro: patient-derived GBM cells (GBM1 and GBM2) were transfected with a pool of 11 miRNAs or with scrambled control, in 2D adherent condition (glioma stem cell medium, matrigel-coated plastic). Cells were then cultured in speroid-permissive condition (glioma stem cell medium, uncoated plastic) for 3 days. In vivo: orthotopic tumors (GBM2 Luc+) were established in NOD/SCID mice striatum; after 42-49 days, animals received intratumoral injection of ApoE-coated LNP loaded with either the 11 miRNA pool or scrambled control. Animals were sacrificed 14 days after treatment and brain dissected to harvest tumoral tissue.

first: Silvia

last: Rancati

first: Rui

middle: C

last: Pereira

first: Michele

last: Schlich

first: Stefania

last: Sgroi

last: Beatini

first: Letizia

last: La Rosa

first: Lidia

last: Giantomasi

first: Roberta

last: Pelizzoli

first: Clarissa

last: Braccia

first: Carlotta

last: Spattini

first: Amanda

last: Lo Van

first: Meritxell

last: Pons-Espinal

first: Anna Lisa

last: Palange

first: Adriana

last: Bajetto

first: Antonio

last: Daga

first: Andrea

last: Armirotti

first: Tullio

last: Florio

first: Paolo

last: Decuzzi

first: Davide

last: De Pietri Tonelli


title: The opposing effect of TET2-derived clonal hematopoiesis in stemness and immunity

city: LONDON

first: Giuseppe

middle: Alessandro

last: D'Agostino

state: ENGLAND

address: 1 Midland Road

summary: The expansion of HSC clones and their immune progeny carrying somatic mutations is defined as clonal hematopoiesis (CH) with its prevalence increasing with age. Individuals with CH are at higher risk of developing hematological malignancies but also other pathologies, such as cardiovascular or inflammatory pulmonary diseases. Understanding which environmental conditions favor mutant clones and the CH-immune system response to such environments is key to designing therapeutic strategies to stall the expansion of mutant clones and the development of CH-associated pathologies. In this work, we characterized how mutations in TET2, one of the most frequent in CH, increase the competitive advantage of human HSCs upon environmental stress conditions and at the same time shape the immune response of their myeloid progeny. Employing a humanized mouse model, we show that environmental conditions that drive emergency myelopoiesis promote human TET2-derived CH. We unravel an opposite and cell-specific impact of TET2 mutations on HSCs and their myeloid progeny to inflammatory stress. In such environments, TET2Mut HSCs have a mitigated transcriptional upregulation of inflammatory pathways, and by contrast, TET2Mut myeloid cells have an exacerbated inflammatory response. A distinct monocyte-macrophage trajectory derived from TET2Mut HSCs contributes to pathological inflammation. These findings provide evidence at the human cell level and reconcile the notion that TET2-derived CH is associated with both increased stemness at the HSC compartment and promoted inflammatory environments from the myeloid progeny.

overall_design: We performed single cell RNA sequencing in human TET2-WT and TET2-Mut hematopoietic stem and progenitor cells and immune cells isolated from bone marrow of humanised mice after a regimen of LPS injections to induce systemic inflammation.

first: Hector

last: Huerga Encabo

first: Dominique

last: Bonnet


title: Improved spike-in normalization clarifies the relationship between active histone modifications and transcription

city: La Jolla

first: Alon

last: Goren

address: 9375 Gilman Dr

department: Department of Medicine

institute: University of California San Diego

summary: Spike-in normalization enables quantitative analysis of ChIP-sequencing (ChIP-seq) signal. Here we introduce a novel robust dual-spike-in normalization approach for ChIP-seq (ChIP-wrangler) and revisit recent claims that active histone marks are dependent on transcription. Concerned that previous studies improperly used spike-in normalization to arrive at their conclusions, we used ChIP-wrangler to show that acute depletion of RNA polymerase II has only a modest impact on the levels of H3K4me3 and H3K27ac. In line with other studies, our results provide proof that the maintenance of histone acetylation is not merely a consequence of ongoing transcription. Our innovative ChIP-seq normalization approach provides increased rigor and “guardrails” for successful spike-in normalization, and as applied here refines the understanding of the intricate crosstalk between RNA polymerase II activity and histone marks associated with transcription

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: HCT116-EF1a-OsTIR1-Rpb1 cells were treated with either 500nM Triptolide (Sigma-Aldrich T3652) or 500uM auxin (3-indoleacetic acid, Sigma-Aldrich #I3750) to degrade Rpb1 for 0, 1, and 4 hours. HeLa-S3 cells were treated with 500nM Triptolide (Sigma-Aldrich T3652) for 0, 1, or 4 hours. Sheared chromatin from D. melanogaster and S. cerevisiae were added as spike-in controls to all samples prior to immunoprecipitation. From every biological sample, a ChIP-input sample was prepared to assess the relative amounts of spike-in chromatin. ChIP-seq was perfomed using antibodies for H3K27ac, H3K4me3, H3K4me1, H3K36me3, and Rpb1 in HCT116-EF1a-OsTIR1-Rpb1 cells, and antibodies for H3K27ac, H3K4me3, H3K4me1, and Rpb1 in HeLa-S3 cells.

first: Lauren

last: Patel

first: Yuwei

last: Cao

first: Tianyao

last: Xu

first: Eduardo

last: Modolo

first: Tamar

last: Dishon

first: Lingzhi

last: Zhang

first: Eric

last: Mendenhall

first: Sven

last: Heinz

first: Itamar

last: Simon

first: Chris

last: Benner


title: Stress controls heterochromatin inheritance through histone H3 ubiquitylation [ChIP-Seq]

city: Bethesda

first: Shiv

last: Grewal

state: MD

address: 9000 Rockville Pike

department: National Cancer Institute

institute: National Institutes of Health

summary: Heterochromatin, marked by histone H3 lysine 9 methylation, can be inherited through cell divisions, maintaining gene repression that preserves cell identity and enables adaptation to environmental challenges. Studies in S. pombe have shown that heterochromatin propagation depends on the read-write mechanism wherein sufficient density of H3K9me3-modified nucleosomes, stabilized by histone deacetylases (HDACs), concentrate Clr4SUV39H on chromatin to promote further deposition of H3K9 methylation. Whether additional mechanisms control heterochromatin propagation via Clr4SUV39H, a part of the E3 ubiquitin ligase complex ClrC, was unknown. Here we uncover a ubiquitin-dependent heterochromatin-heritability regulatory hub (HRH) that broadly governs heterochromatin propagation, even without HDAC activity. The HRH is tuned by the limiting factor Raf1DDB2, a substrate receptor for ClrC ubiquitin ligase. In addition to linking Clr4SUV39H to other ClrC components on chromatin, Raf1DDB2 acts in a dosage-dependent manner to promote ubiquitination of histone H3 at lysine 14 (H3K14ub), which is critical for heterochromatin self-propagation. Importantly, HRH is intricately linked to environmentally responsive pathways, including nonsense-mediated decay (NMD) and TOR signaling, allowing cells to adapt to changing conditions. By modulating heterochromatin propagation, cells leverage the HRH to gain resistance to antifungal agents and to adapt to high temperature. Thus, heterochromatin self-propagation is actively regulated by the HRH in response to external stimuli, with broad implications for understanding mechanisms governing rapid changes in the epigenetic landscape in physiology and disease.

overall_design:
Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) in fission yeast. Input control and immunoprecipitation samples are provided for each experiment.
ChIP-Seq of H3K9me3 and H3K14ub histone modification in Schizosaccharomyces pombe cells

first: Bharat

last: Bhatt

first: Yi

last: Wei

first: Ashis

last: Pradhan

first: Jothy

last: Dhakshnamoorthy

first: Martin

last: Zofall

first: Hua

last: Xiao

first: Drisya

last: Vijayakumari

first: Shweta

last: Jain

first: Hernan

middle: D

last: Folco

first: Hongyun

last: Qi

first: David

middle: A

last: Ball

first: Tatiana

last: Karpova

last: Wheeler

first: Jiemin

last: Wong

middle: I


title: Single-cell spatial transcriptomic profiling defines a pathogenic inflammatory niche in chronic active multiple sclerosis lesions - snRNA-seq

city: Munich

first: Ruoqing

last: Feng

address: Feodor-Lynen-Strasse 17

country: Germany

institute: German Center for Neurodegenerative Diseases (DZNE)

summary: Compartmentalized inflammation is considered a critical factor in driving the progression of multiple sclerosis (MS). Yet, the mechanisms sustaining its persistence remain poorly understood. A hallmark of this persistent and slowly evolving inflammatory process are chronic active MS lesions. In this study, we created a high-resolution, single-cell molecular and spatial atlas of chronic inflammation in MS. To accomplish this, we combined single-nucleus RNA sequencing (snRNA-seq) with single-cell spatial transcriptomics using multiplexed error-robust fluorescence in situ hybridization (MERFISH) to examine MS lesions, specifically focusing on those exhibiting chronic active immune pathology characterized by lymphocyte presence. Our integrative profiling uncovered the molecular landscape of glial and immune cells, their disease-associated states, and the surrounding microenvironments. Within the lesion rim, we identified CD8+ T cell niches with inflamed, foamy microglia, characterized by an interferon response and dysfunctional lipid metabolism. To investigate the function of these microglia, we deleted ATP-binding cassette transporters A1 and G1 (ABCA1/G1) in microglia of mice and induced experimental autoimmune encephalomyelitis (EAE). Our findings revealed that inhibiting cholesterol efflux increased the formation of lipid-storing phagocytes, which actively drove inflammatory processes in EAE. Moreover, we found that pharmacologically targeting sterol metabolism presents a promising therapeutic strategy for mitigating inflammation in EAE. Thus, we have created a high-resolution map of immune niches in chronic active MS lesions, leading to the discovery of lipid-associated microglia as key drivers of neuroinflammation.

