title: Mapping of Rpo21 RNA-binding sites in Saccharomyces cerevisiae with abrogated binding of Nab3 to PIC2

summary: Using CRAC, we compared the transcriptomic occupancy of Rpo21 in a Saccharomyces cerevisiae BY4741 parental strain (PIC2-GFP) and two derived mutants lacking Nab3 RNA-binding sites in PIC2. The purpose of the experiment was to determine that changes in the abundance of differentially expressed transcripts, which had been previously identified by RNA-sequencing, were not caused by alterations in Rpo21 occupancy and transcription. Sequencing outputs were processed using the pyCRAC pipeline and peak calling was performed with DBPeaks, our newly developed package for identification and comparison of binding sites defined by RNA-binding footprinting techniques (e.g., CRAC, iCLIP, PAR-CLIP, etc.).

overall_design: Two biological replicates of CRAC experiments performed in a parental Saccharomyces cerevisiae strain (PIC2-GFP) and two derived mutants: mut-Nab3-BS and mut-NNS-BS. While mut-Nab3-BS lacks Nab3 RNA-binding sites in PIC2, the other strain has no Nab3 or Nrd1 RNA-binding sites in PIC2 (mut-NNS-BS). All strains encoded RPO21-HTF (HIS6-TEV-3xFLAG). When SC-Ura cultures containing 1% (w/v) raffinose reached an OD600 of 1, cells were UV cross-linked (254 nm; 500 mJ/cm2) in the Vari-X-linker (UVO3) and rapidly filtered on 0.8 μm membranes (Merck) using a filtration device connected to a vacuum pump. Membranes were collected in 50 mL tubes, flash frozen by submersion into liquid nitrogen and stored at -80ºC until the day of the experiment.  On the day of the experiment, pellets were resuspended in a volume of lysis buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40, 1U/mL of RQ1 RNase-free DNAse (Promega) and 1 EDTA-free protease inhibitor cocktail (Roche)/ 50 mL and 5 mM β-mercaptoethanol) corresponding to twice their mass. Lysates were then transferred to a fresh 50 mL tubes, where they were mixed with 2 volumes of 0.5 mm zirconia beads (Thistle Scientific), and vortexed at maximum speed for five 1-minute intervals alternated with 1-minute incubations on ice. An additional volume of lysis buffer was pipetted into each sample before partitioning the cell debris by centrifugation at 4ºC (4000 rpm, 15 minutes) and spinning down lighter impurities in an additional centrifugation step (10000 rpm, 20 minutes) at 4ºC. Immunoprecipitation of the labelled protein from the supernatant was achieved during a two-hour incubation of the sample with 75 µL of anti-FLAG® M2 magnetic beads (Sigma-Aldrich, M8823-5ML) which had been previously washed three times with 1 mL TN150 buffer. Immobilised beads underwent three 5-minute washes with TN1000 buffer (50 mM Tris pH 7.8, 1 M NaCl, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol) and three additional 5-minute washes in 2 mL TN150 buffer (150 mM NaCl, 50 mM Tris pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol). Afterwards, beads were resuspended in 550 µL of TN150 and incubated with 1 µL of a 1:100 dilution of RNace-It™ for exactly 5 minutes at 37 °C. To prevent overdigestion of co-immunoprecipitated transcripts and elute the bait proteins from the anti-FLAG beads, the