title: Dual Modes of Gene Regulation by CDK12

first: Apoorva

last: Baluapuri

address: 45 Shattuck St.

department: Biological Chemistry and Molecular Pharmacology

institute: Harvard Medical School

summary: The process of transcription is driven forward by the activity of kinases including CDK7, CDK9 and CDK12. Accordingly, acute inhibition of any of these kinases results in profound downregulation of gene expression. Here,we discover that loss or inhibition of CDK12 also significantly upregulates a set of coding and non-coding loci, whose activation could contribute to the anti-proliferative effects of CDK12 inhibitors. Mechanistically, CDK12 inhibition impairs transcription elongation, leading to increased RNA polymerase II termination or arrest in long genes. However, short genes such as MYC and enhancer RNAs are highly transcribed in the absence of CDK12 activity. Indeed, in HER2+ breast cancer, a malignancy where CDK12 is invariably co-amplified with HER2 and its expression correlates with disease status, CDK12 inhibition markedly elevates MYC expression to induce lethality. The dual effects of CDK12 inhibition elucidated herein clarify its role in transcriptional control and have significant translational implications.

overall_design: To investigate the impact of CDK12 inhibition on RNAPII behavior, PRO-seq was performed over HCC1954 cells +/- THZ531 (N=2, 200 nM, 2h). Cells were permeabilized as in Mimoso et al. 2024; run-ons were performed with biotinylated NTPs and then purified. Purified biotinylated RNAs were ligated to sequencing adapters and amplified by PCR.

first: Yubao

last: Wang

first: Cherubin

last: Manokaran

first: Karen

last: Adelman

first: Jean

first: Thomas

last: Robertts


title: BCL-xL as a therapeutic target in cetuximab-refractory colorectal cancer

status: Public on Jan 27 2026

city: Stuttgart

first: Steven

middle: A

last: Johnsen

address: Auerbachstrasse 112

country: Germany

institute: Robert Bosch Center for Tumor Diseases

type: Expression profiling by high throughput sequencing

summary: Despite recent medical advances, colorectal cancer (CRC) remains the second-leading cause of cancer-related death worldwide. For patients with KRAS wild-type metastatic CRC, the monoclonal antibody cetuximab, which targets the epidermal growth factor receptor (EGFR), is an approved treatment option. However, therapeutic success is often limited by the emergence of drug-resistant cancer cell populations within a few months. Therefore, alternative strategies to effectively target cetuximab-refractory CRC are urgently needed. Here, we sought to identify second-line therapeutic strategies using a CRC cell line with acquired cetuximab resistance as a model. Transcriptomic profiling of the resistant cells identified the apoptosis pathway as a potential therapeutic target, which was supported by their increased susceptibility to BH3-mimetics targeting the anti-apoptotic proteins MCL-1 and BCL-xL under both 2D and 3D culture conditions. These findings were validated in organotypic CRC slice cultures generated from cetuximab- resistant patient-derived xenografts (PDXs). Multiplex immunofluorescence staining demonstrated that BCL-xL inhibition effectively triggered apoptosis in heterogeneous PDX tumor slice models, including models harboring oncogenic BRAF mutations. Our findings suggest that cetuximab-resistant CRC retains apoptotic competence, and that BCL-xL inhibition serves as a robust alternative therapeutic strategy that is largely independent of the tumor mutational profile.

overall_design: To investigate resistance to cetuximab in colorectal cancer, we performed RNAseq in parental and resistant LIM1215 cells.

first: Stella

last: Asmanidou

middle: L

last: Ekstrom

first: Monilola

last: Olayioye


title: Astrocyte Elevated Gene-1/Metadherin (AEG-1/MTDH) in Myeloid Cells Regulates Metabolic Dysfunction-Associated Steatohepatitis (MASH) [Spatial Transcriptomics]

city: Richmond

first: Wright Center for Clinical and Translational Research

last: Bioinformatics Core

state: VA

address: 203 E Cary St

country: USA

institute: Virginia Commonwealth University

summary: Astrocyte elevated gene-1/Metadherin (AEG-1/MTDH) plays an important role in regulating lipid metabolism and inflammation in hepatocytes, and promotes metabolic dysfunction-associated steatohepatitis (MASH). Myeloid cells, such as macrophages, play a key role in regulating inflammation. Here we investigated the role of AEG-1 in myeloid cells in regulating high fat/high sugar diet (HF/HSD)-induced MASH. Both male and female AEG-1MAC mice were significantly protected from MASH development compared to AEG-1fl/fl mice.

overall_design: Littermates of myeloid cell-specific AEG-1 knockout mice (AEG-1DMAC) and AEG-1 floxed mice (AEG-1fl/fl) were fed either control diet (CD) or HF/HSD for 20 weeks.

first: Devanand

last: Sarkar

first: Suchismita

last: Raha

first: Rachel

last: Mendoza

first: Xufeng

last: Qu

first: Jinze

last: Liu


title: Astrocyte Elevated Gene-1/Metadherin (AEG-1/MTDH) in Myeloid Cells Regulates Metabolic Dysfunction-Associated Steatohepatitis (MASH)


title: Paired-end RNA Sequencing on primary human umbilical endothelial cells (HUVEC) exposed to uninfected or bacterially-infected PMA-differentiated macrophage-like U937 for 8 or 24 h

city: Tübingen

first: Effie

last: Bastounis

state: Baden-Württemberg

address: Auf der Morgenstelle 28/E7

department: Interfaculty Institute of Microbiology and Infection Medicine

institute: University of Tübingen

summary: We sought to identify changes in gene expression that occur when endothelial cells in monolayer interact with either uninfected or Listeria monocytogenes-infected macrophages. To that end we seeded HUVEC in monolayer and either exposed them to nothing, or to uninfected PMA-differentiated macrophage-like U937, or to Listeria monocytogenes-infected PMA-differenitated U937. At 8 or 24 hours post U937 exposure (hpe), we washed the samples thoroughly to remove U937 and then collected the mRNA to to perform RNA-Sequencing and gene expression profiling for the six conidtions described. By analyzing the differentially expressed genes between all conditions we find a significant number of up- or down-regulated genes related to innate immune signaling in both (un)infected U937 exposed HUVEC as compared to control unexposed HUVEC, irrespective of the time post-exposure. For example we found significant upregulation of genes related IL-17, NF-kB and TNF related signaling pathways, though to a lesser degree for HUVEC exposed to uninfected U937 as compared to bacterially infected. Interestingly, we found that only HUVEC exposed to uninfected but not infected U937 and only at 8hpe showed significant upregulation of genes related to focal adhesions and regulation of the actin cytoskeleton. We found that consistent with alterations in the dynamics of HUVEC under this condition, which show a significant rise in the traction on monolayer stresses they exert. At later times post-exposure (i.e., 24 hpe) were these changes in gene expression are not present anymore, HUVEC dynamics are also identical irrespective of whether they were exposed or not to (un)infected U937. Thus the RNA sequnecing results at 8 hpe that reveal upregulation of genes related to focal adhesions and the actin cytoskeleton for uninfected U937 exposed HUVEC match with the results of traction force and monolyaer stress microscopy that reveal strong enhancement of these forces over the first 10 hpe and which are absent in the other two conditions or at the later time point of 24 hpe.

overall_design: Primary endothelial cells HUVEC were seeded in monolayer and exposed to: (1) nothing (unexposed HUVEC, UE), (2) uninfected PMA-differentiated U937 macrophage-like cells (uninfected macrophage exposed HUVEC, UI), or (3) Listeria monocytogenes-infected U937 macrophage-like cells (infected macrophage exposed HUVEC, I). After 8 or 24 hours post-exposure (hpe) to U937, the samples were washed to remove U937, and mRNA was collected for RNA sequencing and gene expression profiling across these six conditions. For each condition four samples were analysed, using Illumina NovaSeq 6000 using the NovaSeq 6000 S4 Reagent Kit (300 cycles). Equipment: Illumina with 2x150 read length and 40 million reads per sample.

first: Marie

last: Muenkel


title: An Early Burst of Cytokine Production Before the First Cell Division Influences CD8 T Cell Differentiation

city: Birmingham

first: Rob

last: Welner

state: AL

address: 1824 6th Ave S

department: Medicine - Hem/Onc

institute: University of Alabama at Birmingham

summary: The differentiation of CD8 T cells into effector and memory populations is guided by a combination of antigenic, costimulatory, and cytokine signals. Here we show that within 24 hours of activation naïve CD8 T cells undergo a rapid, dynamic, and heterogeneous burst of IL-2 and IFN-γ synthesis. This divergent early response develops with every CD8 T cell specificity analyzed, manifests in vivo and in vitro, and occurs prior to the first cell division. Nevertheless, how the intrinsic manufacture of distinct cytokines forecasts and influences the properties and fates of the producer cell itself are not well defined.  We demonstrate that the initial cell intrinsic synthesis of IL-2 attenuates IL-2-dependent STAT5 signaling, but that this is not due to differences in the surface expression of the IL-2 receptor complex. These functionally discrete subsets are transcriptionally distinct and begin to display differences in the expression of hallmark effector and memory associated genes.  Using cytokine reporter systems, we reveal that these early functional differences are consequential for establishing fate biases and directing the gain of effector and memory T cell properties. The bifurcation between the abilities of IL-2-producing and non-producing subsets to elaborate STAT5 signaling are consistent with a model in which non-IL-2-producing CD8 T cells are more receptive to extrinsic IL-2 signals. This enables a more terminally differentiated series of effector CD8 T cells that are licensed by cytokine signals to rapidly form.  However, both IL-2-producing and non-producing CD8 T cells are capable of acquiring memory traits.

overall_design: Splenocytes from naive P14 IFN-γ.Thy1.1 IL-2.GFP cytokine reporter mice were stimulated with the LCMV GP33 peptide epitope for 20 hours.  P14 CD8+ Thy1.1+ GFP+ (IFN-γ+IL-2+), Thy1.1+ GFP- (IFN-γ+IL-2-), Thy1.1- GFP+ (IFN-γ-IL-2+), Thy1.1- GFP− (IFN-γ-IL-2-) cells, as well as control unstimulated P14 IFN-γ.Thy1.1 IL-2.GFP CD8+ T cells, were sorted directly into TRIzol LS.  RNA was isolated and submitted for quality assessment, RNA sequencing, and analyses.

first: Shannon

middle: M

last: Kahan

first: Jennifer

middle: T

last: Ingram

first: Robert

middle: S

first: Casey

last: Weaver

first: Laurie

middle: E

last: Harrington

first: Allan

middle: J

last: Zajac


title: MitoPerturb-Seq identifies gene-specific single-cell responses to mitochondrial DNA depletion and heteroplasmy [DamID]

city: Cambridge

first: Stephen

middle: Peter

last: Burr

address: Mitochondrial Biology Unit, Keith Peters Building, Cambridge Biomedical Campus, Hills Road

department: Clinical Neurosciences

country: United Kingdom

institute: University of Cambridge

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Mitochondria contain their own genome, the mitochondrial DNA (mtDNA), which is under strict control of the cell nucleus. mtDNA occurs in many copies in each cell, and mutations often only affect a proportion of them, giving rise to heteroplasmy. mtDNA copy number and heteroplasmy level together shape the cell- and tissue-specific impact of mtDNA mutations, ultimately giving rise to rare mitochondrial and common neurodegenerative diseases. However, little is known about how copy number and heteroplasmy interact within single cells, and how this is regulated by the nuclear genes and pathways that sense and control them. Here we describe MitoPerturb-Seq for CRISPR/Cas9-based high-throughput single-cell interrogation of the impact of nuclear gene perturbation on mtDNA copy number and heteroplasmy. We screened a panel of nuclear mtDNA maintenance genes in cells with heteroplasmic mtDNA mutations. This revealed both common and perturbation-specific aspects of the integrated stress-response to mtDNA depletion, that were only partially mediated by Atf4, and caused cell-cycle stage-independent slowing of cell proliferation. MitoPerturb-Seq thus provides novel experimental insight into disease-relevant mito-nuclear interactions, ultimately informing development of novel therapies targeting cell- and tissue-specific vulnerabilities to mitochondrial dysfunction.