overall_design:
Frozen autopsy brain tissue from fourteen multiple-sclerosis donors and three neurologically healthy controls was sectioned at –20 °C into 80–100 µm slices, and an 8 mm punch (~25 mg) of white matter or matched control tissue was collected from each region of interest . Nuclei were liberated with the 10x Genomics Chromium Nuclei Isolation Kit, using an extended lysis–debris-removal protocol tailored to lipid-rich white matter; the final suspension was stained with propidium iodide and counted before loading. Single-nucleus libraries were prepared with Chromium 3′-v3.1 reagents and sequenced on an Illumina NovaSeq 6000 to ~30 000 read pairs per nucleus, generating ~20 000 quality-filtered nuclei per sample and 306 731 nuclei in total . Reads were aligned to the GRCh38 reference with CellRanger 7.1.0 ( --include-introns ), and nuclei were retained if they contained 700–25 000 UMIs, 500–7 500 genes and < 5 % mitochondrial transcripts. Per-sample objects were log-normalised in Seurat v5, highly variable genes selected, and batch effects removed with the HarmonyIntegration workflow; clustering was performed in PCA–UMAP space and the cleaned layers were merged with JoinLayers to yield a unified atlas. Finally, CytoSPACE was used to project the transcriptomic clusters onto matched MERFISH sections, assigning each nucleus to demyelinated, perilesional, or healthy white-matter domains and thereby linking cell states to their spatial lesion context.

*rds files: slimmed Seurat object with normalized expression values, essential cell-level metadata, and the final dimensional reductions used in the associated publication.

first: Janos

last: Groh


title: A Single-cell Transcriptomic Profiling Reveals the Effects of Chronic House Dust Mite Exposure on the Murine Lung Microenvironment

city: La Jolla,

first: Samuel

last: Bertin

address: 9500 Gilman Drive

department: Division of Rheumatology, Autoimmunity, and Inflammation

institute: UC San Diego Department of Medicine

summary: House dust mite (HDM) is a common environmental aeroallergen linked to the development of allergic airway inflammation and asthma. While the effects of acute HDM exposure on T helper 2 (Th2)-driven allergic responses and pulmonary eosinophilia are well established in murine models, the impact of chronic HDM exposure on other inflammatory responses, particularly those involving interleukin-1β (IL-1β) and neutrophilia, remains poorly defined. To address this gap, we performed single-cell RNA sequencing (scRNA-seq) to examine lung immune responses in wild-type and IL-1β-deficient mice following chronic HDM exposure. Our data reveal that HDM promotes the recruitment of diverse immune cell populations in the lungs, including neutrophils, alternatively activated (M2-polarized) macrophages, B-2 (follicular) B cells, and multiple CD4 regulatory and effector T cell subsets. IL-1β signaling was essential for supporting neutrophil and Th17 responses, whereas its absence enhanced Th2-skewed immune polarization in the lung. These immune populations differently contribute to the development or resolution of lung inflammation in an IL-1β-dependent or -independent manner. This study offers a detailed characterization of the cellular and molecular alterations induced by chronic HDM exposure and reveals previously unrecognized roles for IL-1β in orchestrating chronic lung inflammation.

overall_design: scRNA-seq profiles of 3-5 months old male C57BL/6 WT or Il1b–/– mice with or without HDM intranasal treatment (3-4 mice/group). The mice were first sensitized intranasallly treated with HDM (50 ug/50 uL) or with the control vehicle (VEH; 50uL, 0.9% sodium chloride solution) on day 0, and were then challenged intranasally three times a week for the subsequent 4 weeks with HDM (12.5 ug/50 uL) or with VEH (50uL). Lungs were harvested and pooled, and the pooled sample was split into two for library preparation and sequencing.

first: Han

last: Chang

first: Chanond

last: Nasamran

first: Scott

last: Herdman

first: Maripat

last: Corr

first: Kathleen

middle: M

last: Fisch

first: Nicholas

last: Webster

first: Eyal

last: Raz


title: Stress controls heterochromatin inheritance through histone H3 ubiquitylation [RNA-Seq]

first: Shiv

last: Grewal

overall_design: RNA-Seq of transcripts in Schizosaccharomyces pombe cells

first: Bharat

last: Bhatt

first: Yi

last: Wei

first: Ashis

last: Pradhan

first: Jothy

last: Dhakshnamoorthy

first: Martin

last: Zofall

first: Hua

last: Xiao

first: Drisya

last: Vijayakumari

last: Jain


title: Gene expression profile on dermal fibroblasts treated with melanocortin compounds

first: Trinidad

last: Montero

address: Charterhouse Square

department: Biochemical Pharmacology

institute: Whilliam Harvey Research Institute, Bart's and The London School of Medicine; Queen Mary University of London

summary: Fibroblasts have emerged as new therapeutic targets for the treatment of multiple conditions, ranging from rheumatoid arthritis to fibrosis. Hence, the development of novel therapies targeting this cell type offers new opportunities for diseases with high medican needs.In this study, human dermal fibroblasts obtained from healthy volunteers were cultured in vitro and treated with various compounds to determine their gene expression profile compared to vehicle-treated fibroblasts.

overall_design: Cells were plated on day 0 and drug treatments (BMS-470539 -10uM-, alphaMSH -10uM-, itraconazole -1uM- or vehicle -PBS-) applied on days 1, 3 and 5. Cells were collected on day 6 and RNA extracted.

last: Montero-Melendez

first: Camilla

last: Davan-Wetton

first: David

middle: J

last: Abraham


title: Chemical reprogramming of fibroblasts into functional retinal pigment epithelium cells (cut&tag)

city: Wenzhou

first: zhengbiao

last: zhu

state: Zhejiang

address: Ouhai District

department: The Affiliated Eye Hospital of Wenzhou Medical University

institute: Wenzhou Medical University

summary: Restoring retinal pigment epithelium (RPE) cells is crucial for treating retinal degenerative (RD) diseases, with chemical reprogramming offering a transformative, scalable solution. However, identifying key compounds for generating functional RPE cells remains challenging. Here, we present a two-step chemical reprogramming strategy to convert fibroblasts into functional RPE (ciRPE) cells. Leveraging the Single-Cell Reprogramming Compound Finder (scRCF), which integrates transcriptomics-guided predictions with advanced screening, we identified compound cocktails that precisely and efficiently reprogram fibroblasts through an intermediate state into ciRPE cells. These ciRPE cells closely mimic the structure and function of native RPE cells, and upon transplantation into RD rats, they seamlessly integrate into host tissue, protect photoreceptors, and restore visual function. Omics and mechanistic analyses revealed that the identified compounds synergistically activate core transcription factors, including Ascl1 and Olig2, orchestrating the reprogramming process. This work provides a scalable platform for RPE cell generation, advancing cell replacement therapies for RD diseases.

overall_design: Using multi-omics sequencing to examine cellular changes during the chemical reprogramming of fibroblasts into RPE cells.

first: JianZhong

last: Su

first: ShaSha

last: Li

first: Hui

last: Liu

first: ZhengBiao

last: Zhu


title: Adar1 as a placental innate immune rheostat to prevent endogenous dsRNA-induced integrated stress response-dependent embryonic lethality [bulk RNA-seq]

city: xiamen

first: Xiaogang

last: Chen

address: xiangan Campus，Xiamen University

institute: xiamen university

summary: The mechanisms that balance a robust antiviral defense with tissue tolerance at the maternal-fetal interface under physiological conditions remain largely unknown.

overall_design: Adar1PKO and Adar1f/f Ifnar-/-Elfcre/wtplacentas using the MGI-2000 platform (BGI, China). Paired-end reads were quality-filtered using Trim Galore (v0.6.10) and aligned to the mouse genome (mm39) with STAR (v2.7.9a).


title: Adar1 as a placental innate immune rheostat to prevent endogenous dsRNA-induced integrated stress response-dependent embryonic lethality

overall_design: Nuclei from Adar1f/f and Adar1PKO placental tissues were processed using the Chromium Single Cell 3’ Reagent V3 Kit (10X Genomics) and sequenced on a NovaSeq 6000 S4 platform.