solution was mixed with 0.4 g of guanidium hydrochloride (Gu-HCl) and the necessary volume of NaCl and imidazole (pH 8.0) to reach a final concentration of 300 mM and 10 mM respectively. After removing the beads, samples were pipetted onto 50 µL of Ni-NTA agarose resin (Qiagen) equilibrated with wash buffer I (6 M Gu-HCl, 10 mM imidazole, 300 mM NaCl, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40 and 5 mM β-mercaptoethanol) and incubated at 4 °C overnight while rotating at 12 rpm.  Upon Ni-NTA binding, beads were transferred to Pierce columns (Thermo Fisher Scientific) and washed twice with 500 µL of wash buffer I before undergoing three additional washes of 500 µL with NP-PNK buffer (10 mM MgCl2, 50 mM Tris-HCl pH 7.8, 0.1% (v/v) Nonidet P-40, 5 mM β-mercaptoethanol). Alkaline phosphatase treatment was performed by resuspending immunoprecipitated RNA-protein conjugates in 80 µL of NP-PNK buffer containing 4 units of FastAP thermosensitive alkaline phosphatase (Thermo Fisher Scientific) and 80 units of RNasin® ribonuclease inhibitor (Promega) and incubating the samples at 37 °C for 1 hour. Dephosphorylation was stopped upon addition of 500 µL of wash buffer. Having equilibrated the Ni-NTA agarose with three washes of 500 µL with NP-PNK, we performed adapter ligation by resuspending the beads 80 µL of reaction mix containing 0.6 µM of the applicable App-PE 3′ adapter (5’App-NAGATCGGAAGAGCACACGTCTG-ddC 3’), 30 units of T4 RNA ligase 2 truncated K227Q (New England Biolabs), 60 units RNasin® (Promega) and 10% (w/v) polyethylene glycol 8000 (PEG 8000). The reaction took place at 25 °C for six hours.  After washing once with 500 µL of wash buffer I and three times with 500 µL of NP-PNK buffer, we 5’end radiolabelled co-immunoprecipitated RNAs by incubating the Ni-NTA resin with 60 µL of NP-PNK buffer containing 30 µCi 32P-γATP (PerkinElmer) and 30 units of T4 polynucleotide kinase (New England Biolabs). After a 40-minute incubation of the reaction at 37ºC, ATP (Roche) was supplemented to a final concentration of 1 mM and the reaction continued for another 20 minutes. The reaction was stopped by washing three times with 500 µL of wash buffer I and three 500 µL washes of NP-PNK buffer followed before the addition of the 5′ linker ligation mix. The reaction took place in a volume of 80 µL of NP-PNK buffer supplemented with 1.25 µM of the fitting adapter, 40 units of T4 RNA ligase 1 (New England Biolabs), 80 units of RNasin® and 10 mM ATP (Roche).  5’ adapter ligation was left to proceed overnight at 16 °C for all bait proteins. The adapter sequences were 5’-ACACGACGCTCTTCCGATCTNNNTAAGCN-3’  and 5’-ACACGACGCTCTTCCGATCTNNNCGCTTAGCN-3’ for PIC2-GFP repeats 1 and 2 respectively; 5’-ACACGACGCTCTTCCGATCTNNNGTGAGCN-3’ and 5’-ACACGACGCTCTTCCGATCTNNNTAGTGCN-3’ for mut-Nab3-BS replicates 1 and 2 respectively;  and 5’-ACACGACGCTCTTCCGATCTNNNACTCAGCN-3’ and 5’-ACACGACGCTCTTCCGATCTNNNGTGACAN-3’ for mut-NNS-BS libraries 1 and 2 respectively. After pooling suitable libraries together, the samples and its corresponding traces were submitted to Novogene, where paired-end sequencing of 150 bp-reads was performed in a NovaSeq 6000 system (Illumina).