overall_design: DamID-sequencing analysis of Dam-ATF4 (for ATF4 binding) and Dam-only (control), 48 hours after transient transfection in immortalized m.5024C>T mouse embryonic fibroblasts (MEFs)

middle: P

first: Kathryn

last: Auckland

first: Angelos

last: Glynos

first: Abhilesh

last: Dhawanjewar

first: Wei

last: Wei

first: Cameron

last: Ryall

first: Antony

last: Hynes-Allen

first: Malwina

last: Prater

first: Matylda

last: Sczaniecka-Clift

first: Julien

last: Prudent

first: Patrick

middle: F

last: Chinnery

first: Jelle

last: van den Ameele


title: MitoPerturb-Seq identifies gene-specific single-cell responses to mitochondrial DNA depletion and heteroplasmy [RNA-Seq]

overall_design: Mouse Embryonic Fibroblasts (MEFs) carrying a heteroplasmic mitochondrial (mt)DNA mutation in mt-Ta (m.5024C>T) and stably expressing Cas9 were transduced in biological duplicate with CRISPR gRNAs to Tfam, Opa1 & a non-targeting gRNA. After six days, cells from each transduced population were flow sorted to select 50,000 transduced cells based on mRFP expression. Total RNA was isolated from each sample using the Quick-RNA Microprep Kit (Zymo Research) and RNAseq libraries were prepared using the NEBNext Single Cell/Low Input RNA Library Prep Kit (NEB).


title: MitoPerturb-Seq identifies gene-specific single-cell responses to mitochondrial DNA depletion and heteroplasmy [multiome]

overall_design: Mouse Embryonic Fibroblasts (MEFs) carrying a heteroplasmic mitochondrial (mt)DNA mutation in mt-Ta (m.5024C>T) and stably expressing Cas9 were transduced with a pooled lentiviral CRISPR library targeting 19 genes (three guides per gene) plus three non-targeting control gRNAs using the CROPseq method. 10 days post-transduction cells were flow sorted to select transduced cells based on expression of mRFP, and sorted cells were fixed and permeabilized before being submitted for 10X Genomics single-cell MultiOme ATAC and gene expression (GEX) sequencing. Following library preparation, CROPseq guide mRNA sequences were enriched from the GEX cDNA library using the TAP-seq protocol, and mtDNA sequences were enriched from the final ATACseq library by hybridization capture using a bespoke probeset from IDT


title: Characterization of gRNA-Dependent and gRNA-Independent Off-Target Binding Sites of PspCas13b and RfxCas13d in Mammalian Cells [RNA-Seq]

city: Shanghai

first: Li

last: Zhixue

address: 320 Yueyang Road, Xuhui District

country: China

institute: CAS Center for Excellence in Molecular Cell Science

summary: CRISPR-Cas13 systems, harnessed for RNA-guided transcriptome editing, hold significant promise for clinical and in vivo therapeutic applications. However, understanding their in vivo target specificity and recognition rules remains a challenge. In this study, we employed the uSpyCLIP method, which enhances sensitivity and specificity for identifying RNA-binding protein (RBP) binding sites, to map the transcriptome-wide binding sites of catalytically inactive PspCas13b (dPspCas13b) and RfxCas13d (dRfxCas13d) in HEK293T cells, using a variety of single guide RNAs (gRNAs). Surprisingly, we identified both gRNA-dependent and gRNA-independent off-target binding sites for both dCas13 complexes. These off-target sites exhibited distinct RNA structural and sequence signatures: dPspCas13b's gRNA-independent binding was associated with specific RNA structural features, while dRfxCas13d's was linked to unique sequence motifs. Analysis of gRNA-dependent off-target sites revealed the crucial role of the DR-distal and middle regions of the gRNA in determining binding specificity. mRNA sequencing and biochemical assays further indicated that while off-target binding does not directly affect mRNA levels, it can perturb the expression of certain endogenous proteins. Therefore, our findings provide important insights into the characteristics of gRNA-dependent and gRNA-independent off-target binding for PspCas13b and RfxCas13d, offering valuable guidance for optimizing Cas13 and gRNA design in future applications.

overall_design: Lenti-X 293T cells transfected with empty vector, Lenti-X 293T cells transfected with dPspCas13b expression plasmid,  Lenti-X 293T cells co-transfected with dPspCas13b expression plasmid and  a non-targeting gRNA (gNT) constructs,  Lenti-X 293T cells co-transfected with dPspCas13b expression plasmid and a targeting gRNA (EZH2-g1, EZH2-g2 or TP53-g1) constructs, Lenti-X 293T cells transfected with dRfxCas13d expression plasmid,  Lenti-X 293T cells co-transfected with dRfxCas13d expression plasmid and  a non-targeting gRNA (gNT) constructs,  Lenti-X 293T cells co-transfected with dRfxCas13d expression plasmid and a targeting gRNA  (EZH2-g1, EZH2-g2 or TP53-g1) constructs were prapared for RNA-seq.

first: Huanhuan

last: Feng

first: Zhixue

last: Li

first: Ligang

last: Wu

first: Lin

last: Zou


title: RNA sequencing of dystrophin- and utrophin-deficient HeLa cells reveals overlapping transcriptomic alterations

city: Poznań

first: Paulina

last: Kościelniak-Wawro

address: Uniwersytetu Poznańskiego 6

department: Faculty of Biology, Institute of Human Biology and Evolution

country: Poland

institute: Adam Mickiewicz University

summary: CRISPR/Cas9 and iRNA approaches were used to knock-out and knock-down expression of the dystrophin (DMD) and utrophin (UTRN) genes. RNA-sequencing analyses revealed overlapping transcriptomic alterations in HeLa cell lines relating to the observed phenotypic abnormalities, including increased membrane permeability and intracellular calcium levels, aggregation of mitochondria, elevated reactive oxygen species, increased cyto- and genotoxicity, and apoptosis.

overall_design: RNA-seq profiling of wild-type HeLa cells after control siRNA (siCtrl) delivery, DMD knock-out HeLa cells after siUTRN delivery.

first: Patryk

last: Konieczny

first: Sylwia

last: Szwec

first: Alicja

last: Durska


title: Characterization of gRNA-Dependent and gRNA-Independent Off-Target Binding Sites of PspCas13b and RfxCas13d in Mammalian Cells [CLIP-seq]

overall_design: uSpyCLIP-seq of PTBP1 with 10,000,000 cells, 100,000 cells, 10,000 cells, or 1000 cellls; uSpyCLIP-seq of RBFOX2 with 10,000,000 cells;  uSpyCLIP-seq of dPspCas13b and dPspCas13b-gRNA;  uSpyCLIP-seq of dRfxCas13d and dRfxCas13d-gRNA; uSpyCLIP-seq for comparing the efficiency of Spycatcher V1.0 and  Spycatcher V2.0.


title: Context-dependent translation inhibition as a novel oncology therapeutic modality

city: SOUTH SAN FRANCISCO

first: Paige

middle: D

last: Diamond

state: California

address: 201 HASKINS WAY

institute: Interdict Bio

summary: Inhibitors of protein synthesis, including anisomycin, homoharringtonine, and other natural products bind in the peptidyl-transferase center (PTC) of the eukaryotic ribosome to inhibit translation. Recent work has demonstrated that some PTC-binding antibiotics act in a sequence-selective manner, inhibiting translation elongation at specific amino acids while the polypeptide is engaged in the PTC. However, this phenomenon has yet to be documented for compounds that inhibit translation by the human ribosome. Here we use structure-based design to guide synthesis of molecules called interdictors that bind to the human ribosome PTC and act in a context-selective manner to inhibit translation elongation. Using ribosome profiling, in combination with in vitro biochemistry and cryo-electron microscopy, we characterize the context selectivity of unique analogues and observe their preferred interactions with nascent chain residues with complementary properties. Furthermore, we present a structure for an interdictor bound to a portion of the MYC protein at ~ 1.9 Å resolution and identify resulting structural rearrangements in both the nascent chain and ribosomal RNA. Finally, we document how these compounds differentially impact the ribotoxic stress response pathway which monitors ribosome collisions and can lead to apoptosis. Together, our data establish sequence-selective inhibition of translation as a novel small-molecule therapeutic modality for historically difficult to address cancers by targeting translation of oncogenic dependency factors in the human ribosome PTC.

overall_design: To determine the impact and context selectivity of Interdictors on translation, we treated HCC1143 TNBC cells with compound for 1 hour and performed ribosome profiling. DMSO treated samples were used as a control.

first: Paul

middle: V

last: Sauer

first: Mikael

last: Holm

first: Canessa

last: Swanson-Swett

first: Lucas

last: Ferguson

first: Natalie

last: Bratset

first: Grant

middle: W

last: Wienker

first: Justin

last: Sim

first: Hailey

middle: K

last: Adams

first: Lillian

last: Kenner

first: Margot

last: Meyers

first: David

last: Gygi

first: Zef

last: Könst

first: Sogole

last: Bahmanyar

first: Lawrence

middle: G

last: Hamann

first: Anthony

last: Schuller


title: Matrix stiffness modulated release of spheroid-derived extracellular vesicles and discovery of Piezo1 cargo [mRNA]

city: Cincinnati

first: Claire

last: Chougnet

state: OH

address: 240 Albert Sabin Way

department: Immunobiology

institute: Cincinnati Children's Hospital Medical Center

summary: Augmented extracellular matrix (ECM) stiffness is a mechanical hallmark of cancer. Mechanotransduction studies have extensively probed the mechanisms by which ECM stiffness regulates intracellular communication. However, the influence of stiffness on intercellular communication aiding tumor progression in three-dimensional microenvironments remains unknown. Small extracellular vesicles (EVs) are communicators of altered biophysical cues to distant sites through EV-ECM interactions and EV-mediated recipient cell-ECM interactions. Here we demonstrate stiffness-mediated modulation of small EVs secretion and cargo from three-dimensional oral squamous cell carcinoma spheroids. Using a spheroid culture platform with varying matrix stiffness properties, we show that small EVs carry parental biomolecular cargo, including mechanosensitive Piezo1 ion channel and adhesion molecule CD44. We comprehensively validate the presence of both markers in our EV populations using proteomic and genetic analysis. Transcriptomic analysis of microRNA and long non-coding RNA cargo of small EVs released from soft and stiff ECM spheroids revealed enrichment of tumorigenic and metastatic profiles in EVs from stiff ECM cultures compared to that of soft ones. Gene set enrichment analysis of a comparative dataset obtained by overlaying spheroid mRNA and EV miRNA profiles identified key oncogenic pathways involved in cell-EV crosstalk in the spheroid model.

overall_design: Cal27 spheroids were cultured in soft (3 mg/mL Matrigel) and stiff (12 mg/mL Matrigel) using the liquid overlay technique. Spheroid culture media was used to extract small extracellular vesicles. Total RNA was extracted from EV samples and sequenced for mRNA, miRNA, and lncRNA.

first: Maulee

last: Sheth

first: Manju

last: Sharma

first: Leyla

last: Esfandiari


title: The E-protein Daughterless regulates protein translation and memory in adult Drosophila nervous system

city: Tallinn

first: Tõnis

last: Timmusk

address: Akadeemia tee 15

department: Chemistry and Biotechnology

country: Estonia

institute: Tallinn University of Technology

summary: Daughterless (Da), the Drosophila melanogaster homolog of mammalian E-protein transcription factor 4 (TCF4), is well studied in fruit fly embryonic development but its functions in adult nervous system are poorly understood. Mutations in human TCF4 gene lead to intellectual disabilities such as Pitt-Hopkins syndrome and TCF4 has also been linked to schizophrenia. Here, to explore the roles of Da in the Drosophila mature brain, we map Da DNA binding sites and study the transcriptomics of the brains where Da function is inhibited by pan-neuronal Extramacrohaete (Emc) overexpression, in both male and female Drosophila Our transcriptome analyses reveal that in the adult brain Da regulates the expression of genes involved in behavior, memory, synaptic signaling, protein translation, and metabolic processes. Moreover, combining the RNA sequencing data with Da ChIP sequencing results indicates that genes associated with neuronal projection guidance, metabolism, and translation are direct targets of Da. In addition, we validate the involvement of Da in memory formation. Overall, our results provide valuable information about the functions of Da in the adult brain and aid in better understanding the mechanisms of TCF4-related disorders.