title: therapy

first: LIGUO

last: WANG

state: MN

address: 200 1st St SW

department: Division of Computational Biology

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

overall_design: Refer to individual Series


title: The effect of dual radiation and antiandrogen therapyt on prostate cancer cell lines [Myc-CaP]

summary: Immunotherapy has been effective in many cancer types, but failed in multiple clinical trials in cancers of the prostate, an organ largely influenced by androgen action. However, the prostate propensity cannot be explained by the immune suppressive activity of androgen receptor (AR) in T cells as reported recently.The OPeRATIC trial (NCT02816983) showed significantly improved outcomes in patients with oligometastatic castration-resistant prostate cancer (CRPC) treated with metastasis-directed radiation (IR) and ARPI who experienced increased levels of CD8+CD11ahigh T cells in peripheral circulation. Here we report that AR pathway inhibitor (ARPI) plus radiation triggers robust anti-cancer immunity in prostate cancer from mice.

overall_design: Myc-CaP cells treated with irradiation (6 Gy), enzalutamide (10 μM, 48 h) or combination.Then we used RNA seq data from different treatments for gene expression analysis

first: Haojie

last: Huang

first: Xiang

first: Jacob

last: Orme

first: Liguo

last: Wang


title: T cell help transiently unlocks a high plasticity state in germinal center B cells during the humoral immune response [ActB_RNA-seq]

city: New York

first: Prokopios Alexandros

last: Polyzos

address: 413 E 69th St

institute: Weill Cornell Medicine

summary: During cellular differentiation, phenotypic plasticity is gradually lost due to the strengthening of epigenetic barriers, although it may be re-acquired upon disease or injury. Here, we report an unprecedented physiological gain of epigenetic plasticity among mature B cells during the humoral immune response. Acquisition of plasticity was strictly dependent on T follicular helper (TFH) cells and restricted to germinal center (GC) but not activated B cells, indicating both non- and cell-autonomous contributions to this phenotype. Importantly, GC plasticity was not dependent on proliferation and could not be solely explained by the activation of MYC programs. Instead, GC plasticity was associated with the reciprocal weakening of B cell identity and the de- repression of progenitor and stem cell-like programs induced by NFκB and other signals, downstream of TFH signaling. Therefore, physiological acquisition of GC B cell plasticity is tightly controlled by the affinity maturation positive selection checkpoint. Notably, Histone 1 loss of function, which results in aberrant stem cell gene reactivation through chromatin decompaction, allowed GC B cells to bypass this gatekeeping mechanism. Finally, patients with Diffuse LargeB Cell Lymphoma (DLBCL) that enriched for GC plasticity signatures had significantly worse outcomes, implicating a role for this mechanism in immune regulation and fitness and potential relevance to lymphomagenesis.

overall_design: Splenic B cells from mice immunized with SRBC for 9 days were pre-enriched for B220pos cells by negative magnetic enrichment. Naive and GC B cells were sorted from 3 x 2 individual 3-month-old male mice before culture with mIL4 and aCD40,CpG or LPS for 5 days. Cells were then collected for RNA extraction using the RNAeasy Qiagen kit according to the provider specifications. RNAs were QC using Agilent Bioanalyzer 2100 and Libraries were prepared using the NEB Ultra II Directional RNA Library Prep (plus Poly A isolation module), and clustered on an Illumina NovaSeq 6000

first: Laurianne

last: Scourzic

first: Alexander

first: Effie

last: Apostolou


title: Changes in peripheral blood mononuclear cells (PBMCs) of 10 patients who underwent continued ADT and stereotactic body radiation therapy (SBRT).

summary: Immunotherapy has been effective in many cancer types, but failed in multiple clinical trials in cancers of the prostate, an organ largely influenced by androgen action. However, the prostate propensity cannot be explained by the immune suppressive activity of androgen receptor (AR) in T cells as reported recently. We use single-cell sequencing to analyze the changes in blood PBMCs of patients who respond good or poorly with ADT and SBRT.

overall_design: Separate PBMCs from the patient's blood and analyze them through single-cell sequencing.


title: Integrated Multiomics Profiling Uncovers miRNA-Guided Regulatory Networks After Spinal Cord Injury in Rats

city: Vestec

first: Ruslan

last: Klassen

address: Průmyslová 595

department: Institute of Biotechnology

country: Czech Republic

institute: Czech Academy of Science


title: Circadian regulator REV-ERBα is a master regulator of tumor lineage plasticity and an effective therapeutic target. [ATAC-Seq]

city: sacramento

first: Xiong

last: Zhang

address: 4645 2nd Ave

institute: University of California, Davis

summary: Epigenetic and transcriptional dysregulation plays a fundamental role in tumor lineage plasticity (LP). However, the underlying mechanisms, especially for the initial events of LP development, are still poorly understood. Here, we report that in progression of prostate cancer from adenocarcinoma to treatment-induced neuroendocrine prostate cancer (t-NEPC), anti-AR signaling inhibitors (ARSIs) reprogram the function of a circadian regulator/nuclear receptor REV-ERBα by switching its target gene programs from kinase signaling and metabolic programs to programs of LP, which includes neurogenesis, stem cell and epithelia to mesenchymal transition (EMT) as well as over fifteen LP drivers including POU3F2/BRN2, ASCL1, FOXA2, ONECUT2 and MYCN. Unexpectedly, REV-ERBα facilitates the chromatin occupancy of BRN2, ASCL1 and FOXA1 in their activation of LP programs, thus functioning as a master regulator of ARSI-induced LP driver network. Mechanistically, REV-ERBα induces chromatin accessibility and H3K27ac modification at promoters of LP genes through its recruitment of BRD4 and p300. Overexpression of REV-ERBα alone is sufficient to induce LP and neuroendocrine phenotype and confers cell resistance to ARSI in adenocarcinoma cells. Loss of REV-ERBα potently inhibits NEPC cell growth and abolishes the expression of LP drivers and gene programs. Pharmacological inhibition of REV-ERBα exhibits high potency in blocking the growth of NEPC tumors including patient-derived xenografts.  Our findings reveal that therapy-induced LP development entails a coordinated induction of a network of LP drivers and that REV-ERBα is a novel master regulator of the network and a promising therapeutic target for treatment of advanced prostate cancer such as NEPC.

overall_design: ATAC-seq profiling of circadian regulator REV-ERBα inhibition in ARSI-resistant prostate cancer cells.

first: Yatian

last: Yang

first: Ronald M

last: Evans

first: Hongwu


title: T cell help transiently unlocks a high plasticity state in germinal center B cells during the humoral immune response [Foxo1_RNA-seq]

summary: Germinal Center (GC) B cellsconstitutekey effectorsofthehumoral immune responseandundergoiterativerounds ofproliferation, somatic hypermutationof their immunoglobulin genesand eventuallycompete for T follicular helper (TFH) cellshelpin order togenerate cells with high affinity B cell receptor (BCR).While selected cells passing the critical TFHfitness checkpointcan differentiate toplasma cells,memory B cellsor go through additional rounds of proliferation and mutagenesis, the mechanisms enabling fully differentiated GCs to re-acquire plasticity–usually lost upon cell fate specification–arestill largely unknown.Herein,we report that matureGCsundergo a physiologicaltransientstate characterized byanincreased functional plasticity andinduced pluripotent stem cell (iPSC)reprogramming capacity.This transition state isreminiscent of anaplasisdue to theconcomitantpartial activation of embryonic stem cellandattenuation of the B cell programs.We furthermore demonstrate thatGC anaplasisis dependent on TFHcells sincein vivointerference of TFH:GC B cells communication or lymphoma driver mutationsaffectingGCsresponse to TFHcell help were shown to modulate their reprogramming efficiency accordingly. Importantly, GC stem-like gene signatures, as revealed by single-cell Multiome analysis of thistransient anaplastic state, were often enriched inDiffuse Large B cell lymphoma (DLBCL)patients and associate with poor outcomes, suggesting that this rare preexisting plastic state might be hijacked and further enhancefitnessduring lymphomagenesis.

overall_design: Splenic B cells from mice immunized with SRBC for 9 days were pre-enriched for B220pos cells by negative magnetic enrichment and stained for naive B cell markers before flow sorting. Naive B cells were sorted from 10 individual 3-month-old male mice from 2 independent experiments and pooled before culture with mIL4 and aCD40 for 5 days. On day 3, cells were either treated with DMSO or with 1uM of the Foxo1 inhibitor (Cpd10) for 48h. Cells were then collected for RNA extraction using the RNAeasy Qiagen kit according to the provider specifications. RNAs were QC using Agilent Bioanalyzer 2100 and Libraries were prepared using the NEB Ultra II Directional RNA Library Prep (plus Poly A isolation module), and clustered on an Illumina NovaSeq 6000


title: small RNA seq of patient-derived glioma cells (GBM1,GBM2, GG3)

first: Kiril

last: Tuntevski

summary: Glioblastoma (GBM) has a poor prognosis due to infiltrative and therapy-resistant glioma stem-like cells. MicroRNAs (miRNAs) synergistically modulate biological pathways by repressing tumor-related genes, a mechanism lost in cancer.