last: Esteban Serna


title: Systemic miR-26a deficiency attenuates pulmonary fibrosis via PTEN upregulation and downstream TIMP-1 suppression

status: Public on Nov 15 2025

city: Hiroshima

first: Arisa

last: Hamada

state: Hiroshima

address: Kasumi, Minami-ku,

department: Department of Molecular and Internal Medicine

country: Japan

institute: Graduate School of Biomedical and Health Sciences, Hiroshima University

type: Expression profiling by high throughput sequencing

summary: To identify factors that suppressed fibrosis in miR-26a KO mice, we performed a comprehensive comparison of total lung mRNA expression levels between the WT and miR-26a KO groups using RNA sequencing analysis.

overall_design: WT and KO mice were euthanized on day 0 (prior to BLM OA) and day 7 after BLM OA.  Both the day 0 and day 7 groups were subjected to identical procedures from sample collection through RNA sequencing.

first: Kiyofumi

last: Shimoji

first: Taku

last: Nakashima


title: Tramadol Triggers Paraptotic Cell Death through ATF4 in Breast Cancer Cells

city: Taipei

first: Zih-Syuan

last: Wu

address: No.161, Sec. 6, Minquan E. Rd., Neihu Dist., Taipei City 114201, Taiwan

country: Taiwan

institute: National defense medical center

summary: Breast cancer treatment has significantly improved, achieving a five-year survival rate of 90%. However, 30% to 50% of survivors continue to suffer from persistent pain, a common long-term side effect. Tramadol, an analgesic often prescribed for postsurgical pain, has recently been shown to have anticancer effects on breast cancer cells. The use of tramadol in postoperative care has been associated with reduced recurrence and improved survival rates, but the mechanisms involved in its anticancer activity are not fully understood. This study explored how tramadol induces paraptosis, a form of nonapoptotic cell death, by focusing on endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) pathways in breast cancer cells. The findings revealed that tramadol treatment increased ROS levels, triggering ER stress and activating the p-eIF2α/ATF4/CHOP signaling axis. This stress response led to cytoplasmic vacuolization and mitochondrial dysfunction, both of which are key features of paraptosis. Inhibiting ROS production or blocking the ATF4 pathway reduced tramadol-induced paraptosis, confirming the critical role of these mechanisms in breast cancer cells. In summary, tramadol induces paraptosis in breast cancer cells by activating ROS and ER stress, offering potential insight into its anticancer effects. These findings could have implications for improving both pain management and cancer treatment outcomes in breast cancer patients.

overall_design: MDA-MB-231 and MCF-7 cells were treated with 0 or 1 mg/ml of tramadol for 4 h. Total RNA was isolated and subjected to RNAseq analyses.

first: Yi-Hsuan

last: Huang

first: Shih-Ming


title: Spatial and single cell transcriptomics in the human Entorhinal Cortex across diverse risk of Alzheimer’s disease

status: Public on Nov 14 2025

city: Baltimore

first: Keri

last: Martinowich

state: Maryland

address: 855 N Wolfe St Suite 300

country: USA

institute: Lieber Institute for Brain Development

summary: We present the first spatially-resolved transcriptomics and single-nucleus RNA-sequencing integrated dataset from the entorhinal cortex (ERC) of postmortem human brain donors. ERC is implicated in early progression of Alzheimer’s Disease (AD) and we chose this brain region to study gene expression changes associated with AD risk by comparing APOE E2 and E4 allele carriers. Donors with no AD diagnosis or widespread neurodegeneration were exclusively selected. As AD risk is increased in African Ancestry in contrast to European Ancestry backgrounds, and AD risk is also increased in women compared to men, our dataset included donors from these different backgrounds. We identified a pre-myleinating oligodendrocyte sub-cluster with transcriptomic changes strongly associated with APOE E4 carriers. These changes were present in both ancestry backgrounds, although they were more pronounced in African Ancestry donors. To facilitate future studies based on this dataset, we provided the raw and processed data, and created interactive web applications.

overall_design: We obtained tissue blocks of postmortem human brain entorhinal cortex (ERC) from 31 neurotypical adult brain donors carrying either E2 or E4 APOE alleles from both African and European ancestry. For each donor, a sample was generated for both spatially resolved transcriptomics with 10x Visium, and single nucleus RNA-seq with 10x Chromium. Visium data showed 9 transcriptional distinct spatial domains. snRNA-seq data was clustered into 38 fine subtypes across 8 broad cell types. Differential expression between APOE carrier status (E2+ vs. E4+) was performed at the cluster level in both data types.

first: Louise

middle: A

last: Huuki-Myers

first: Heena

middle: R

last: Divecha

first: Svitlana

middle: V

last: Bach

first: Madeline

last: Valentine

first: Nicholas

middle: J

last: Eagles

first: Bernard

last: Mulvey

first: Rahul

last: Bharadwaj

first: Ruth

last: Zhang

first: James

last: Evans

first: Melissa

last: Grant-Peters

first: Ryan

last: Miller

first: Joel

middle: E

last: Kleinman

first: Shizhong

last: Han

first: Thomas

middle: M

last: Hyde

first: Stephanie

middle: C

last: Page

first: Danie

last: Weinberger

first: Mina

last: Ryten

first: Kristen

last: Maynard

first: Leonardo

last: Collado-Torres


title: Endothelial IL-36 receptor activation promotes vascular stability to limit pathological microvessel permeability in the CNS