overall_design: Anti-FLAG (Sigma-Aldrich F1804) ChIP-seq from adult Drosophila heads of 3xFLAG-Da and Control (white*) flies

first: Laura

last: Tamberg

first: Carl Sander

last: Kiir

first: Jürgen

last: Tuvikene

first: Käthy

last: Rannaste

first: Mari

last: Palgi

first: Indrek

last: Koppel


title: Characterizing age-dependent changes in histone methylation and determine the role of Trr and Trx in histone methylation in adult fly photoreceptors

city: WEST LAFAYETTE

first: Weake

middle: Marie

last: Vikki

state: IN

address: 175 S University st

department: Biochemistry

institute: Purdue University

summary: Histone modifications are important for DNA-templated activities, such as transcription. In the first part of this study, we profiled H3K4me2/3, H3K36me3. H3K9me3 and H3K27me3in young (D10) and old (D40) fly photoreceptors using CUT&RUN to characterize global changes in histone methylation during aging. Our data show that both active and repressive marks exhibit consistently decreased levels in photoreceptors cells of old flies compared to young flies. This global decrease in histone methylation suggests an overall reduction in methylation potential in old photoreceptors. In the second part of the same manuscript, we expressed shRNA against mCherry (control), Trr and Trx, two H3K4 methyltransferases, and profile H3K4me1, H3K4me2, and H3K4me3 using CUT&RUN. Our data show that the knockdown of Trr and Trx leads to pronounced decrease in H3K4me3 levels and modest decrease in H3K4me2 and H3K4me1 in adult photoreceptors.

overall_design: Drosophila were maintained on standard fly food at 25°C under 12-h:12-h light:dark conditions. Male flies were collected at indicated age (days post-eclosion, D) ± 1 day and ZT12 ± 15 minutes. For D10/D40 CUT&RUN, Rh1-Gal4>UAS-GFPKASH flies that express GFP on the photoreceptor nuclei membrane were collected on either D10 or D40 in quadruplicates, 400 flies per replicate. Photoreceptor nuclei were immuno-enriched (NIE) by anti-GFP coated magnetic beads (Jauregui-Lozano et al., 2021). CUT&RUN was performed on bead-bound photoreceptor nuclei as described in (Meers et al., 2019). Antibodies used are: anti-H3K4me2/3 (Abcam #ab8580), anti-H3K36me3 (Abcam #ab9050), anti-H3K9me3 (Abcam #ab176916) and anti-H3K27me3 (Epicypher #13-0055) and anti-IgG negative control (Epicypher #13-0042). pAG-MNase activation/ cleavage was performed for 20 minutes upon the addition of CaCl2.  For knockdown CUT&RUN, Rh1-Gal4>UAS-GFPKASH were crossed to either control (mCherry) RNAi, Trr RNAi (BDSC #36916), or Trx RNAi (BDSC #33703) and progenies were collected on D10 in triplicates for CUT&RUN. Antibodies used are: anti-H3K4me1 (Abcam #ab8895), anti-H3K4me2 Thermo Fisher #710796), anti-H3K4me3 (EpiCypher #13-0041) and anti-IgG negative control (Epicypher #13-0042). pAG-MNase activation/ cleavage was performed for 30 minutes. CUT&RUN libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® kit. Detailed protocols for NIE-based CUT&RUN are available: dx.doi.org/10.17504/protocols.io.kxygxp1d4l8j/v2.

first: Gaoya

last: Meng

first: Makayla

middle: N

last: Marlin

first: Vikki

last: Weake


title: H3K27me3 epigenetic mark crucial for callus cell identity and regeneration capacity [ChIP-seq]

city: Rehovot

first: Leor

middle: Eshed

last: Eshed Williams

address: P.O.Box 12

department: Faculty of Agriculture

country: Israel

institute: Hebrew university

summary: Plant callus cells possess a remarkable ability to regenerate organs that often differ from their respective origins or even entire new individuals. Yet, the mechanisms underlying their pluripotent state remain elusive. We propose a strategy that involves two independent mechanisms to endow callus cells with pluripotency: (1) maintaining a unique transcriptional profile, characterised by the expression of genes from diverse developmental pathways that allows rapid response to developmental cues; (2) preventing premature differentiation through H3K27 methylation-mediated silencing of key transcription factors such as WUCHEL and SPEECHLESS. This strategy relies on a mechanism to silence the pluripotency network upon regenerative stimuli, enabling a single developmental pathway to dominate. Our study reveals that the EMF2 complex, a key regulator of H3K27 tri-methylation, plays a crucial role in this process. Callus derived from the emf2 mutant, deficient in H3K27me3, exhibits severely impaired regeneration. Comparative analyses of chromatin states and transcription profiles between wild-type and emf2 calli revealed that the loss of EMF2 leads to upregulation of key transcription factors in callus, and identified the genes regulated solely by EMF2. Our findings suggest that suppressing pluripotency networks through H3K27me3 is essential for executing specific developmental programs to ensure effective regeneration.

overall_design: ChIP-seq profiling of wild-type Arabidopsis callus, and emf2 mutant-derived callus


title: Allosteric effectors of PRC2 synergise to restrict Polycomb domains spread

city: Clayton

first: Evan

last: Healy

state: VIC

address: 23 Innovation Way, Building 77,, Monash University

country: Australia

institute: Monash University

summary: Broad chromatin marks are often spread through read-write loops, where chromatin modifiers bind to their products. Polycomb domains are broad repressive chromatin regions in metazoan species, defined by the repressive chromatin mark H3 lysine 27 tri-methyl (H3K27me3) of the Polycomb repressive complex 2 (PRC2). PRC2 is an allosteric enzyme: it switches into a stimulated state by its own H3K27me3 product in a positive feedback loop that maintains Polycomb domains. Yet, it is unknown what prevents this PRC2-H3K27me3 positive feedback loop from spreading H3K27me3 indefinitely. Here we show that mutant mouse embryos, defective in the allosteric activation of PRC2 by its accessory subunits, exhibit a homeotic transformation characteristic of a Polycomb gain-of-function. The same mutations in mouse embryonic stem cells lead to a global gain of H3K27me3 and its spread beyond Polycomb domains. Collectively, we show that the allosteric effector activity of PRC2 accessory subunits restricts H3K27me3 deposition and prevents its spread beyond Polycomb domains.

overall_design: Quantitative ChIP sequecning for H3K27me3, H3K4me3, RING1B & H2AK119ub1 performed in wild type and SOF (JARID K116R, PALI1 K1241R + JARID2 K116R) mutant mouse embryonic stem cells. Independent replicates included. RNA sequencing of total RNA in wild type and JARID2 K116R mutant mouse embryonic tailbud tissue.

first: Samuel

middle: C

last: Agius

first: Marion

last: Boudes

first: Yoona

last: Kim

first: Yi-Cheng

last: Chang

first: Melanie

last: Murray

first: Jan

last: Manent

first: Jeanette

last: Rientjes

first: Zhihao

last: Lai

first: Sophie

last: Nixon

first: Jihane

last: Homman-Ludiye

first: Vitalina

last: Levina

first: Edwina

last: McGlinn

first: Chen

last: Davidovich


title: A role for condensin-mediator interaction in mitotic chromosomal organization [Hi-C]

city: Sapporo

first: Hideki

last: Tanizawa

state: Hokkaido

address: Kita 15, Nishi 7, Kita-ku

department: Institute for Genetic Medicine

country: Japan

institute: Hokkaido University

summary: Eukaryotic genomes are organized into layers of chromatin domains, such as topologically associating domains (TADs) and A/B compartments.  TADs (i.e., cohesin-mediated chromatin domains) are restricted by CCCTC-binding factor (CTCF) and convergent genes in higher eukaryotes and fission yeast, respectively.  However, molecular mechanisms underlying the formation of condensin-mediated chromatin domains remain largely unclear.  Here, we investigate the role of newly identified interaction between the Cnd1 condensin and Pmc4 mediator subunits.  We develop a condensin mutation, cnd1-K658E, that impairs the condensin-mediator interaction and find that this mutation diminishes condensin-mediated domains and causes defects in chromosomal segregation.  The condensin-mediator interaction is involved in recruiting condensin to highly transcribed genes and mitotically activated genes, the latter of which demarcate condensin-mediated domains.  Our study also predicted that the condensin-mediator interaction and its function in chromosomal segregation are potentially conserved in human cells.  This study provides a novel insight into how genome-wide gene expression is connected to the mitotic chromosomal architecture via the condensin-mediator interaction.

overall_design: (1) Hi-C experiment in mitotic cells with wild-type Cnd1 expression from the plasmid , without exogenous Cnd1 expression , and with exogenous Cnd1-K658E expression.  The endogenous Cnd1 was repressed by culturing cells in EMM medium containing  auxin.  Mitotic cells were prepared by the cdc25-22 G2/M block-release and harvested at the 60-minute time point after the release. (2) Hi-C experiment in mitotic cells with wild-type Pmc4 expression from the plasmid  and without exogenous Pmc4 expression.  The endogenous Pmc4 was degraded by the AID system.  The mitotic cells were prepared using the cdc25-22 block-release. (3) Hi-C experiment in asynchronous culture cells of various mediator deletions. (4) Hi-C experiment in asynchronous culture cells with Cnd2 depletion.  Cells were cultured in EMM liquid medium containing thiamine and auxin to induce transcriptional inhibition of cnd2 gene and post-translational degradation of endogenous Cnd2 proteins  The Cnd2 WT or C703R mutant was expressed from the plasmid. (5) Hi-C experimets in motitic cells with Cnd1 WT or Cnd1 K658E expression from the endogenous locus. Mitotic cells were prepared in YEA using the cdc25-22 G2/M block-release and harvested at the 40-minute time point after the release. (6) Hi-C experiments in mitotic cells with Cnd1, Pmc4 or Cnd1/Pmc4 double depletion. Mitotic cells were prepared in YEA using the cdc25-22 G2/M block-release and harvested at the 40-minute time point after the release. (7) Hi-C experiments in mitotic cells with 5 or 10 % HD treatment for 5min before fixation. Mitotic cells were prepared in YEA using cdc25-22 G2/M block-release and harvested at the 40-minute time point after the release.

first: Osamu

last: Iwasaki

first: Tomomi

last: Hayashi

first: Claire

last: Yik-Lok Chung

first: Sanki

last: Tashiro

first: Gabrielle

last: Tabor

first: Shinya

last: Ohta

first: Xuebing

last: Wang

first: Mikihiro

last: Kawagoe

first: Ken-ichi

last: Noma


title: The E-protein Daughterless regulates protein translation and memory in adult Drosophila nervous system [RNA-seq]

first: Tõnis

last: Timmusk

overall_design: To study the roles of Da in adult Drosophila nervous system, we investigated the brain transcriptome of flies where Da activity was suppressed by pan-neuronal Emc overexpression. For Emc overexpression elavC155-Gal4 was used. Differentially expressed genes in the brains of EmcOE (elavC155-Gal4>emc) flies were analysed compared to Control (elavC155-Gal4xwhite*) flies.