overall_design: GBM1-2 (glioblastoma) and GG3 (glioma grade III) patient-derived cells were cultured in glioma stem cell medium (Griffero et al., 10.1074/jbc.M807111200) and total RNA extracted for small RNA sequencing.

first: Silvia

last: Rancati

first: Rui

last: Pereira

first: Michele

last: Schlich

first: Stefania

last: Sgroi

last: Beatini

first: Letizia

last: La Rosa

first: Lidia

last: Giantomasi

first: Roberta

last: Pelizzoli

first: Clarissa

last: Braccia

first: Carlotta

last: Spattini

first: Amanda

last: Lo Van

first: Meritxell

last: Pons-Espinal

first: Anna Lisa

last: Palange

first: Adriana

last: Bajetto

first: Antonio

last: Daga

first: Andrea

last: Armirotti

first: Tullio

last: Florio

first: Paolo

last: Decuzzi

first: Davide

last: De Pietri Tonelli


title: ERRγ impedes neuroendocrine prostate cancer development

first: Vincent

last: Giguère

address: 1160 Pine Ave West, McIntyre Bldg Room 710


title: ERRγ impedes neuroendocrine prostate cancer development [RNA-Seq]

summary: Neuroendocrine prostate cancer (NEPC) is a lethal subtype of castration-resistant prostate cancer (CRPC). The molecular mechanisms underlying the progression of CRPC toward NEPC remain incompletely understood and effective treatments remain to be discovered. Herein, we report that loss of the nuclear receptor ERRγ promotes neuroendocrine differentiation in a Pten-deficient mouse model of prostate adenocarcinoma. These findings were recapitulated in advanced cellular and xenograft models of human prostate cancer (PCa). Critically, we show that ERRγ gain-of-function can reverse instilled NEPC features accompanied by suppression of growth and oncogenic metabolic reprogramming. Activation of a neuroendocrine transcriptional program enabled by ERRγ deficiency unveiled a targetable vulnerability exploited by combined pharmacological inhibition of EZH2 and RET kinase that effectively inhibited the growth of ERRγ-deficient tumor organoids and cells. Collectively, our findings demonstrate that ERRγ downregulation facilitates PCa adeno-to-neuroendocrine transformation and offer potential therapeutic strategies to prevent/treat the development of poor outcome NEPC.

overall_design: RNA-seq was performed on xenograft tumors (n=3 per condition) derived from four established DU145 cell lines. DU145 cells were engineered to express +/- PTEN and/or ERRγ overexpression. DU145 cells have high PTEN and low ERRγ status defined as PTEN+ERRγlow to which we generated PTEN-ERRγlow cells by stably knocking out PTEN by CRISPR/Cas9 followed by lentivirus-mediated ERRγ re-expression in both PTEN+ERRγlow and PTEN-ERRγlow cell lines to generate PTEN+ERRγ+ and  PTEN-ERRγ+ cells.

first: Ting

last: Li

first: Catherine

last: Dufour


title: B cells transition through a T cell dependent plastic state during the adaptive immune response

first: Prokopios Alexandros

last: Polyzos

summary: During the adaptive immune response, fully mature germinal center (GC) B cells manifest the unique capacity to undergo rapid multidirectional, and dramatic phenotypic shifts and evolve into diverse memory and antibody secreting cells. No other mature cell lineages are known to display such extensive phenotypic plasticity under physiological conditions. Whether this represents a form of differentiation or re-acquisition of stem-like plasticity remain unknown. Herein, we show that GC B cells undergo a transient gain of functional plasticity, which is unprecedented under physiological conditions. Strikingly, this process, which we call “anaplasis”, was spatially and temporally restricted to GC B cells receiving T cell help. Anaplasis involved partial silencing of the B cell program, the reactivation of embryonic stem cell super-enhancers and genes and was dependent on specific transcription factors. Notably, lymphoma mutations that enhance T cell help, further increased GC stem-like functionality, whereas mutations inducing chromatin plasticity bypassed the T cell requirement. Moreover, anaplasis signatures were associated with unfavorable outcome in Diffuse Large B cell lymphoma. Altogether, our findings document that differentiated cells can re-acquire stem-like plasticity under physiological conditions. This potentially harmful phenotype co-evolved with strict non-cell autonomous checkpoints linked to immunological selection processes. GC anaplasis may help to explain why most lymphoid malignancies arise from mature GC B cells and is potentially hijacked, leading to aggressive diseases and highly fit lymphomas.

overall_design: Splenic B cells from mice immunized with SRBC for 9 days were pre-enriched for B220pos cells by negative magnetic enrichment and stained for GC markers as described above before flow sorting. GC Myc-GFPpos and Myc-GFPneg fractions were sorted from 6 individual 3-month-old male mice and pooled before processing for Multiomic sequencing using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit (10X Genomics #N-1000283) according to the provider specifications. gDNA and cDNA Indexed libraries were quantified using Qubit and QC using Agilent Bioanalyzer 2100. Libraries were diluted to 2 nM and clustered on an Illumina NovaSeq 6000

first: Laurianne

last: Scourzic

first: Franco

last: Izzo

first: Effie

last: Apostolou


title: RNASeq of K562 cell clones for DNMT3A-WT, DNMT3A-W305*, DNMT3A-R882H, DNMT3A-R882C

city: London

first: Hector

last: Huerga Encabo

address: The Francis Crick Institute, 1 Midland Rd

institute: The Francis Crick Institute

summary: Donor blood saves lives, yet the potential impact of recurrent large-volume phlebotomy on donor health and hematopoietic stem cells (HSCs) remains largely unexplored. In our study, we conducted a comprehensive screening of 217 older male volunteer donors with a history of extensive blood donation (>100 life-time donations) to investigate the phenomenon of clonal hematopoiesis (CH). No significant difference in the overall incidence of CH was found in frequent donors (FD) compared to sporadic donors (<10 life-time donations, 212 donors). However, upon deeper analysis of mutations in DNMT3A, the most commonly affected gene in CH, we observed distinct mutational patterns between the FD and age/sex matched control donor (CD) cohorts. Functional analysis of FD enriched DNMT3A variants examined in CRISPR-edited human HSCs demonstrated their competitive outgrowth potential upon stimulation with erythropoietin (EPO), a hormone which increases in response to blood loss. In contrast, clones harboring leukemogenic DNMT3A R882 mutations increase upon stimulation with IFNy. Through concurrent mutational and immunophenotypic profiling of primary samples at single cell resolution, a myeloid bias of premalignant R882 mutant HSCs was found, while no significant lineage bias was observed in HSCs harboring EPO responsive DNMT3A variants. The latter exhibited  preferential erythroid differentiation when persistent erythropoietic stress was applied to CRISPR-edited human HSC xenografts. Our data demonstrate a nuanced ongoing Darwinian evolution at the somatic stem cell level, with EPO identified as a novel environmental factor that favors HSCs carrying certain DNMT3A mutations.

overall_design: We performed bulk RNA sequencing in K562 cells after isolation and expansion of single mutant clones for each genotype

first: Miriam

last: Llorian

first: Dominique

last: Bonnet


title: NKT cells contribute to alleviating lung metastases in adenoid cystic carcinoma

city: Beijing

first: Tingyao

last: Ma

address: DongchengNo.1, Dongjiao Min Alley

institute: Beijing Tongren Hospital

summary: Through CD4 and CD8 immunohistochemical analysis, we systematically established immune desert, exclusion, and inflammatory types in SACC primary lesions and lung metastases. Notably, the majority of lung metastases exhibited immune inflammatory types in contrast to the common desert type in primary SACC tissue. Tissue type rather than MYB-NFIB fusion should be a key factor causing differences in the immune environment. At the single-cell level, we demonstrated the presence of a small amount of anergy T cell types and a few elevated cytokines in the primary inflammatory tissues without MYB-NFIB fusion; and a large amount of myeloid-derived suppressor cells (MDSCs), a small amount of activated CD8+ NKT-like cells and substantially elevated cytokines in the primary exclusion tissues with MYB-NFIB fusion. In contrast, in the inflammatory metastatic lung tissues without MYB-NFIB fusion, small numbers of memory CD4+ and CD8+ cells were presented, but anergy CD8+ NKT-like cells and only CCL28 were elevated. Moreover, validated by immunodeficient nude mice, NKT cells are the remarkable effector cells in reducing lung metastasis, but required advance activation by a low invasive SACC cell line with higher human leukocyte antigen (HLA)-A, -B, and C expression induction.

overall_design: Cells from fresh tumour tissues were isolated for the preparation of single-cell suspensions via the ChromiumTM Single Cell 3’ Solution technique and analysed by BGI company (Shenzhen).