city: Dublin 2

first: Jeffrey

last: O'Callaghan

state: Dublin

address: Smurfit Institute

country: Ireland

institute: Trinity College, Dublin

summary: Pathological increases in vascular permeability lead to edema and swelling, causing a host of retinal and neurological disorders. Few barrier-enhancing factors have been discovered to specifically promote barrier integrity and make blood vessels resistant to fluid leakage. In this study, we explore the effects of IL-36 receptor (IL-36R) activation on vascular permeability in vivo in adult mice, ex vivo in tissue explant and in vitro in primary mouse and human microvascular endothelial cells. Using a highly soluble and biologically active DEVD- modified recombinant IL-36β cytokine, we find that processed DEVDIL-36β enhances endothelial barrier function, reducing vascular leakage and thus limiting pathology. Specific knockdown of IL-36R on endothelial cells using the Cre/Lox system demonstrates it is IL-36R signaling in endothelial cells that is responsible for DEVDIL-36β’s barrier-promoting property. Analysis of IL-36R expression and localization on endothelial cells implies IL-36R is mobilized to the plasma membrane in response to loss of endothelial cell-cell contact, in keeping with the IL-1 family role in responding to tissue stress. Mechanistically, IL-36R signaling increases adherens and tight junctions and induces vasculoprotective processes that result in vessel remodeling and stabilization. These functional assays are further supported by RNA sequencing data. In summary, our data present IL-36R signaling as a novel regulator of vascular integrity, with barrier-enhancing properties that prevent pathological vascular leakage.

overall_design: RNA-Seq profiling of primary mouse brain microvascular endothelial cells (BMVECs) treated with DEVD mIL-36β (100 ng/ml) for either 24 hours or untreated.

first: Erin

last: Fahey

first: Lucia

last: Celkova

first: Valentina

last: Frezza

first: Graeme P.

last: Sullivan

first: Kieran

last: Byrne

first: Avril

last: Watson

first: Yasmina E.

last: Hernandez-Santana

first: Kieva

first: Rachel

last: Dalton

first: Hande

last: Efe-Cicek

first: Conor

last: Delaney

first: Ema

last: Ozaki

first: Patrick T.

last: Walsh

first: Seamus J.

last: Martin

first: Matthew

last: Campbell

first: Sarah L.

last: Doyle


title: Spatial and single cell transcriptomics in the human Entorhinal Cortex across diverse risk of Alzheimer’s disease [Visium]

first: Keri

last: Martinowich

first: Louise

last: Huuki-Myers

first: Heena

last: Divecha

first: Svitlana

last: Bach

first: Madeline

last: Valentine

first: Nicholas

last: Eagles

first: Bernard

last: Mulvey

first: Rahul

last: Bharadwaj

first: Ruth

last: Zhang

first: James

last: Evans

first: Melissa

last: Grant-Peters

first: Ryan

last: Miller

first: Joel

last: Kleinman

first: Shizhong

last: Han

first: Thomas

last: Hyde

first: Stephanie

last: Page

first: Daniel

last: Weinberger

first: Mina

last: Ryten

first: Kristen

last: Maynard

first: Leonardo

last: Collado-Torres


title: Cholesterol efflux protein, ABCA1, supports anti-cancer functions of myeloid immune cells.

status: Public on Nov 16 2025

city: Urbana

first: Erik

middle: Russell

last: Nelson

state: IL

address: 407 S. Goodwin Ave

department: Molecular and Integrative Physiology

institute: University of Illinois at Urbana-Champaign

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Breast and other solid tumors respond poorly to immune therapy. Myeloid cells (MCs) such as macrophages contribute to resistance. Established clinical evidence links cholesterol to cancer outcomes, with MC function being regulated by cholesterol metabolism. We screened MC-expressed regulators of cholesterol homeostasis linked to survival and identified the cholesterol efflux protein ABCA1. ABCA1 activity increases anti-cancer functions of macrophages: enhancing tumor infiltration, decreasing angiogenic potential, reducing efferocytosis, and improving support of CD8+ T cell activity. Mechanistically, different AKT isoforms are involved, through both PI3K-dependent and independent mechanisms. Highlighting the clinical relevance of our findings are correlations between ABCA1 in macrophages and angiogenic potential, VEGFA, and CD8 T cell abundance and activity. The culmination of these activities was demonstrated through increased tumor growth and metastasis in mice lacking MC-expressed ABCA1. Tumors grown in these mice were also more resistant to immune therapy. Therefore, modulating ABCA1 activity within MCs may represent a novel approach to immune therapy.