first: Laura

last: Tamberg

first: Carl Sander

last: Kiir

first: Jürgen

last: Tuvikene

first: Käthy

last: Rannaste

first: Mari

last: Palgi

first: Indrek

last: Koppel


title: A role for condensin-mediator interaction in mitotic chromosomal organization [ChIP-Seq]

first: Hideki

last: Tanizawa

overall_design: (1) Genome-wide distributions of condensin were determined by ChIP-seq using mitotic cells with wild-type Cnd1 expression (Cnd1 WT-Pk) and with Cnd1-K658E expression (Cnd1-K658E-Pk) from the plasmids.  The endogenous Cnd1 was repressed by culturing cells in EMM medium containing auxin.  Mitotic cells were prepared by the cdc25-22 G2/M block-release and harvested at a 60-minute time point after the release.   (2) In addition, genome-wide distributions of Cnd1-Pk in cells with wild-type and Pmc4 depletion are prepared.  (3) Genome-wide distributions of the Cnd2-Flag (WT and C703R) expressed from the plasmids were determined using cells depleting the endogenous Cnd2. (4) Genome-wide distributions of Cnd1-WT-Myc and Cnd1-K658-Myc expressed from the endogenous locus. Mitotic cells were prepared in YEA using the cdc25-22 G2/M block-release and harvested at a 40-minute time point after the release. (5) Genome-wide distributions of Cnd1, Pmc4, Pol II in mitotic cells with or without 5% HD treatment for 5min. Mitotic cells were prepared in YEA using the cdc25-22 G2/M block-release and harvested at a 40-minute time point after the release.


title: RNAseq analysis of bone marrow plasma cells from KD-treated mice

city: Guangzhou

first: Yiming

last: Zhu

address: No. 135, Xingang Xi Road

institute: Sun Yat-Sen University

summary: Humoral immunological memory is essential for the protection against pathogens post primary infection or vaccination. This protection relies on the long-term survival of pathogen-specific memory B cells and lone-lived plasma cells. We found that ketogenic diet (KD) impairs the humoral immunological memory by depleting the plasma cells. We performed functional analysis of these bone marrow plasma cells from control mice or KD-treated mice.

overall_design: Mice were provided with regular diet or ketoenic diet for three weeks, respectively. Then 10,000 plasma cells(CD138high CD267high) in bone marrow of these mice were sorted using BD FACSAria Ⅲ. mRNA profiles of sorted bone marrow plasma cells were generated in triplicates by Illumina Platform.

first: Chunliang

last: Xu


title: RNAseq analysis of bone marrow plasma cells from fasted mice

summary: Humoral immunological memory is essential for the protection against pathogens post primary infection or vaccination. This protection relies on the long-term survival of pathogen-specific memory B cells and lone-lived plasma cells. We found that fasting impairs the humoral immunological memory by depleting the plasma cells. We performed functional analysis of these bone marrow plasma cells from Ad Lib mice or fasted mice.

overall_design: Mice were subjected to a prolonged fasting treatment (72 hours)  or had free access to food, respectively. Then 10,000 plasma cells (CD138high CD267high) in bone marrow of these mice were sorted using BD FACSAria Ⅲ. mRNA profiles of sorted bone marrow plasma cells were generated in triplicates by Illumina Platform.


title: S-nitrosoglutathione reductase GSNOR drives age-related obesity by promoting adipose tissue whitening through de-nitrosation of Beclin-1

status: Public on Jan 26 2026

city: Beijing

first: Xinhua

last: Qiao

state: 北京市

address: 15# Datun Road,Chaoyang District, Chaoyang District

institute: Institute of Biophysics，Chinese Academy of Sciences

summary: Age-related obesity is a growing public health concern linked to various metabolic disorders, yet its underlying mechanisms remain incompletely understood. Here we report that S-nitrosoglutathione reductase (GSNOR), a pivotal denitrosation enzyme, increases in adipose tissue of both male mice and humans from middle-age. GSNOR knockout protects against age-related weight gain and enhances metabolism, whereas adipose-specific GSNOR knock-in mice promotes obesity and metabolic decline.  Further investigation reveals that aged GSNOR KO mice maintain higher mitochondrial content and more beige adipocytes, whereas adipose-specific GSNOR overexpression promotes adipose tissue whitening. Mechanistically, GSNOR denitrosates Beclin-1 at cysteine 351 and mutation of this site (Beclin-1C351A) increases autophagy by enhancing Beclin-1 and ATG14 interaction, thereby accelerating beige-to-white adipocyte conversion. Together, our findings reveal that GSNOR regulates adipose tissue remodeling during aging through Beclin-1 S-nitrosation, pointing to a potential therapeutic target for age-related obesity.

overall_design: To determine the transcriptional basis for the enhanced lipid metabolism in aged GSNOR KO mice, we performed RNA sequencing (RNA-seq) on inguinal white adipose tissue (iWAT) from 20-month-old wild-type (WT) and GSNOR knockout (KO) mice.

first: Xinhao

first: Ting

last: Xie

first: Chang

last: Chen


title: Differential Circadian Time Modulates of Dentate Gyrus and CA1 Excitation-Inhibition Balance and Seizure Threshold.

city: Hefei

first: daoming

last: cheng

state: Anhui

address: No. 96 Jinzhai Road, Baohe District, Hefei, Anhui, 230026, P.R. China.

institute: 中国科学技术大学附属第一医院的英文名称是 The First AfUniversity of Science and Technology of China

summary: The circadian rhythm modulates seizure threshold and neuronal excitability. Seizures in temporal lobe epilepsy are more likely to occur during the diurnal phase compared to the nocturnal phase. However, the intrinsic excitability of dentate gyrus granule cells and the state of synaptic transmission in CA1 pyramidal neurons exhibit relatively high excitability during the nocturnal phase. To investigate how circadian time modulates neuronal excitability and seizure threshold in the dentate gyrus and CA1, we utilized video-electroencephalography monitoring, systemic administration of pilocarpine, optogenetically induced seizures, mRNA sequencing, and whole-cell patch clam techniques.Our findings demonstrate that circadian time exerts differential modulation on the seizure threshold in dentate gyrus and the CA1.Furthermore, the circadian modulation of the CA1 seizure threshold is similar to that observed with systemic administration of pilocarpine-induced seizures. We also found that circadian rhythms differentially modulate excitatory and inhibitory synaptic transmission, as well as the intrinsic excitability of principal neurons in both the dentate gyrus and CA1. This study emphasizes the significance of circadian regulation in different brain regions concerning seizure threshold and the excitation-inhibition balance. Understanding how circadian rhythms influence hippocampal physiology and seizure threshold can enhance the translational relevance of therapeutics and inform the optimal timing for seizure management。

overall_design: Performed RNA-seq analysis on different hippocampal samples from  mice during the day and night.

first: Jiandong

last: Yu

first: Rongjing

last: Ge

first: Menghan

last: Zhang

first: Daoming

last: Cheng

first: Bingjing

last: Sun

first: Hong

last: Ni

first: Hao

last: Qian


title: Defining transcriptome signature consistent between cell types in blood and brain and related to the severity of Prader-Willi syndrome.

city: Parkville

first: Shokouh

last: Shahrokhi Sabzevar

address: Royal Children's Hospital, 50 Flemington Rd

institute: MCRI

summary: This study defined genetic subtype and cell type-specific gene expression changes and dysregulated pathways in the prefrontal cortex (PFC) of individuals with Prader-Willi syndrome (PWS). Genes identified to be differentially expressed consistently between all cell types of the PFC, were examined in blood with their mRNA levels related to the severity of PWS including intellectual functioning and behavioral problems. It was hypothesised that genes identified to be consistently differentially expressed across all cell types in the brain tissues examined from individuals with Prader-Willi syndrome (PWS) would be also affected in blood and with their mRNA levels related to the severity of PWS.

overall_design: single-nucleus RNA-sequencing was performed on the PFC tissue from 8 donors with PWS (4 15q11-q13 deletion and 4 non-deletion subtypes) and 4 age and sex-matched neurotypical controls. The shortlisted differentially expressed genes (DEGs) that were shared across all cell types of the brain, were examined using droplet digital PCR (ddPCR) in peripheral blood mononuclear cells (PBMCs) from an independent cohort of 36 individuals with PWS (16 deletion and 20 non-deletion).

first: David

last: Godler

last: Shahrokhi

first: Michael

last: See

first: Fernando

last: Rossello


title: SAGA/ATAC complexes sustain aberrant chromatin regulation and metabolic programs in diffuse midline glioma

city: Houston

first: Jamie

middle: N.

last: Anastas

state: TX

address: 1 Baylor Plaza

department: Neurosurgery

institute: Baylor College of Medicine

summary: Diffuse midline gliomas (DMG) are aggressive pediatric brain tumors characterized by chromatin and transcriptional dysregulation induced by H3K27M mutations. Strategies for overcoming epigenetic dysfunction to reduce DMG tumorigenesis remain limited. We identified multiple components of the SAGA and ATAC chromatin regulatory complexes as DMG genetic dependencies and found that genetic or pharmacological inhibition of the SAGA/ATAC-associated chromatin reader, SGF29, reduces DMG proliferation. Small molecules targeting SAGA/ATAC-associated histone acetylation, ubiquitination, and methylation similarly suppressed DMG growth. Further chromatin profiling and RNAseq analyses reveal that SGF29 controls H3K9ac and H3K4me3 dynamics at both H3K27M-bound and H3K27M-independent target genes linked to proliferation, differentiation, and metabolism. Finally, we find that SAGA/ATAC inhibition may reduce DMG viability by repressing cholesterol metabolism gene expression and show that combinations of cholesterol- and SAGA/ATAC-targeting drugs synergistically reduce DMG growth. These findings reveal a functional link between SAGA/ATAC-dependent chromatin regulation and both transcriptional and metabolic dysregulation underlying DMG malignancy.

overall_design: In one experiment, SU-DIPGXIII+Cas9 cells were transduced with control or SGF29-targeting sgRNAs and selected with 250 ug/mL hygromycin for 7 days 48 hours later. Equal live cell numbers were collected for RNAseq analysis at day 16 following the start of hygromycin selection and processed for bulk RNAseq of polyA transcripts. In another experiment, SU-DIPGXIII cells were treated with small molecules targeting either the bromodomains of KAT2A/2B (GSK4027) or the acetyltransferase activity of KAT2A/2B (CPTH2), with DMSO serving as a vehicle control.  Equal live cell numbers were collected for RNAseq analysis 25 hours after starting treatment with these inhibitors.

first: Rosemary

last: Richard

first: Akdes

last: Serin-Harmanci

last: N. Anastas


title: Sex-Based Differences in Cell Types and Gene Expression within the Anterior Cruciate Ligament

city: San Francisco

first: Brian

middle: T

last: Feeley

state: CA

address: 1700 Owens St

department: Orthopedic Surgery

institute: University of California, San Francisco

summary: ACL tear rates in females is significantly higher compared to their male counterpart. We utilized 11 patient samples to explore the biological and transcriptomic differences that exist in male and female ACL.

overall_design: Human anterior cruciate ligament was mechanically and enzymatically digested. Live cells were sorted by FACS and then sequenced. All cells were analyzed by 10x Genomics 3' single cell RNA sequencing.

first: Justin

last: Lau

first: Steven

middle: M

last: Garcia

first: Xuhui

last: Liu

first: Stephanie

last: Wong


title: Transcriptomic profile of B cells in response to IL-2 signal in vitro

city: Rennes

first: Simon

last: Leonard

address: 2 avenue du Prof Léon Bernard

country: France

institute: Université Rennes

summary: IL-2 is one of the first cytokine discovered but its complex roles in T cell effector functions has shadowed its function in B cell responses. B cells transiently expressed IL-2 receptor upon activation but it is unclear whether all mature B cell subsets have an equal dependence upon IL-2 and how IL-2 dictates B cell fate. Using our newly generated B-cell-specific Il2rb conditional knockout (KO) model of mice lacking IL2RB signaling in mature B cells, our objectives were to characterize the transcriptomic profile of B cells in response to IL-2 signal. We found that IL-2 acts intrinsically on IL2RB expressing B cells to promote Il10 expression and plasma cell reprogramming in context dependent manner in vitro. In contrast, an inflammatory response was observed in KO B cells.