first: Ge-xuan

last: Feng

first: Meng-jiao

last: Zhou


title: Chemical reprogramming of fibroblasts into functional retinal pigment epithelium cells (scRNA-seq)

first: zhengbiao

last: zhu

first: JianZhong

last: Su

first: ShaSha

first: Hui

last: Liu

first: ZhengBiao

last: Zhu


title: RNASeq of TET2-mutant human HSPCs and monocytes

summary: The expansion of HSC clones and their immune progeny carrying somatic mutations is defined as clonal hematopoiesis (CH) with its prevalence increasing with age. Individuals with CH are at higher risk of developing hematological malignancies but also other pathologies, such as cardiovascular or inflammatory pulmonary diseases. Understanding which environmental conditions favor mutant clones and the CH-immune system response to such environments is key to designing therapeutic strategies to stall the expansion of mutant clones and the development of CH-associated pathologies. In this work, we characterized how mutations in TET2, one of the most frequent in CH, increase the competitive advantage of human HSCs upon environmental stress conditions and at the same time shape the immune response of their myeloid progeny. Employing a humanized mouse model, we show that environmental conditions that drive emergency myelopoiesis promote human TET2-derived CH. We unravel an opposite and cell-specific impact of TET2 mutations on HSCs and their myeloid progeny to inflammatory stress. In such environments, TET2Mut HSCs have a mitigated transcriptional upregulation of inflammatory pathways, and by contrast, TET2Mut myeloid cells have an exacerbated inflammatory response. A distinct monocyte-macrophage trajectory derived from TET2Mut HSCs contributes to pathological inflammation. These findings provide evidence at the human cell level and reconcile the notion that TET2-derived CH is associated with both increased stemness at the HSC compartment and promoted inflammatory environments from the myeloid progeny.

overall_design: We performed bulk RNA sequencing following our competitive assays, where TET2WT and TET2Mut cells are present within the same environment (Figure 2A). For the analysis of HSPCs, we isolated Lineage-CD34+ cells on day 4 of the LTC assay, 6 hours after the LPS treatment (Figure 2B-C). For the analysis of the myeloid compartment, we isolated monocytes (CD14+ cells) on day 14 of the LTC assay, also 6 hours after the last LPS treatment (Figure 2D-E) to compare between the two cell types the impact of TET2 mutations in the transcriptional adaptation to stress.

first: Huerga Encabo

last: Hector

first: Llorian Sopena

last: Miriam

first: Bonnet

last: Dominique


title: Fibulin-7 in progenitor cells promotes adipose tissue fibrosis and disrupts metabolic homeostasis in obesity [RNA-seq]

status: Public on Oct 28 2025

city: Shanghai

first: Cheng

last: Hu

address: No.600, Yishan Road, Shanghai, China

institute: Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China

summary: Fibrosis, resulting from excess extracellular matrix deposition, is a feature of adipose tissue (AT) dysfunction and obesity-related insulin resistance. In this study, through a comparative analysis of single-cell RNA-sequencing data from stromal vascular cells in the AT of lean and obese mice, we identify Fibulin-7 (FBLN7), a newly recognized member of the fibulin family of secreted glycoproteins, as a promoter of fibrosis. Moreover, we found that FBLN7 levels are upregulated in visceral fat among obese individuals, showing a correlation with clinical metabolic traits. However, the precise role of FBLN7 in AT and the underlying regulatory mechanisms remain to be elucidated. Here, we demonstrate that loss and overexpression of FBLN7 in adipogenic stem and precursor cells (ASPCs) inhibits and promotes, respectively, TGF-β-induced fibrogenic responses. In vivo, in response to caloric excess, ASPCs-specific FBLN7 knockout mice display a diminished state of AT fibrosis-inflammation state, coupled with improved systemic metabolic health. Mechanistically, FBLN7 binds to thrombospondin-1 (TSP1) via its EGF-like calcium-binding domain, enhancing TSP1 protein stability. This interaction, in turn, facilitates the conversion of latent TGF-β to its bio-active form, thereby promoting TGFBR1/Smad signaling. Furthermore, we develop an anti-FBLN7 neutralizing antibody, which could dramatically alleviate diet-induced AT fibrosis. Conclusively, our findings imply that FBLN7 produced by ASPCs exerts a major influence in AT fibrosis and may represent a potential target for therapeutic intervention.

overall_design: RNA-seq  on WT ASPCs infected with lentiviral sh-FBLN7 versus those infected with sh-Ctrl after TGF-β1 stimulation

first: Hairong

last: Yu

first: Fan

last: Yang

first: Dandan

last: Yan

first: Wei

last: Chen

first: Jian

last: Zhang

first: Jing


title: A 3D in vitro mouse ovarian follicle culture system preserves molecular signatures of ovulation for studying ovulation biology and novel non-hormonal discovery

city: Piscataway

first: Shuo

last: Xiao

state: NJ

address: 170 Frelinghuysen Rd

department: Department of Pharmacology and Toxicology

institute: Rutgers University

summary: We previously established an alginate hydrogel-based 3D encapsulated in vitro follicle growth (eIVFG) system to culture mouse and human immature follicles in vitro. The alginate encapsulation maintained the 3D architecture of follicles and support their growth from the secondary to the antral stage to acquire maturation. Upon the stimulation of human chorionic gonadotropin (hCG), a LH analog, the grown antral follicle from eIVFG was able to rupture, undergo cumulus cell expansion, and ovulate a fertilizable metaphase II (MII) oocytes. We have also demonstrated that mouse follicles cultured using eIVFG preserved dynamic transcriptomic profile of many key genes that are essential for gonadotropin-dependent folliculogenesis, such as genes related to gonadotropin hormone receptors and ovarian steroidogenesis. However, it is unknown whether these follicles preserve molecular signatures of ovulation, which would enable this system to serve as a truly scalable and highly controllable system for nominating therapeutic and contraceptive candidates. In the present study, we treated mouse mature follicles from eIVFG with hCG to induce in vitro ovulation. Follicles were collected at 0, 1, 4, and 8 hours post-hCG for single-follicle RNA sequencing (RNA-seq) analysis. Our results showed that follicles grown from eIVFG preserve key ovulatory genes and signaling pathways.

overall_design: After 8 days of eIVFG culture, follicles were stimulated for ovulation by hCG, and we collected time-course samples for RNA-seq analysis.Among all 46 samples collected in two batches, 9, 13, 13 and 11 follicles were collected at  0, 1, 4, and 8 hours post-hCG treatment, resepectively.

first: Jiyang

first: Brittany

last: Goods

first: Pawat

last: Pattarawat

first: Yingzheng

last: Wang

first: Tessa

last: Haining

first: Qiang

first: Alex

middle: K

last: Shalek

first: Francesca

middle: E

last: Ducan

first: Teresa

last: Woodruff


title: A single-cell resolution spatial transcriptomics atlas of human multiple sclerosis lesion (MERSCOPE)

first: Ruoqing

last: Feng

summary: In this study, we conducted an in-depth investigation into the complex intercellular immune reactions in multiple sclerosis (MS). To accurately map the spatial transcriptomic landscape of MS, we designed a gene panel comprising 500 key genes and collected samples from the brain tissues of 14 patients with chronic active MS. Using MERFISH and 10x single-cell sequencing technologies, we successfully created detailed spatial transcriptional maps. Through comprehensive analysis of the data obtained, we identified several disease-specific glial cell subtypes associated with MS. Furthermore, we explored how these disease-related glial cells interact with peripheral immune cells, revealing their potential roles in the progression of the disease. This research not only provides new insights into the pathological mechanisms of multiple sclerosis but also offers significant scientific basis for the development of future therapeutic strategies.

overall_design:
In this study, our experimental design focused on the detailed analysis of lesion areas in the brain tissues of patients with multiple sclerosis (MS). First, we collected brain tissue samples from 14 MS patients. Using specialized staining techniques, we accurately identified the locations of MS lesions within the brain tissues. Subsequently, we used a brain puncher to extract the brain tissue regions containing lesions from each sample. These precisely cut samples were then analyzed on the MERSCOPE platform by Vizgen.
*rds files: slimmed Seurat object with normalized expression values, essential cell-level metadata, and the final dimensional reductions used in the associated publication.


title: Chemical reprogramming of fibroblasts into functional retinal pigment epithelium cells (RNA-seq)

first: zhengbiao

last: zhu

summary: Restoring retinal pigment epithelium (RPE) cells is crucial for treating retinal degenerative (RD) diseases, with chemical reprogramming offering a transformative, scalable solution. However, identifying key compounds for generating functional RPE cells remains challenging. Here, we present a two-step chemical reprogramming strategy to convert fibroblasts into functional RPE (ciRPE) cells. Leveraging the Single-Cell Reprogramming Compound Finder (scRCF), which integrates transcriptomics-guided predictions with advanced screening, we identified compound cocktails that precisely and efficiently reprogram fibroblasts through an intermediate state into ciRPE cells. These ciRPE cells closely mimic the structure and function of native RPE cells, and upon transplantation into RD rats, they seamlessly integrate into host tissue, protect photoreceptors, and restore visual function. Omics and mechanistic analyses revealed that the identified compounds synergistically activate core transcription factors, including Ascl1 and Olig2, orchestrating the reprogramming process. This work provides a scalable platform for RPE cell generation, advancing cell replacement therapies for RD diseases.

overall_design: Using multi-omics sequencing to examine cellular changes during the chemical reprogramming of fibroblasts into RPE cells.