overall_design:
To investigate the role of IL-2 on B cell fate we developed a new mice model invalidated for IL2 signaling in B cells only (Il2rb KO)
We recovered spleen from Ctrl and Il2rb KO mice, isolated B cells and cultured them 3 days with aBCR + IL+5 +/- IL-2 (n=4)
We then extracted RNA and performed RNA-sequencing on these sixteen samples with illumina technology
We performed comparative gene expression profiling analysis of RNA-seq data

first: Juliette

last: Gauthier

first: Celine

last: Delaloy


title: Transcriptome analysis of Arabidopsis thaliana seedlings after combined isoxaben and PEP3 treatment

city: Marburg

first: Timo

last: Engelsdorf

address: Karl-von-Frisch-Str. 8

institute: Marburg University

summary: Cell wall damage causes the induction of plant signaling. Here, we used isoxaben treatment to inhibit cellulose biosynthesis and cause cell wall damage. PEP3 is detected by PEPR receptors and contributes to both plant defense signaling and the regulation of isoxaben-induced responses.

overall_design: Arabidopsis thaliana Col-0 seedlings (10 days-old) were treated with DMSO (mock control), 600 nM isoxaben (ISX), 10 nM PEP3 and a combination of ISX and PEP3. Samples were taken at 6 h after treatment and 3 biological replicates were analyzed per treatment.

first: Richard

last: Morton


title: Nanoluciferase-CD63 labeling reveals extracellular vesicle kinetics in a mouse intraductal model of ductal carcinoma in situ

city: Oklahoma city

first: Bethany

last: Hannafon

state: OK

address: 975 NE 10th Street

department: Obstetrics and Gynecology

institute: University of Oklahoma Health Sciences Center

summary: Background. Ductal carcinoma in situ (DCIS) is a non-invasive precursor to breast cancer that increases lifetime risk for invasive disease and, in rare cases, causes distant metastases without local recurrence. Tumor-derived small extracellular vesicles (sEVs), or exosomes, mediate tumor progression, systemic signaling, and metastasis; however, the timing of release and biodistribution of sEVs from early preinvasive lesions remains unclear. Methods. Here, we investigated the release kinetics and systemic dissemination of sEVs secreted by DCIS cells confined to the mammary ducts using an orthotopic mouse mammary intraductal xenograft model with MCF10DCIS cells engineered to express NanoLuc (NLuc)-tagged CD63, an sEV marker. Tumor growth was monitored using bioluminescent imaging (BLI) in vivo and ex vivo; DCIS outgrowth and focal invasion were confirmed by fluorescent immunohistochemistry; and NLuc activity was assayed in tissue lysates. Next-generation RNA sequencing of brain tissue was used to evaluate gene expression changes at 28 days post-implantation (dpi). Results. Longitudinal BLI revealed CD63-NLuc signals in mammary glands (P < 0.05), and distant organs, including spleen (P < 0.05), liver (P < 0.05), lungs (P < 0.01) and brain (P < 0.01) by day 14 followed by detection in heart tissue (P < 0.001) and in the circulation by day 21, preceding histological evidence of invasion. Further, CD63-NLuc activity was significantly and positively correlated with BLI levels in mammary glands and plasma (P < 0.05). Immune, metabolic, and stress response pathways were downregulated in brain tissue at 28 dpi compared to 3 dpi. Conclusions. These findings demonstrate systemic dissemination of tumor-derived sEVs during DCIS progression, suggesting roles in early microenvironmental conditioning and metastatic preconditioning prior to overt invasion.

overall_design: RNA sequencing was performed to evaluate gene expression changes in brain tissue collected at days 3 and 28 from mice implanted with MCF10DCIS CD63-NLuc cells.

first: Hladik

last: Cole

first: Hannafon

last: Bethany


title: Microbiome analysis of control, radiation enteritis model, and treatment groups in rat stool samples using 16S rRNA sequencing

city: Nanning

first: Lingzhang

last: Meng

state: Guangxi

address: Qingxiu District

institute: Guangxi Academy of Medical Sciences

summary: This study investigates the gut microbiome composition and diversity in three groups of rats: control, radiation enteritis model, and treatment (TG) groups. Total DNA was extracted from stool samples, PCR-amplified targeting 16S rRNA gene variable regions, and sequenced using Illumina MiSeq or NovaSeq platforms. Downstream bioinformatics analyses included sequence quality control, denoising (DADA2/OTU clustering), taxonomic classification, alpha and beta diversity evaluation, differential species abundance analysis, and functional prediction. The processed data include ASV/OTU tables, taxonomy assignments, and sample metadata.

overall_design: Comparative microbiome profiling of three groups (control, model, TG) to assess microbial composition and diversity changes following disease induction and treatment. Each group includes 6–7 biological replicates. 16S rRNA amplicon sequencing was performed, followed by bioinformatics analyses including denoising, OTU clustering, taxonomic annotation, diversity analyses, and functional prediction.

first: Donglin

last: Zheng


title: TCR avidity governs CD8 T cell competitive fitness in an antigen dose-dependent manner but does not reliably predict fate

city: Aurora

first: Tonya

last: Brunetti

state: CO

address: 12800 East 19th Avenue

department: Immunology and Microbiology

institute: University of Colorado Anschutz Medical Campus

summary: T cell receptor (TCR) avidity for cognate peptide–MHC (pMHC) has been proposed to influence both the magnitude and differentiation of CD8 T cell responses, but its effects have been difficult to isolate from confounding factors such as epitope specificity and antigen presentation kinetics. To address this, we generated TCR-transgenic mice (CoVT1lo and CoVT1hi) expressing low- or high-avidity receptors for the same conserved H2-Kb–restricted epitope (VVLSFELL) derived from SARS-CoV-1/2 Spike. We show that TCR avidity governs expansion and differentiation through distinct mechanisms. Higher avidity consistently confers a competitive advantage only when antigen is abundant, with inter-clonal competition increasing in a dose-dependent manner. In contrast, the impact of avidity on differentiation varied with inflammatory context: across peptide vaccines, mRNA vaccination, and viral infection, higher avidity was associated with divergent effector versus memory precursor outcomes. These findings support a model in which TCR avidity determines competitive fitness in an antigen-dependent manner, whereas fate decisions integrate avidity with context-specific innate signals.

overall_design: vaccine_1, vaccine_2, and vaccine_3, are biological replicates of immunized C57BL/6 mice followed by isolation of CD8+ T cells enriched and sorted from the spleen 7 days post-immunization; naive_1, naive_2, and  naive_3 are biological replicates of C57BL/6 mice of isolated CD8+ T cells enriched and via  sorted from the spleen

first: Ross

last: Kedl

first: John

last: Manalastas

first: Cameron

last: Manes

first: Michael

last: Harbell

first: Bennett

last: Davenport

first: Liyen

last: Loh

first: Thomas

middle: E

last: Morrison

first: Jennifer

middle: L

last: Matsuda

first: Laurent

last: Gapin

first: Jared

last: Klarquist


title: Single cell transcriptomic profile of splenic B and T cells from  B-cell specific Il2rb deficient mice

first: Simon

last: Leonard

summary: IL-2 is one of the first cytokine discovered but its complex roles in T cell effector functions has shadowed its function in B cell responses. B cells transiently expressed IL-2 receptor upon activation but it is unclear whether all mature B cell subsets have an equal dependence upon IL-2 and how IL-2 dictates B cell fate. Using our newly generated B-cell-specific Il2rb conditional knockout (KO) model of mice lacking IL2RB signaling in mature B cells, our objectives were to characterize the heterogeneity of splenic B cell early response to in vivo immunization (SRBC) comparing scRNAseq data from control and KO mice. In parallel, we analyzed T cell subsets.

overall_design:
Il2rb KO and Ctrl mice were immuninized intraperitoneally with sheep red blood cells (SRBC).
Spleen of unimmunized mice (KO and Ctrl) and day 4 SRBC-immunized mice (KO and Ctrl) were recovered. Splenic B and CD4+ T cells were magnetically isolated.
We then extracted RNA and performed scRNA-sequencing.
ADT/HTO: barcode sequence: hashtagged sample
TotalSeq-C0301 anti-mouse Hashtag1: ACCCACCAGTAAGAC: KO D4
TotalSeq-C0302 anti-mouse Hashtag2: GGTCGAGAGCATTCA: Cre D4
TotalSeq-C0303 anti-mouse Hashtag3: CTTGCCGCATGTCAT: KO D0
TotalSeq-C0304 anti-mouse Hashtag4: AAAGCATTCTTCACG: Cre D0

first: Juliette

last: Gauthier

first: Celine

last: Delaloy


title: In vitro cartilage tissues following overexpression of TFs

city: Boston

first: April

last: Craft

address: 300 Longwood Ave

institute: Boston Children's Hospital

summary: Cartilage plays a crucial role in skeletal development and function, and abnormal development contributes to genetic and age-related skeletal disease. Compromises to the integrity of joint-lining articular cartilage in particular lead to debilitating chronic degenerative diseases including osteoarthritis. To better understand how human articular cartilage develops in vivo, we jointly profiled the transcriptome and open chromatin regions in individual nuclei recovered from distal femora at 2 fetal timepoints. We used these multiomic data to predict transcription factor-based regulatory networks that are important for articular chondrocyte differentiation. We developed a human pluripotent stem cell platform for interrogating the function of predicted transcription factors during chondrocyte differentiation and identified new biological roles for CREB5. We expect new regulatory networks we uncovered using multiomic data to be important for promoting cartilage health and treating disease, and our platform to be a useful tool for studying cartilage development in vitro.

overall_design: bulk RNA seq of in vitro derived chondrocytes following modulation of NFATC2 or CREB5

first: Divya

last: Venkatasubramanian

first: Gayani

last: Senevirathne

first: Andrew

last: Zhu

first: Terence

last: Capellini

first: Sarah

last: Jachim


title: Identifying the impact of H3K4 methylation on the circadian clock in aging Drosophila photoreceptors

first: Weake

last: Vikki

summary: To synchronize with environmental stimuli, predominantly light, the circadian clock directs the physiological and behavioral cycles with a periodicity of approximately 24 hours. The circadian clock is progressively dysregulated during aging, correlating with the development of many neurodegenerative diseases. Circadian disruption in the eye affects retinal development and accelerates photoreceptor degeneration during aging, which implies a protective effect of the functional circadian clock on photoreceptor neurons. At the core of the molecular clock, Clock (CLK) and Cycle (CYC), a pair of transcription activators, regulate the rhythmic transcription of output genes, including a substantial fraction of phototransduction genes. In the aging photoreceptors, CLK:CYC exhibit differential activity while the mechanisms that contribute to this change and its impact on downstream gene expression are poorly understood. H3K4me3 is a ubiquitous chromatin modification that is present at actively transcribed gene promoters. By profiling nuclear transcriptome throughout the 24h day, we identified that knockdown of any of the H3K4 methyltransferase significantly alter the rhythmic gene expression, and collectively, H3K4 methyltransferases (Set1, Trr and Trx) contribute to 90% of the rhythmic transcriptome in Drosophila photoreceptors. In addition, all of the core clock genes showed decreased expression amplitude in any of the KD conditions, indicating H3K4 methylation elevates clock-controlled transcription activation. Given that a global decrease of H3K4me3 is observed in the aging eye, our study favors the scenario where decreased H3K4me3 levels during aging reduce clock-controlled transcription.

overall_design: To knockdown H3K4 methyltransferase in Drosophila photoreceptors, we cross Rh1-Gal4 flies with UAS-RNAi lines, and use F1 progenies for RNA-seq. mCherry knockdown is used as a negative control. F1 flies are raised in 12-hour light: 12-hour dark incubators to Day 10. 400 males are collected every 4 hours spanning the 24- hour day (from ZT0 to ZT20) with a replicate of three each time point. Photoreceptor cells are extracted using nuclear-immuno enrichment (NIE) technique developed in our lab (Jauregui-Lozano et al., 2021, Genetics). Nuclear RNA is purified using ZYMO Direct-zol RNA Microprep Kits (#R2061). RNA library is prepared using Ovation® SoLo RNA-Seq System (#0502-32). mCherry and Set1 KD cDNA libraries are split into 3 laned and sequenced on HiSeq PE150. Trr and Trx KD cDNA libraries are sequenced using NovaSeq X Plus PE150.