first: JianZhong

last: Su

first: ShaSha

last: Li

first: Hui

last: Liu

first: ZhengBiao

last: Zhu


title: Adar1 as a placental innate immune rheostat to prevent endogenous dsRNA-induced integrated stress response-dependent embryonic lethality [Adar1 PKO RNA-seq]

first: Xiaogang

summary: The mechanisms that balance a robust antiviral defense with tissue tolerance at the maternal-fetal interface under physiological conditions remain largely unknown.

overall_design: Adar1f/f and Adar1PKO placentas using the MGI-2000 platform (BGI, China). Paired-end reads were quality-filtered using Trim Galore (v0.6.10) and aligned to the mouse genome (mm39) with STAR (v2.7.9a).


title: Fibulin-7 in progenitor cells promotes adipose tissue fibrosis and disrupts metabolic homeostasis in obesity [scRNA-seq]

first: Cheng

last: Hu

overall_design: ScRNA-seq on  SVFs of eWAT from NCD- and HFD-fed male C57BL/6J mice

first: Hairong

last: Yu

first: Fan

last: Yang

first: Dandan

last: Yan

first: Wei

first: Jian

first: Jing


title: Difference analysis of microRNAs in abdominal aorta of ApoE-/- AngII-induced abdominal aortic aneurysm mices

city: Shenyang

first: Qingbin

last: Hou

address: wenhua road 83

institute: General Hospital of Northern Theater Command

summary: Abdominal aortic aneurysm (AAA) is a vascular disease with high incidence at present.There is no effective drug treatment. MicroRNAs(miRNAs) play a regulatory role in the occurrence and development of AAA,and miRNA therapy is a promising treatment for AAA. The purpose of this study was to explore the differential expression of miRNAs in abdominal aorta of  ApoE-/- AngII-induced abdominal aortic aneurysm mices. We used miRNA array to analyze miRNA differences in abdominal aorta between mices with ApoE-/- AngII-induced abdominal aortic aneurysm and ApoE-/-.

overall_design: To explore the differences in miRNA in abdominal aorta of mices  with AngII-induced abdominal aortic aneurysm and control , we collected abdominal aorta samples of  AngII-induced abdominal aortic aneurysm mices and control mices , There were three mice in each group,extracted total RNA, and analyzed them using miRNA array

first: Xiaozeng

first: Chenghui

first: Yaling

last: Han


title: Clinical, physiologic, imaging, and molecular responses to cannabis smoking: the Canadian Users of Cannabis Smoke (CANUCK) Study

city: Vancouver

first: Janice

last: Leung

state: BC

address: Room 166, 1081 Burrard Street

institute: Centre for Heart Lung Innovation, St. Paul’s Hospital, University of British Columbia

summary: Background. The growing popularity of cannabis smoking in an era of legalization has prompted concerns about respiratory health. Objective. To investigate clinical and airway epithelial transcriptomic features associated with cannabis smoking.  Methods. This cross-sectional study analyzed 139 cannabis-smoking participants categorized by joint-year exposure (low: ≤5; moderate: >5-20; high: >20), and 57 never-smokers. We evaluated respiratory symptom questionnaire scores, lung function measurements, and chest computed tomography and hyperpolarized 129Xe pulmonary magnetic resonance imaging measurements across groups. We compared the expression of immune response signatures and mucin genes in airway epithelial brushings collected from bronchoscopy. Using air-liquid interface (ALI) cell cultures, we quantified epithelial MUC5AC protein and correlated its expression with clinical outcomes. Results. Among cannabis-smoking individuals (48% male and median age of 27 years), 84% reported current or former cigarette smoking or vaping. Cannabis-smoking groups reported worse respiratory symptoms than never-smokers. High joint-year cannabis-smoking participants showed lower pre-bronchodilator FEV1/FVC and FEF25-75, more radiographic emphysema, and more ventilation abnormalities than never-smokers. Airway epithelial brushings from cannabis-smoking individuals demonstrated increased type 2 immune response, decreased type 17 immune response, and higher MUC5AC gene expression than non-cannabis-smoking individuals. Epithelial MUC5AC protein expression in cell cultures correlated with worse clinical outcomes and imaging abnormalities. Conclusions. Cannabis smoking, particularly at high exposures, is associated with worse respiratory symptoms, lower lung function, functional imaging abnormalities, and dysregulated immune responses in the airway epithelium. These observations suggest respiratory harm associated with cannabis smoking and underscore the concerns for future respiratory morbidities related to persistent cannabis use.

overall_design: RNA-seq profiling of airway epithelial samples from cannabis-smoking and non-cannabis individuals.

first: Don

last: Sin


title: RNA-seq of gubernaculum and testis from wild-type, cetacean INSL3 knock-in, and INSL3 knockout mice across developmental stages

city: Nanjing

first: Yu

last: Zheng

state: Jiangsu Sheng

address: No.1 Wenyuan Road, Xianlin University Town, Qixia District, Nanjing, Jiangsu Province

institute: NANJING NORMAL UNIVERSITY

summary: This dataset contains high-throughput RNA sequencing (RNA-seq) data generated from gubernaculum and testis samples of wild-type (WT), cetacean INSL3 knock-in (KI), and INSL3 knockout (KO) mice at selected postnatal developmental stages. The purpose of the study was to investigate the molecular consequences of replacing the endogenous mouse Insl3 coding sequence with the cetacean INSL3 ortholog, and to compare transcriptomic profiles among WT, KI, and KO mice to elucidate the functional role of INSL3 in testicular descent and cryptorchidism. RNA-seq was performed on gubernaculum tissue at postnatal day 8 (P8) and testis tissue at P8, P23, and P40 (with some groups excluded due to degeneration or non-specific damage). The dataset supports integrative analyses of developmental stage–specific and genotype–specific gene expression changes in reproductive tissues.

overall_design: Male C57BL/6J background mice of three genotypes were used: wild-type (WT), cetacean INSL3 knock-in (KI), and INSL3 knockout (KO). In the KI model, the endogenous mouse Insl3 coding sequence was precisely replaced with the cetacean ortholog at the native genomic locus, preserving all mouse promoter and regulatory elements. For WT and KI mice, RNA-seq was performed on gubernaculum at postnatal day 8 (P8) and on testes at P8, P23, and P40. For KO mice, RNA-seq was performed on gubernaculum at P8 and testes at P8 and P23.

first: Simin

last: Chai

first: Cuijuan

last: Zhong

first: Yixuan

last: Sun

first: Guang


title: Cell cycle regulator MYBL2 is a distinct vulnerability in acute myeloid leukemia

city: Dresden

first: Sandra

last: Küchler

address: Fetscherstraße 74/PF 64

institute: National Center for Tumor Diseases (NCT) Dresden

summary: We were interested in transcriptional changes after depletion of MYBL2 in acute myeloid leukemia. Therefore, we utilized THP-1 and NOMO-1 as models, and analyzed global alterations of gene expression.

overall_design:
Transcriptomic changes in THP-1 and NOMO-1 after CRISPRi-based MYBL2 knockdown.
RNA-Sequencing from THP-1/NOMO-1 cells stably expressing dCas9-KRAB-GFP after transduction with either a sgRNA against a non-targeting luciferase (luc) or MYBL2. Experiment was performed in biologically independent triplates.

first: Alexander

last: Wurm


title: Modeling SMAD2 mutations in iPSCs provides insights into cardiovascular disease pathogenesis [RNA-seq]

city: Boston

first: Jon

last: Seidman

state: Massachusetts

address: 77 Avenue Louis Pasteur

department: Genetics

institute: Harvard Medical School

summary: Rationale: SMAD2 is a co-regulator that binds a variety of transcription factors and modulates activities during human development. Heterozygous SMAD2 loss-of-function (LoF) variants and missense variants are identified in patients with complex congenital heart disease (CHD), and in patients with arterial aneurysms and dissections. Mechanisms that account for distinct cardiovascular phenotypes caused by SMAD2 variants remain unknown. We aimed to define the transcriptional and epigenetic effects of heterozygous SMAD2 loss-of-function (LoF) and missense variants and identify the involvement of transcription factor networks and genes that contribute to CHD. Compared to LoF variants, we assessed the function of rare SMAD2 missense variants of uncertain significance. Methods: We constructed isogenic induced pluripotent stem cells (iPSCs) with heterozygous or homozygous LoF and rare (MAF ≤ 10-5) missense SMAD2 variants identified in CHD probands. Wildtype and mutant iPSCs were studied by bulk RNA sequencing (RNAseq) and chromatin accessibility (ATACseq). Datasets were integrated with published SMAD2/3 chromatin immunoprecipitation (ChIPseq) studies. Cardiomyocyte differentiation and contractility of mutant iPSCs was assessed. Results: SMAD2 haploinsufficiency reduced open chromatin across promoter regions and altered the expression of 385 direct SMAD regulated genes, including ten previously identified as dominant CHD genes. Motif analyses in regions with differential ATAC peaks predicted that SMAD2 haploinsufficiency disrupts interactions with at least five transcription factors, NANOG, ETS, TEAD3/4, CREB1 and AP1. Compared to SMAD2-haploinsufficient cells, iPSCs with heterozygous R114C or W274C variants exhibited shared and distinct patterns of altered chromatin accessibility, transcription factor binding motifs, and dysregulated genes. Conclusions: SMAD2 haploinsufficiency disrupts chromatin interactions and perturbs transcription factor binding, disrupting normal cardiovascular development. Differences in the molecular consequences of LoF and missense variants likely contribute to clinical phenotypic heterogeneity. Additionally, these data indicate opportunities for molecular functional analyses to improve re-classification of SMAD2 variants of uncertain clinical significance.