first: Gaoya

last: Meng


title: A role for condensin-mediator interaction in mitotic chromosomal organization [PRO-Seq]

first: Hideki

last: Tanizawa

overall_design: Nascent RNA profiles in the following conditions were determined by PRO-seq.  The endogenous Cnd1 expression was repressed by culturing cells in EMM medium containing thiamine and auxin, and the exogenous Cnd1 proteins (WT and K658E) were provided from the plasmids.  For Pmc4 depletion, the endogenous Pmc4 was degraded by the AID system, and the exogenous Pmc4 protein was provided from the plasmid (Pmc4 WT).

first: Osamu

last: Iwasaki

first: Tomomi

last: Hayashi

first: Claire

last: Yik-Lok Chung

first: Sanki

last: Tashiro

first: Gabrielle

last: Tabor

first: Shinya

last: Ohta

first: Xuebing

last: Wang

first: Mikihiro

last: Kawagoe

first: Ken-ichi

last: Noma


title: Multiscale analysis and functional validation of the cellular and genetic determinants of skeletal disease

city: SYDNEY

first: Peter

last: Croucher

state: NSW

address: 384 VICTORIA ST, DARLINGHURST NSW 2010

institute: Garvan Institute of Medical Research

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: The effect of lncRNA GAS5 on lens epithelial cells

city: zhengzhou

first: Deqian

last: Kong

state: henan

address: No.1 Longhu Zhonghuan Road, Jinshui District

institute: The First Affiliated Hospital of Zhengzhou University

summary: Through transcriptome sequencing screening, it was found that knocking down GAS5 significantly disrupted the mRNA expression of key factors related to aging secretion phenotype, cell cycle inhibitory proteins, and antioxidant enzymes.

overall_design: This suggests that GAS5 may maintain the homeostasis of lens epithelial cells and delay aging by regulating the aging related gene network.


title: Exercise-Derived Exosomal miR-151-3p: An Innovative Anti-inflammatory and Antioxidant Exerkine for Spinal Cord Injury

city: Shanghai

first: Tiansheng

last: Xu

state: Shanghai

address: Shanghai

institute: OE

type: Non-coding RNA profiling by high throughput sequencing

summary: Exercise (Exe) training serves as the hub of the multimodal treatment system for spinal cord (SC) injury (SCI), with well-established efficacy. However, the forms in which it exerts its effects and the potential key mechanisms still require in-depth research. Exosomes (Exos) have demonstrated great potential in disease treatment and the elucidation of disease mechanisms. This experiment aims to investigate the functions and action targets of Exe-derived Exos (Exe-Exos) in SCI, providing a theoretical foundation for developing treatment strategies targeting Exos pathways post-SCI. Circulating blood Exos were extracted from normal Sprague-Dawley (SD) rats after 4-week treadmill Exe and from the control group (Sed). Hydrogel (Hyd) microneedles were employed to target and deliver the Exos to the T10 segment of the SC in rats with SCI. By constructing corresponding in vitro and in vivo cell and SCI animal models, we found that Exe-derived Exo miR-151-3p (Exe-Exos-miR-151-3p) mediated functional repair following SCI in rats. Furthermore, Exos were found to carry protective signals, with Exo-miR-151-3p as the key molecule. For the first time, we discovered that Exo-miR-151-3p produced anti-inflammatory (NF-kB signaling pathway), anti-apoptotic (Caspase3 and JNK), and anti- oxidative stress (Oxs)oxidative (Nrf2 and HO-1) effects by inhibiting the mitochondrial membrane gene ROMO1.

overall_design: Then, we analyzed the gene expression profiles of RNA-seq from serum exosomes of 20 rats in Exercise group and 20 rats in Sedentary group.

first: Xinwang

last: Ying

first: Jiamen

last: Shen

first: Yanfang

last: Zhao

first: Tiantaixi

last: Tu

first: Yi

last: Jiang

first: Zhiyang

last: Huang

first: Qingfeng

last: Xie


title: The Rpd3 histone deacetylase is critical for temperature-mediated morphogenesis and virulence in the human fungal pathogen Histoplasma

first: Mark

last: Voorhies

address: 513 Parnassus, S472

department: Microbiology and Immunology

institute: University of California


title: RNA-Seq Results of Hearts from Mice in the Sham Group, Sepsis-Induced Cardiomyopathy Group, and AAV-GATA4 Group

city: Xi’an

first: He

last: Sun

state: Shaanxi

address: Changle West Road

institute: Air Force Medical University

summary: Sepsis-induced cardiomyopathy (SIC) is a clinical emergency. To identify potential intervention targets, we established an SIC model in mice and performed RNA-Seq on the mice’s hearts.

overall_design: Eight-week-old male specific pathogen-free (SPF) C57BL/6J mice were purchased from the Laboratory Animal Center of Air Force Medical University. For the Sham group, only laparotomy and suture procedures were performed; for the SIC group, the model was established via the cecal ligation and puncture (CLP) method; and for the SIC + AAV-GATA4 group, mice were given a tail vein injection of AAV9-cTNT-GATA4 4 weeks prior to CLP. All mice were euthanized at 24 hours after the Sham operation or CLP, and their hearts were harvested for RNA-Seq analysis, with 3 mice in each group.


title: Transcriptome analysis of Arabidopsis thaliana treated with valinomycin

city: Seoul

first: NAYEON

last: YOO

address: 145 Anam-ro, Seongbuk-gu,

country: South Korea

institute: Korea Univ.

summary: The goal of this study is to compare NGS-derived transcriptome profiles of Arabidopsis thaliana with or without valinomycin treatment.

overall_design: Arabidopsis thaliana seedlings were infiltrated with valinomycin and/or Pseudomonas syringae pv. tomato DC3000. mRNA was extracted from the plants  6 h post infiltration.

first: Nayeon

last: Yoo

first: Jaeyoung

last: Kim

first: Jongyeun

last: Park

first: Sung Wook

last: Chi

first: Eui-Hwan

last: Chung


title: The Rpd3 histone deacetylase is critical for temperature-mediated morphogenesis and virulence in the human fungal pathogen Histoplasma – HDAC mutant TF ChIP-seq

summary: Adaptive responses to environmental stimuli are integral to the survival and virulence of microbial pathogens. The thermally dimorphic human fungal pathogen Histoplasma senses temperature to transition between a mold form in soil and a pathogenic yeast in mammalian hosts. The contributions of chromatin-modifying enzymes to the ability of Histoplasma to appropriately respond to temperature have never been explored. Through chemical inhibition and genetics, we determined that the class I histone deacetylase (HDAC) RPD3 is required for normal Histoplasma yeast morphology at 37 ºC. Rpd3 regulated the expression of key morphology-specific genes, including critical virulence factors and transcription factors (TFs), was required for normal DNA-binding activity of yeast-promoting TFs, and influenced histone acetylation levels at the loci of putative pro-filamentation TFs. Furthermore, Rpd3 was required for virulence in a macrophage model of infection. Taken together, Rpd3 is a critical regulatory component that both activates the pathogenesis program and represses the filamentation program to enable thermal dimorphism in Histoplasma. This work uncovers the crucial role that chromatin regulation plays in temperature response of this ubiquitous pathogen.

overall_design: Ryp1, Ryp2, and Ryp3 ChIP-seq of WT or rpd3 mutant Histoplasma yeast cells with three bioreps per genotype.

first: Nebat

last: Ali

first: Anita

last: Sil


title: The Rpd3 histone deacetylase is critical for temperature-mediated morphogenesis and virulence in the human fungal pathogen Histoplasma – HDACi TF ChIP-seq

overall_design: Ryp1, Ryp2, and Ryp3 ChIP-seq of WT Histoplasma yeast cells treated with DMSO or 50uL Entinostat with three bioreps per treatment.


title: Matrix stiffness modulated release of spheroid-derived extracellular vesicles and discovery of Piezo1 cargo [miRNA-seq]

city: Cincinnati

first: Claire

last: Chougnet

address: 240 Albert Sabin Way

institute: Cincinnati Children's Hospital Medical Center

summary: Augmented extracellular matrix (ECM) stiffness is a mechanical hallmark of cancer. Mechanotransduction studies have extensively probed the mechanisms by which ECM stiffness regulates intracellular communication. However, the influence of stiffness on intercellular communication aiding tumor progression in three-dimensional microenvironments remains unknown. Small extracellular vesicles (EVs) are communicators of altered biophysical cues to distant sites through EV-ECM interactions and EV-mediated recipient cell-ECM interactions. Here we demonstrate stiffness-mediated modulation of small EVs secretion and cargo from three-dimensional oral squamous cell carcinoma spheroids. Using a spheroid culture platform with varying matrix stiffness properties, we show that small EVs carry parental biomolecular cargo, including mechanosensitive Piezo1 ion channel and adhesion molecule CD44. We comprehensively validate the presence of both markers in our EV populations using proteomic and genetic analysis. Transcriptomic analysis of microRNA and long non-coding RNA cargo of small EVs released from soft and stiff ECM spheroids revealed enrichment of tumorigenic and metastatic profiles in EVs from stiff ECM cultures compared to that of soft ones. Gene set enrichment analysis of a comparative dataset obtained by overlaying spheroid mRNA and EV miRNA profiles identified key oncogenic pathways involved in cell-EV crosstalk in the spheroid model.

overall_design: Cal27 spheroids were cultured in soft (3 mg/mL Matrigel) and stiff (12 mg/mL Matrigel) using the liquid overlay technique. Spheroid culture media was used to extract small extracellular vesicles. Total RNA was extracted from EV samples and sequenced for mRNA, miRNA, and lncRNA.

first: Maulee

last: Sheth

first: Manju

last: Sharma

first: Leyla

last: Esfandiari


title: Notch signaling in the embryonic ectoderm promotes periderm cell fate and represses mineralization of vibrissa hair follicles

first: Hong

last: Li

department: Craniofacial Biology

institute: University of Colorado Denver Anschutz Medical Campus

summary: The Notch signaling pathway is a critical means to regulate cell face choice in animals.  Appropriate regulation of this pathway is also required for human face formation as both loss and gain of function mutations of Notch signaling can cause craniofacial syndromes.  Here we examine the consequences of manipulation of Notch signaling in the early mouse embryonic ectoderm by either removing the transcriptional effector Rbpj or expressing a constitutively active form of the Notch1 intracellular domain.  Loss of Rbpj resulted in cleft secondary palate but strikingly was also associated with the ectopic mineralization of vibrissal follicles.  In contrast, activation of Notch signaling resulted in multiple embryonic defects including a fully penetrant bilateral cleft lip and palate.  Further, single cell RNA-seq data indicated a switch from a basal epithelial cell identity towards periderm when Notch signal transduction was elevated.  These cell fate changes were accompanied by misregulation of genes and pathways known to impact human and mouse orofacial clefting including Grhl3, Irf6, and the Wnt pathway.  Together, these findings provide insight into human craniofacial conditions caused by misregulated Notch activity.

overall_design: Single-cell RNA sequencing was performed on microdissected regions of the upper face where the nasal and maxillary prominences are juxtaposed, including the lambdoid junction, nasolacrimal groove, and nasal fin, from E11.5 control and NotchGOF mutant embryos. For each condition, tissue from two embryos with 43 somites was pooled prior to library preparation. Control and mutant samples were processed separately and sequenced as independent datasets. A total of 11,618 cells were obtained for the control dataset (NotchGOF_Control) and 12,220 cells for the mutant dataset (NotchGOF_mutant). Cells were sequenced to an average depth of 38,111 reads per control cell and 36,122 reads per mutant cell.


title: The Rpd3 histone deacetylase is critical for temperature-mediated morphogenesis and virulence in the human fungal pathogen Histoplasma – HDACi histone acetyl ChIP-seq (SE)

overall_design: ChIP-seq with antibodies to the histone modifications H4K16Ac or H3K9Ac for Hisoplasma yeast cells treated with Entinostat or DMSO control with three bioreps per treatment.


title: Non-viral in situ gene editing effectively and safely rescues congenital ichthyosis-causing mutations in human skin

city: Berlin

first: Benedikt

last: Obermayer

address: Chariteplatz 1

department: Core Unit Bioinformatics

institute: Berlin Institute of Health

summary: Autosomal recessive congenital ichthyosis (ARCI) comprises a heterogeneous group of rare, severe genodermatoses that profoundly reduce patients’ quality of life and for which no curative therapies are currently available. In this study, we demonstrate clinically relevant in situ correction of the most prevalent ARCI-associated mutation, TGM1 c.877-2A>G, a pathogenic splice-site variant, using human disease models. Transient modulation of the skin barrier followed by topical delivery of the cytosine base editor eTD encapsulated in lipid nanoparticles resulted in restoration of approximately 30% of wild-type transglutaminase 1 activity in treated skin tissue. Repeated dosing was well tolerated and revealed an excellent safety profile, with no evidence of systemic exposure to either the lipid nanoparticles or the genetic payload, as confirmed by highly sensitive analyses including desorption electrospray ionization (DESI) metabolic imaging. Together, these data provide a comprehensive preclinical framework supporting the feasibility of in situ gene correction for genodermatoses and highlight its potential to enable curative, next-generation therapies for severe inherited skin disorders.

overall_design:
single-cell RNA sequencing of human skin tissue treated with eGFP-mRNA LNP following laser microporation; and Bulk-RNA-Seq of primary (healthy) and immortalized (ARCI) human keratinocytes with base editor mRNA and sgRNA delivered vis electroporation targeting a TGM1 Mutation in ARCI

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raw data are unavailable due to patient privacy restriction.
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first: Dilem

last: Apaydin

first: Gaurav

last: Sadhnani

first: Sarah

last: Hedtrich


title: The Rpd3 histone deacetylase is critical for temperature-mediated morphogenesis and virulence in the human fungal pathogen Histoplasma – HDAC mutant histone acetyl ChIP-seq

first: Mark

last: Voorhies

overall_design: ChIP-seq with antibodies to the histone modifications H4K16Ac or H3K9Ac for WT or rpd3 mutant Hisoplasma cells with three bioreps per treatment.


title: The Rpd3 histone deacetylase is critical for temperature-mediated morphogenesis and virulence in the human fungal pathogen Histoplasma – HDACi histone acetyl ChIP-seq (PE)

overall_design: ChIP-seq with antibodies to the histone modifications H4K16Ac or H3K9Ac for Hisoplasma cells treated with Entinostat or DMSO control at 37C or RT with three bioreps per treatment.


title: Differentially expressed genes in human monocyte-derived macrophages overexpressing TRIM21, NFATc3, or ASK1

city: Guangzhou

first: Sijia

last: Liang

address: 74 Zhongshan 2 Rd

department: Pharmacology

institute: Sun Yat-Sen University

summary: We hypothesize that the TRIM21-NFATc3/ASK1 axis plays a key role in steatohepatitis. TRIM21, NFATc3, and ASK1 plasmids were transfected separately in  human monocyte-derived macrophages(HMDMs), with the pCMV6 expression vector used as a control. Subsequently, RNA sequencing analysis was performed to obtain common feature genes for Connectivity Map screening.

overall_design: RNA-seq was performed to compare the mRNA expression level in HMDMs overexpressing TRIM21, NFATc3, or ASK1.

first: Si-Jia


title: The Rpd3 histone deacetylase is critical for temperature-mediated morphogenesis and virulence in the human fungal pathogen Histoplasma – HDAC mutant RNA-seq

overall_design: Expression profiling by RNA-seq of WT, rpd3, and hos2 mutant Histoplasma yeast cells at 37C or room temperature with three bioreps per treatment.

first: Ali

last: Nebat

first: Voorhies

last: Mark

first: Sil

last: Anita


title: The Rpd3 histone deacetylase is critical for temperature-mediated morphogenesis and virulence in the human fungal pathogen Histoplasma -- HDACi RNA-seq

overall_design: Expression profiling by RNA-seq of Hisoplasma yeast cells treated with two concentrations of Entinostat or control or room temperature with three bioreps per treatment and sampled after 1 or 2 days.

first: Nebat


title: Hypercholesterolemia impairs the expression of angiogenic microRNAs in extracellular vesicles within ischemic skeletal muscles

city: Montreal

first: Alain

last: Rivard

state: Qc

address: 900 rue St-Denis

country: Canada

institute: CRCHUM

summary: Background/Objectives: In severe peripheral artery disease (PAD) with limb ischemia, hypercholesterolemia (HC) is associated with impaired neovascularization. Extracellular vesicles (EVs) are present within ischemic muscles, and they contain microRNAs (miRs) involved in several biological functions, including angiogenesis and neovascularization. Methods: We used a mouse model of PAD and compared the response to hindlimb ischemia in hypercholesterolemic ApoE−/− vs. normocholesterolemic mice. Next generation sequencing (NGS) was used to perform a complete profiling of miR expression in ischemic skeletal muscles, and in EVs of varying sizes — large EVs (lEVs) and small EVs (sEVs) — within these muscles. Results: We identified several miRs with potential pro-angiogenic effects (angiomiRs) that are reduced by HC in lEVs (Let-7b-5p, miR-151-3p, Let-7c-5p) or sEVs (miR-21a-5p, miR-196b-5p, miR-340-5p). As proof of principle, we show that overexpression of Let-7b-5p in lEVs, or miR-21a-5p in sEVs, can significantly increase the angiogenic capacity of these EVs in vitro. HC also impaired the enrichment of specific angiomiRs in lEVs (miR-100-5p), sEVs (miR-142a-3p), or in both lEVs and sEVs (miR-146b-5p). In silico approaches including prediction of functional miR targets, pathway unions and gene unions, identified predictive resulting effects of HC-modulated miRs in EVs on processes having key roles in the modulation of angiogenesis and neovascularization, such as the regulation of actin cytoskeleton and focal adhesion, and HIF-1, MAPK, AMPK and PI3K-Akt signaling pathways. Conclusions: Our results constitute an important first step towards the identification of miRs that could be targeted to improve the angiogenic function of EVs in atherosclerotic conditions and reduce tissue damage in patients with severe PAD.

overall_design: small RNAseq profiling of ischemic muscles and of sEVs and lEVs from ischemic muscles from ApoE ko mice on western diet chow for 4 weeks prior to ischemia induction and from control C57Bl/6 on regular chow.

first: Nozha

last: Raguema

first: Sylvie

last: Dussault

first: Kevin

last: Sawaya

first: Michel

last: Desjarlais

first: Eric

last: Boilard

first: Sylvain

last: Chemtob


title: Smyd5 mediates IL-4 epigenetic control of macrophage antigen presentation to drive cooperative T cell-mediated tumor suppression [Crispr screen]

city: Xiamen

first: dang

last: buyun

state: Fujian

address: Xiang 'an District Xiang Annan Road

institute: xiamen university

summary: IL-4 is classically associated with “M2” macrophages considered tumor-immunosuppressive, yet marker-based labels poorly reflect macrophage function in tumors. Reports of a non-canonical, IL-4–imprinted macrophage state (M2inf) with trained-immunity features prompted us to test whether such imprinting contributes to antitumor immunity in vivo. Here we show that, in B16F10 melanoma, IL-4 programs macrophages through the lysine methyltransferase Smyd5 to deposit promoter-proximal H3K36me3, increase MHC class II, and potentiate T cell activation. Myeloid Il4ra deletion increased tumor burden and reduced MHC class II and intratumoral T cell activation, whereas co-transfer of IL-4–primed macrophages suppressed tumors and expanded activated CD4 and CD8 T cells. Antitumor benefit required adaptive lymphocytes and macrophage-intrinsic MHC class II, as Rag1 deficiency or macrophage Rfx5 deletion abrogated tumor suppression. These findings identify a non-canonical, IL-4–imprinted macrophage program that enables antigen presentation and cooperative T cell immunity in this tumor context.

overall_design: Firstly,  BMDMs were treated with IL-4 for 24 hours, then BMDMs were stimulated with LPS for another 24 hours

first: Buyun

last: Dang

first: Yuling

last: Hong

first: Qingxiang

last: Gao

first: Shih-Chin

last: Cheng


title: Smyd5 mediates IL-4 epigenetic control of macrophage antigen presentation to drive cooperative T cell-mediated tumor suppression [RNA-Seq]

overall_design: The BMDM macrophages of WT or Smyd5 KO were treated with IL-4 for 24 hours and then ATAC library construction was performed


title: RORγt⁺ Dendritic Cells Are a Distinct Lymphoid-Derived Lineage

city: St. Louis

first: Marco

last: Colonna

state: MO

address: 660 S. Euclid Ave.

department: Pathology and Immunology

institute: Washington University School of Medicine in St. Louis

summary: How tolerogenic dendritic cell (DC) lineages are established to prevent inappropriate immune responses to commensals and food antigens remains unclear. We identify RORγt⁺ DCs in mice as a distinct lymphoid-derived lineage to safeguard intestinal tolerance. Using lineage tracing and single-cell transcriptomics, we unveiled bone marrow–resident Rorc(t)⁺ progenitors, which include a RORγt⁺ innate lymphoid progenitor (RILP) that generates both ILC3s and RORγt⁺ DCs, and a pre-RORγt⁺ DC precursor committed exclusively to the RORγt⁺ DC lineage. RORγt⁺ DC development required the Rorc +7 kb enhancer, whose accessibility was ensured by the repressors REV-ERBα and REV-ERBβ, and depended on the transcription factors PRDM16 and PU.1 for lineage commitment. Loss of any of these regulators abrogated RORγt⁺ DC differentiation, reduced peripheral regulatory T (Treg) cell induction, and skewed toward T helper 2 responses. Together, these findings define murine RORγt⁺ DCs as a lymphoid-derived lineage whose enhancer- and transcription factor–driven development is essential for peripheral Treg cell-mediated immune homeostasis.

overall_design: We performed single-cell RNA sequencing on B220– CD3– Ly6C– tdTomato+ hCD2+ cells (ILC3s and RORγt+ DCs) isolated from bone marrow cultures of 15-day-old Gm38411 iCre-hCD2 R26-tdTomato mice after 8 days of culture.

first: Patrick

last: Rodrigues


title: The site-mutated Ehd3 failed to rescue the aberrant H3K36me2/me3 distribution patterns in the ehd3-1 mutant background.

first: Jiachen

last: Wang

address: No. 2005, Songhu Road

department: Institute of Plant Biology

institute: Fudan University

summary: We examined the genome-wide occupancy of H3K36me2/H3K36me3 in the transgenic plants HA-Ehd3/ehd3-1-1 and HA-Ehd3mu/ehd3-1-1.

first: Jiabing

last: Wu

first: Kangxi

last: Du

first: Wenhao

last: Xie

first: Xiang

last: Li

first: Xiaolong

first: Aiwu

last: Dong


title: A hierarchical Rorc(γt) cis-regulatory cascade orchestrates differentiation of RORγt⁺ innate immune cells [ChIP-seq]

city: Fukuoka

first: Shinichiro

last: Sawa

address: 3-1-1 Maidashi, Higashiku

institute: Kyushu University

summary: Beyond its well-established role in type 3 immunity, RORγt+ innate immune cells are also essential for secondary lymphoid organ (SLO) formation and gut homeostasis. However, the transcriptional regulatory mechanisms governing RORγt expression in these cells, including ILC3s, LTi cells, and antigen-presenting cells (APCs), remain largely unknown. Here, we identify two key cis-regulatory elements within conserved non-coding sequences (CNS) 9 and 11 in the Rorc locus, which are sequentially utilized during their differentiation. Initially, Runx-binding sites in CNS11 establish chromatin accessibility as early as the HSC stage. Notably, disruption of this chromatin priming prevents subsequent transcriptional activation, thereby abolishing the initial induction of RORγt in these cells. At later stages, CNS9 plays a critical role, particularly in the development of RORγt⁺ APCs, contributing to colonic pTreg induction. This hierarchical transcriptional regulation is essential for SLO formation, postnatal type 3 immunity, and restraining excessive intestinal type 2 immune responses through pTreg induction.