overall_design: Examination of genetically engineered iPS cell lines that carry SMAD2 mutations. Two biological replicates for SMAD2 HET LoF, W274C, R114C cell lines and two technical replicates for each

first: Tarsha

last: Ward

first: Warren

last: Tai

first: Sarah

last: Morton

first: Joshua

last: Gorham

first: Christine


title: Adar1 as a placental innate immune rheostat to prevent endogenous dsRNA-induced integrated stress response-dependent embryonic lethality [RIP-seq]

first: Xiaogang

last: Chen

overall_design: Placental tissues from E14.5 Adarf/f and AdarPKO mice were processed to extract input RNA. A seperate portion was lysed in dsRIP buffer with protease inhibitors and RNase inhibitor. Lysates were incubated with Mda5 antibody followed by Protein G Dynabeads capture. The RNA samples were fragmented before sequencing on the NovaSeq platform.


title: The effect of dual radiation and antiandrogen therapyt on prostate cancer cell lines [C4-2]

city: Rochester

first: LIGUO

last: WANG

institute: Mayo Clinic

summary: Immunotherapy has been effective in many cancer types, but failed in multiple clinical trials in cancers of the prostate, an organ largely influenced by androgen action. However, the prostate propensity cannot be explained by the immune suppressive activity of androgen receptor (AR) in T cells as reported recently.The OPeRATIC trial (NCT02816983) showed significantly improved outcomes in patients with oligometastatic castration-resistant prostate cancer (CRPC) treated with metastasis-directed radiation (IR) and ARPI who experienced increased levels of CD8+CD11ahigh T cells in peripheral circulation. Here we report that AR pathway inhibitor (ARPI) plus radiation triggers robust anti-cancer immunity in prostate cancer from patients.

overall_design: C4-2 cells treated with irradiation (6 Gy), enzalutamide (10 μM, 48 h) or combination.Then we used RNA seq data from different treatments for gene expression analysis

first: Haojie

last: Huang

first: Xiang

last: Li

first: Jacob

last: Orme

first: Liguo

last: Wang


title: RNASeq of DNMT3A mutant K562

first: Hector

last: Huerga Encabo

summary: Donor blood saves lives, yet the potential impact of recurrent large-volume phlebotomy on donor health and hematopoietic stem cells (HSCs) remains largely unexplored. In our study, we conducted a comprehensive screening of 217 older male volunteer donors with a history of extensive blood donation (> 100 life-time donations) to investigate the phenomenon of clonal hematopoiesis (CH). No significant difference in the overall incidence of CH was found in frequent donors (FD) compared to sporadic donors (< 10 life-time donations, 212 donors). However, upon deeper analysis of mutations in DNMT3A, the most commonly affected gene in CH, we observed distinct mutational patterns between the FD and control donor (CD) cohorts. Functional analysis of FD enriched DNMT3A variants examined in CRISPR-edited human HSCs demonstrated their competitive  outgrowth potential upon stimulation with erythropoietin (EPO). In contrast, clones harboring leukemogenic DNMT3A R882 mutations exhibited a stronger response to inflammatory cues rather than EPO. Through concurrent mutational and immunophenotypic profiling of primary samples at single cell resolution as well as experiments in humanized mice, a myeloid bias of R882 mutant HSCs was detected. Remarkably, no significant lineage bias was observed in HSCs harboring EPO responsive DNMT3A variants. Our data demonstrate a nuanced ongoing Darwinian evolution at the somatic stem cell level, with EPO identified as a novel environmental factor that favors HSCs carrying certain non-malignant DNMT3A mutations. Crucially, our findings robustly demonstrate that frequent blood donation over many years does not promote the selection of malignant clones, thus strongly reaffirming its safety.

overall_design: Using CRISPR-mediated genetic engineering of K562 we generated K562 clones harbouring DNMT3A W305*, R882H or R882C mutations and compare their trnascriptome with wild type K562 cells.

middle: H

last: Encabo

first: Miriam

middle: L

last: Sopena

first: Dominique

last: Bonnet


title: EZH2 directs breast cancer progression through the modulation of epithelial cellular plasticity

city: Montreal

first: William

last: Muller

state: Quebec

address: 1160 Av des Pins O

department: Biochemistry

institute: McGill University

summary: Breast cancer remains a leading cause of death among women, encompassing diverse molecular subtypes with distinct clinical outcomes. The HER2+ subtype is particularly aggressive, with many patients eventually developing resistance to HER2-targeted therapies, underscoring the urgent need for alternative treatment strategies. Enhancer of Zeste Homolog 2 (EZH2), the catalytic subunit of Polycomb Repressive Complex 2, represses the expression of genetic programs crucial for differentiation, control of proliferation, and apoptosis through histone H3 lysine 27 trimethylation (H3K27me3). To investigate the role of EZH2 in HER2+ tumor progression, we crossed a genetically engineered mouse model (GEMM) of HER2-driven breast cancer with a conditional Ezh2 knockout strain. The results showed that Ezh2 is essential for accelerating tumor initiation and metastatic dissemination in HER2-driven breast cancer. Combined bulk and single cell RNA sequencing analyses revealed a significant downregulation of basal cell populations in the absence of Ezh2, characterized by the loss of Tp63 expression, and an upregulation of luminal progenitor cell populations, driven by crucial transcription factors such as Esr1, thereby driving luminal lineage commitment. Further, inhibition of EZH2 in vitro resulted in increased expression of ER in HER2+ human breast cancer cell lines and rendered them susceptible to tamoxifen. Altogether, these findings demonstrate that EZH2 dictates breast cancer plasticity through control of cellular identity and provides rationale for combining EZH2 inhibitors with endocrine therapies to improve outcomes in HER2+ breast cancer.

overall_design: Single-nucleus RNA sequencing (snRNA-seq) of wild-type EIC/EZH2 tumor cells and EIC/EZH2 knockout tumor cells collected from mice at endpoint; two biological replicates per group.

first: Linshan

last: Liu

first: Ellie

last: Massey

first: Dongmei

last: Zuo

first: Alain

last: Pacis

first: Mert

last: Demirdizen

first: Jessica

last: Cinkornpumin

first: Yu

last: Gu

first: Elizabeth

last: Podleszanski

first: Hailey

last: Proud

first: Virginie

last: Sanguin-Gendreau

first: Vasilios

last: Papavasiliou

first: Zhengze

last: Jiang

first: Harvey W.

last: Smith

last: Pastor

first: Paolo

last: Ceppi

first: William J.


title: KHSRP depletion in leukemia cells

first: Qingyu

last: Luo

state: MA

address: 450 Brookline Ave

institute: Dana-Farber Cancer Institute

summary: KHSRP was knocked down in CAL-1 cells by CRISPR interference, then RNA-seq was performed in control and KHSRP knockdown cells to assess differentially expressed genes.

first: Andrew

last: Lane


title: Single cell RNA sequencing identifies a novel tenogenic heterologous differentiation in endometrial carcinosarcomas

city: Madrid

first: Silvia

last: Gonzalez-Martinez

address: M-607, Km. 9, 100, Fuencarral-El Pardo

department: Pathological Anatomy

country: Spain

institute: Ramon y Cajal University Hospital

summary: This study presents a single-cell RNA sequencing (scRNAseq) analysis of six carcinosarcomas and two normal endometrial samples, profiling over 96,000 cells. By integrating transcriptomic data with inferred copy number variations (CNVs), immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH), we resolve the complex cellular architecture and differentiation dynamics of these tumors. We identify distinct lineage-specific programs and uncover heterologous differentiation trajectories—including rhabdomyogenic, osteogenic, and a previously undescribed tenogenic lineage defined by SCX, MKX, and TNMD expression. Our results reveal that carcinosarcomas exhibit multilineage plasticity within their mesenchymal component and highlight the power of single-cell analysis to refine tumor classification and uncover hidden developmental programs.

overall_design: Six carcinosarcoma samples and two normal endometrial controls were analyzed using scRNAseq. Tumors were dissociated and processed for droplet-based scRNAseq, followed by integration with inferred CNVs, IHC, and FISH validation. The study aimed to characterize epithelial and mesenchymal lineages, identify novel differentiation programs, and explore the genomic architecture underlying carcinosarcoma plasticity.