overall_design: Using a CBFβ antibody, we performed ChIP-seq analysis on Lin⁻ cells isolated from the E12.5 fetal livers of WT and CNS11mut/mut mice.

first: Takuma

last: Fukui

first: Satoshi

last: Kojo

first: Akihito

last: Harada


title: Ehd3 plays an essential role in recruiting SDG724 to H3K4me1-enriched chromatin regions (CUT&Tag).

summary: We performed CUT&Tag assays to examine SDG724 enrichment in the sdg724 and ehd3 sdg724 mutant backgrounds.


title: MYC modulates TOP2A diffusion to promote substrate detection and activity [SLAM-seq]

city: Solna

first: Vladislav

last: Kuzin

state: Stockholm

address: Solnavägen 9

department: Cell and Molecular Biology

country: Sweden

institute: Karolinska Institute

summary: Topoisomerases are essential enzymes that alleviate DNA supercoiling by cleaving and resealing DNA strands. Regulation of their abundance and activity is crucial for maintaining genomic stability and supporting nuclear functions. Previously, we showed that the oncoprotein MYC recruits and stimulates topoisomerases to remove DNA entanglements generated by oncogenic transcription. Understanding this mechanism may suggest methods to inhibit MYC-driven topoisomerase activation, thereby selectively targeting tumor-specific transcription. Here, we demonstrate that the essential topoisomerase TOP2A in human cells exists in a dynamic equilibrium between sequestration in the nucleolus, substrate searching in transcription hubs, and active engagement on chromatin. This equilibrium is highly responsive to changes in DNA topology, allowing cells to regulate TOP2A levels in the nucleoplasm. Using single molecule tracking, we show that MYC accelerates TOP2A diffusion in cells. In vitro, we demonstrate that MYC decreases the size of TOP2A condensates, likely by limiting non-specific self interactions. By increasing TOP2A diffusion, MYC promotes substrate binding and increases TOP2A engagement on the chromatin genome-wide, revealing the mechanism underlying MYC stimulation of TOP2A activity.

overall_design: SLAM-seq in HCT116 MYC-mAID cells in triplicates.

first: Donald

last: Cameron

first: Kathryn

last: Jackson

first: Evanthia

last: Iliopoulou

first: Laura

last: Baranello


title: Coviretinopathy: COVID-19-induced VEGF-dependent retinopathy

city: Wuxi

first: Xiaolu

state: China

address: 299 Qingyang Road

institute: The Affiliated Wuxi People's Hospital of Nanjing Medical University

summary: COVID-19 has been associated with high prevalences of retinal diseases in humans. However, cellular and molecular mechanisms that underly the COVID-19-associated retinopathy remains unknown. Here, we deployed a mouse COVID-19 model to investigate the causative link between SARS-CoV-2 infection and retinopathy development. Our data showed that COVID-19-induced pulmonary hypoxia triggered systemic hypoxia and markedly augmented VEGF expression levels in the retina and plasma. High VEGF levels altered vascular structures and functions in the retina, resulting in neovascularization, vascular disorganization and increased leakiness. We deployed a new terminology of coviretinopathy to accurately describe these COVID-19-induced pathological changes in the retina. Consequently, blocking VEGF by a specific neutralizing antibody (VEGF blockade) completely ablated the COVID-19-associated vascular changes in the retina. Together, these findings provide new mechanistic insights into the COVID-19-associated retinopathy and propose a new therapeutic paradigm for effective treatment of coviretinopathy.

overall_design: Mouse lung and retinal tissues were dissociated into single-cell suspensions and processed by CapitalBio Technology (Beijing, China) using the 10x Genomics Chromium platform with the Single Cell 3′ Library Kit v2. Libraries were sequenced on an Illumina NovaSeq 6000 (paired-end 150 bp, >100,000 reads/cell). The data were processed with Cell Ranger, and downstream analysis, including clustering (Seurat), pseudotime trajectory (Monocle 2), and functional enrichment (DAVID), was performed using standard pipelines.

first: Yihai

last: Cao


title: X-ray activated platinum complex induces DNA damage and enhances cancer immunotherapy through abscopal effect

city: Hefei

first: Chen

last: Guiyuan

address: Jinzhai Road

institute: University of Science and Technology of China

summary: Radiotherapy is used in more than half of cancer patients, yet most radiosensitisers increase reactive oxygen species (ROS) to enhance cytotoxicity in treated cells. This approach is limited in hypoxic tumours and may cause oxidative injury to healthy tissues. We developed a platinum(II) azido complex (Complex 1) that releases platinonitrene upon X-ray exposure. Platinonitrene reacts with nucleophilic sites on DNA bases, forming covalent adducts that disrupt DNA integrity and cause double-strand breaks, leading to tumour cell death through a mechanism distinct from classical platinum coordination. Computational modelling elucidated this pathway and supported its role in radiosensitisation. Complex 1 was synthesised by sequential ligand exchange of potassium tetrachloroplatinate with cyclohexanediamine, silver nitrate, and sodium azide. In murine models, Complex 1 showed negligible toxicity to major organs and normal immune cells while selectively reducing regulatory T-cell infiltration in tumours. Combined with low-dose radiotherapy and programmed cell death protein 1 blockade, it achieved complete regression of bilateral tumours in forty per cent of mice, demonstrating a strong abscopal effect. This work establishes metallonitrene-based, ROS-independent radiosensitisation for precision radiotherapy.

overall_design: RNA-seq of CT26 tumor tissues from different groups of mice treated with Complex 1 and radiation therapy

first: Guiyuan

last: Chen

first: Xiangxia

first: Yu

last: Huang

first: Cheng

last: Zheng

first: Yuanzeng

last: Min


title: A hierarchical Rorc(γt) cis-regulatory cascade orchestrates differentiation of RORγt⁺ innate immune cells [ChIL-seq]

first: Shinichiro

overall_design: ChIL-seq targeting CBFβ was performed in MNK3 cells cultured with IL-7, IL-2, and IL-15 in the presence of ATRA. A total of 5,000 cells were used per sample for the analysis.

first: Kaori

last: Tanaka


title: RORγt⁺ Dendritic Cells Are a Distinct Lymphoid-Derived Lineage [CITE-seq]

first: Marco

last: Colonna

overall_design: We performed scRNA-seq on B220– CD3– Ly6C– tdTomato+ hCD2+ (Sample 1) and tdTomato+ hCD2- (Sample 2) cells from BM, spleen, cLN, mLN, and sLN of 15-day-old and adult Gm38411 iCre-hCD2 R26tdTomato mice.

first: Patrick

last: Rodrigues


title: Multiomic analysis reveals that polyamines alter G. vaginalis-induced cervicovaginal epithelial cell dysfunction

city: Hanover

first: Olha

last: Kholod

state: NH

address: 15 Thayer Drive

institute: Dartmouth College

summary: An anaerobe-dominant, Lactobacillus-deplete cervicovaginal microbiome is associated with adverse reproductive outcomes. Gardnerella vaginalis, a common cervicovaginal anaerobe, alters cervicovaginal epithelial cell function, resulting in inflammatory immune responses and epithelial barrier breakdown. Specific host-microbial mechanisms inducing this epithelial dysfunction remain unknown. Here we show microbe-specific alterations in cervicovaginal epithelial cell metabolite profiles where G. vaginalis, but not Lactobacillus crispatus, increases polyamine biosynthesis. Pretreatment with polyamines (putrescine, spermidine and spermine) globally shifts G. vaginalis-induced transcriptomic profiles. Alterations in transcripts encoding enzymes responsible for polyamine synthesis and catabolism provides further evidence that G. vaginalis modifies polyamine biosynthesis. Additionally, polyamine-mediated transcriptomic changes include genes related to bacterial defense, inflammation, and epigenetic processes. Polyamines mitigate G. vaginalis-induced inflammatory responses through reduction of cytokines, chemokines, and matrix metalloproteinases. The ability of cervicovaginal metabolites to alter microbe-mediated changes in epithelial cell function suggests that metabolite-microbe interactions are critical mediators of epithelial defense against a Lactobacillus-deplete microbiota.

overall_design: Human cervicovaginal epithelial cell lines (Ectocervical, Endocervical, and Vaginal) were exposed to Gardnerella vaginalis (10⁷ CFU/well) for 24 hours to characterize microbe-induced transcriptional responses. To assess the impact of vaginal polyamines on these responses, cells were pretreated for 4 hours with putrescine (4 mM), spermidine (400 µM), or spermine (400 µM) prior to G. vaginalis exposure. Control conditions included untreated cells and G. vaginalis exposure without polyamines. Total RNA was extracted, ribosomal RNA–depleted, and sequenced using the CORALL total RNA-seq protocol on an Illumina NovaSeq platform.

first: Lauren

last: Anton

first: Rutuja

last: Phatate

first: Briana

last: Ferguson

first: Kristin

last: Klohonatz

first: Brittany

middle: A

last: Goods

last: Gerson


title: Smyd5 mediates IL-4 epigenetic control of macrophage antigen presentation to drive cooperative T cell-mediated tumor suppression [ATAC-seq]

first: dang

last: buyun

first: Buyun

last: Dang

first: Yuling

last: Hong

first: Qingxiang

last: Gao

first: Shih-Chin

last: Cheng


title: RNA-seq analysis of periostin-stimulated human nasal epithelial cells

city: Sakado

first: Yusuke

last: Ando

state: Saitama

address: 1-1, Keyaki-dai

institute: Josai University

summary: This study investigates transcriptomic changes induced by recombinant human periostin in cultured normal human nasal epithelial cells using bulk RNA sequencing.

overall_design: Primary human nasal epithelial cells were cultured in vitro and treated with recombinant human periostin or vehicle control. Total RNA was extracted and subjected to paired-end bulk RNA sequencing to compare gene expression profiles between conditions.

first: Yoshihiko

last: Chiba


title: Deep mutational scan of tapasin-TM, a variant of the MHC class 1 chaperone tapasin

city: Philadelphia

first: Nikolaos

last: Sgourakis

state: PA

address: 3501 Civic Center Blvd

institute: Children's Hospital of Philadelphia

summary: Tapasin is a member of the peptide loading complex which is responsible for stabilizing empty HLA molecules and facilitating the loading of antigenic peptides. Many neuroblastoma cell lines contain low baseline levels of HLA due to defects in antigen processing and presentation machinery. Supplementing the neuroblastoma cell line EBc1 with tapasin-TM, an altered form of tapasin containing the transmembrane domain of HLA-G, enhances surface HLA expression. Here, we have applied our previously used deep mutational scanning library (GSE159247) to the tapasin-TM construct. We then introduced this library into EBc1 cells and isolated cells with the highest surface HLA-A2 expression.

overall_design: EBc1 cells were transduced with lentivirus containing our previously generated site saturation mutagenesis library (GSE159247) encoding 2160 tapasin-TM variants, where 108 tapasin-TM residues were mutated to every other amino acid. Cells were transduced such that the majority of cells contained only one variant, stained for the transduction marker FLAG (DYKDDDDK), and sorted to isolate transduced cells. This population was used as the naïve library. These cells were then expanded, stained for surface HLA-A2 (clone BB7.2) and cells were sorted to isolate those with the highest HLA-A2 expression (top 5%). Both the transduced cell population and the HLA-A2 sorted populations were sequenced. The frequencies of each mutation in the sorted cell library were compared to that of the naïve library.

first: Molly

last: Erdman

first: Kaya

last: Epstein

first: Daniel

last: Hwang