last: González Martínez

first: José

last: Palacios


title: EZH2 alters aggressive variant prostate cancer subtype evolution, but is not required for neuroendocrine prostate cancer [RNAseq]

city: Buffalo

first: David

last: Goodrich

state: NY

address: Elm and Carlton Streets

department: Department of Pharmacology & Therapeutics

institute: Roswell Park Comprehensive Cancer Center

summary: Advanced prostate cancer (PrCa) remains a leading cause of cancer-related death among men because nearly all patients progress on standard therapy that targets androgen receptor (AR) signaling. An important mechanism driving therapeutic resistance is lineage plasticity, the ability of PrCa cells to reprogram into AR independent lineage variants like neuroendocrine (NE) PrCa. The histone methyltransferase EZH2 has been implicated in PrCa lineage plasticity and EZH2 inhibitors are being evaluated clinically for the treatment of PrCa. Here we test how Ezh2 impacts the evolution of PrCa lineage plasticity using genetically engineered mice and human patient data. Ezh2 deficiency in mice diversifies the evolution of PrCa lineage variants, including a newly discovered variant with high expression of neurofilament genes. Analogous PrCa lineage variants are detectable in human specimens where they similarly correlate with EZH2 expression. Lineage variant diversification is associated with activation of transcription factor programs and AR cistrome reprogramming that is normally repressed by Ezh2. These findings advance the understanding of PrCa lineage plasticity and have implications for targeting EZH2 as a PrCa therapy.

overall_design: Bulk RNA-seq profiling of prostate tumor tissue from genetically engineered mouse models (SKO, DKO, TKO) or DKO mouse models with Ezh2 deletions (DKO_EP and DKO_EE). Prostate tissue was collected at 12 weeks of age (intact, pre-castration), endstage (castrated at 12 wks.), or intact endstage. Select TKO mice were treated with either Vehicle or Ezh2-inhibitor (CPI1688) following castration.

first: Justine

last: Jacobi


title: Impacts of DNA methylation on H2A.Z deposition and nucleosome stability

first: Rochelle

middle: Makanani

last: Shih

address: 1230 York Ave

institute: Rockefeller University

type: Methylation profiling by high throughput sequencing

summary: DNA methylation and the histone variant H2A.Z are maintained in mutually exclusive sections of the genome. How this antagonism is established, however, remains an open question. Here, we examined the impacts of DNA methylation on both the intrinsic stability of H2A.Z nucleosomes and chaperone-mediated H2A.Z deposition. Cryo-EM and endonuclease-based assays show that H2A.Z nucleosomes on methylated DNA substrates are more open and accessible compared to their unmethylated counterparts. In Xenopus, H2A.Z preferentially associates with unmethylated DNA of both sperm pronuclei in the transcriptionally silent egg extract as well as in somatic cells, though a substantial proportion of H2A.Z colocalizes with DNA methylation in sperm pronuclei. By monitoring nucleosome assembly on synthetic DNA constructs in egg extract, we find that the H2A.Z bias for unmethylated substrates is dependent on the SRCAP complex. Accordingly, DNA-binding of SRCAP is suppressed by DNA methylation. Altogether, we propose that the SRCAP complex is the major determinant for preferential H2A.Z enrichment on unmethylated DNA, and that DNA methylation destabilizes DNA wrapping in H2A.Z-containing nucleosomes.

overall_design: CUT&Tag-bisulfite sequencing (Li et al. 2021; https://doi.org/10.1016/j.crmeth.2021.100118) on H3 and H2A.Z in Xenopus laevis sperm pronuclei incubated in interphase-cycled Xenopus egg extract as well as XTC-2 cells (a Xenopus laevis fibroblast cell line).

first: Yasuhiro

last: Arimura

first: Hide

last: Konishi

first: Hironori

last: Funabiki


title: Prevotella copri facilitates wound healing in mice through the sphingosine-CerS1-ceramide metabolic pathway

first: Hailiang

last: Liu

address: NO 1239, siping road

institute: Tongji university

summary: Skin wound repair constitutes a sophisticated biological process involving spatiotemporally coordinated molecular cascades, with emerging evidence highlighting the dynamic regulatory role of skin microbiota. Utilizing a broad-spectrum antibiotic-treated (ABX) murine model, we identified Prevotella copri as a core functional commensal in the wound microecosystem that orchestrates tissue regeneration through metabolite-host crosstalk. ABX-induced microbial remodeling significantly enriched P. copri relative abundance, accelerated wound closure, and upregulated pro-regenerative factors VEGF and FGF-2. Metabolomic profiling revealed that P. copri-secreted sphingosine undergoes bioconversion to C18-ceramide via the non-canonical CerS1 pathway, driving keratinocyte hyperproliferation and neoangiogenesis. Pharmacological inhibition of CerS1 with P053 suppressed ceramide synthesis and delayed healing, mechanistically validating the sphingosine-CerS1-ceramide axis. Crucially, P. copri exhibits dual regulatory modalities: ecologically, β-lactamase-mediated antibiotic resistance establishes microbial dominance, while metabolically, sphingolipid-driven spatiotemporal signaling remodels the regenerative microenvironment. This paradigm shifts the pathocentric view of wound microbiota, proposing a synergistic strategy combining "functional microbiota enrichment" and "metabolic axis targeting". Our findings elucidate a microecology-metabolism circuit that transitions wound management from passive anti-infection to precision intervention, providing a molecular blueprint for developing microbiome-reprogramming therapies in regenerative medicine.

overall_design: Traditional wound repair research often focuses on microbial diversity, neglecting the critical role of specific taxa in tissue regeneration. Our study challenges this by highlighting Prevotella copri as a key species in wound healing, operating through the Prevotella copri-sphingosine-CerS1-ceramide signaling pathway. This discovery reshapes the understanding of microbiome-host interactions and paves the way for precision microbial therapies. By showing that a single bacterium can replace complex community dynamics, we connect ecological theory with regenerative applications, offering a strategy to use microbial metabolism for precise wound healing.


title: Piwi-piRNA complex-mediated silencing of transposable elements through transcription termination

city: Tokyo

first: Kensaku

last: Murano

address: 35 Shinanomachi, Shinjuku

department: Department of Molecular Biology

country: Japan

institute: Keio University School of Medicine

summary: Piwi protein, guided by Piwi-interacting RNA (piRNA), establishes sequence-specific heterochromatin on transposable elements (TEs) in animal germ cells, thereby repressing TEs to preserve genomic integrity. We previously reported that TE silencing mediated by the Piwi-piRNA pathway can be divided into two distinct steps: the initiation and maintenance of silencing. During initiation, the Piwi-piRNA complex interferes with RNA polymerase II transcription through the effector complex containing Nxf2 and Panx, preceding chromatin compaction mediated by linker histone H1 and HP1a in the silencing-maintenance. However, the mechanisms underlying silencing initiation remain elusive. Here, we found that SUMOylated Panx recruits Sov, resulting in reporter gene silencing independent of H1 and HP1a. Additionally, Sov associates with PNUTS and Senataxin, both of which are involved in the transcription termination, and are required for Panx-mediated silencing. These findings reveal that transcription termination plays a critical role in the piRNA-guided transcriptional repression preceding heterochromatin formation on TEs.

overall_design:
To investigate the function of SUMO, SUMO-related genes, Piwi, and Setx in the silencing of transposable elements in ovarian somatic cells (OSCs), we performed gene knockdown experiments by siRNA transfection, followed by RNA-seq.
To investigate the function of Panx, PNUTS, and Setx in the silencing of transposable elements in OSCs, we performed ChIP-seq using anti-Pol II Ser5-P antibody.

first: Yaning

last: Wu

first: Toshiya

last: Nakahara


title: FeaSion Decodes the Regulatory Landscape and Functional Diversity of RNA Polymerase II CTD Phosphorylation

first: Junyi

last: Zhu

address: Peking University, No.5 YiHeYuan Road, Haidian District

department: School of Life sciences

institute: Peking University

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

overall_design: Refer to individual Series


title: DNGR-1 signalling limits dendritic cell activation for optimal antigen cross-presentation

first: Probir

last: Chakravarty

address: 1 Midland Road

institute: Francis Crick Institute

summary: Innate immune receptors often induce activation of conventional dendritic cells (cDCs) and enhance antigen (cross-)presentation, favouring immune responses. DNGR-1 (CLEC9A), a receptor expressed by type 1 cDCs (cDC1s) and implicated in immune responses to viruses and cancer, recognises F-actin exposed on dead cell remnants and promotes cross-presentation of associated antigens. Here, we show that recruitment of phosphatase SHIP1, a process governed by a single amino acid residue adjacent to the signalling motif of the receptor, partly explains how DNGR-1 fails to trigger cDC1 activation in vitro. Substituting this residue converts DNGR-1 into an activating receptor but decreases induction of cross-presentation of dead cell-associated antigens. Introducing the reverse mutation into the related receptor Dectin-1 impairs its activation capacity while enhancing its ability to promote cross-presentation. These findings reveal a functional trade-off in receptor signalling and suggest that DNGR-1 has evolved to prioritise antigen cross-presentation over cellular activation, possibly to minimise inflammatory responses to dead cells.

overall_design: DNGR-1 or mutant DNGR-1 expressing MuTuDCs were treated with 12.5 nM DNGR-1 ligand for 6 hours

first: Michael

last: Buck

first: Caetano

last: ReiseSousa