overall_design: Bone marrow derived macrphages were transfected with either a control siRNA or siRNA against ABCA1. 48hrs post transfection cells were dissociated using Accutase (Thermo #A1110501)

first: Shruti

last: Bendre

first: Rajendra

last: K C

first: Kevin

last: VanBortle


title: Transcriptomic analysis of pathway activation after decitabine treatment in EBV+ vs EBV- gastric cancer [RNA-Seq]

city: Boston

first: Yin

last: Wang

address: 181 Longwood Ave

institute: Brigham and Women's Hospital

summary: Epstein-Barr virus associated gastric cancer (EBVaGC) accounts for ∼9–10% of gastric cancers worldwide and is defined by a distinctive molecular profile, including extreme hypermethylation of the DNA. Targeting this aberrant methylation may be a potential therapeutic strategy. EBV ⁺ gastric cancer cell lines (YCCEL1, SNU719) and EBV ⁻ lines (AGS, SNU16, HGC27) were treated with a DNA methyl-transferase inhibitor (DNMT), decitabine (DCB), for three days followed by RNA sequencing to identify EBV-specific responses.

overall_design: EBV+ cell lines (YCCEL1 and SNU719) and EBV- cell lines (AGS, SNU16 and HGC27) were treated with DCB for 72 hours or DMSO as the control.

first: Benjamin

last: Gewurz


title: Phospholipid Scramblase 1 (PLSCR1) Regulates Interferon-Lambda Receptor 1 (IFN-λR1) and IFN-λ Signaling in Influenza A Virus (IAV) Infection

city: PROVIDENCE

first: Alina

middle: Xiaoyu

last: Yang

state: RI

address: 171 Meeting Street, Room 624

department: Molecular Microbiology and Immunology

institute: Brown University

summary: Phospholipid scramblase 1 (PLSCR1) is an antiviral interferon-stimulated gene (ISG) that has several known anti-influenza functions. However, the mechanisms in relation to its expression compartment and enzymatic activity have not been completely explored. Moreover, only limited animal models have been studied to delineate its role at the tissue level in influenza infections. Our results showed that Plscr1 expression was highly induced by influenza A virus (IAV) infection in vivo and in airway epithelial cells treated with IFN-λ. We found that infected Plscr1-/- mice exhibited exacerbated body weight loss, decreased survival rates, heightened viral replication, and increased lung damage. Interestingly, transcriptomic analyses demonstrated that Plscr1 was required for the expression of type 3 interferon receptor (Ifn-λr1) and a large subset of ISGs upon IAV infection. The impaired expression of Ifn-λr1 and downstream ISGs may be responsible for delayed viral clearance and worse lung inflammation in Plscr1-/- mice. PLSCR1 acts as a transcriptional activator of IFN-λR1 by directly binding to its promotor after IAV infection. In addition, PLSCR1 interacted with IFN-λR1 on the cell surface of pulmonary epithelial cells following IAV infection, suggesting it also modulated IFN-λ signaling via protein-protein interactions. The lipid scramblase activity of PLSCR1 was found to be dispensable for its anti-flu activity. Finally, single-cell RNA sequencing data indicated that Plscr1 expression was significantly upregulated in ciliated airway epithelial cells in mice following IAV infection. Consistently, Plscr1floxStopFoxj1-Cre+ mice with ciliated epithelial cell-specific Plscr1 overexpression showed reduced susceptibility to IAV infection, less inflammation and enhanced Ifn-λr1 expression, suggesting that Plscr1 primarily regulates type 3 IFN signaling as a cell intrinsic defense factor against IAV in ciliated airway epithelial cells. Our research will elucidate virus-host interactions and pave the way for the development of novel anti-influenza drugs that target human elements like PLSCR1, thereby mitigating the emergence of drug-resistant IAV strains.

overall_design: To determine if IFN signaling pathways are regulated by Plscr1 during IAV infection, RNA sequencing of a total of 20,700 genes was performed using pooled samples from Wt and Plscr1-/- mouse lungs infected with 300 pfu of IAV. A comprehensive examination of interferons and their receptors revealed that Ifn-λr1 expression was significantly lower in Plscr1-/- mice, despite high expression of Ifn-λ at both 3 and 7 dpi

middle: X

first: Lisa

last: Ramos-Rodriguez

first: Parand

last: Sorkhdini

first: Dongqin

first: Carmelissa

last: Norbrun

first: Sonoor

last: Majid

first: Sanghyun

last: Lee

first: Yong

last: Zhang

first: Michael

last: Holtzman

first: David

middle: F

last: Boyd

first: Yang

last: Zhou


title: SOX2 phosphorylation during mitosis limits genomic damage [ChIP-Seq 2]

city: Edinburgh

first: Charles

middle: A C

last: Williams

state: Midlothian

address: 5 Little France Dr, Bioquarter, Scottish Ctr For Regenerative Medicine, Scottish Ctr For Regenerative Medicine

department: Institute for Regeneration and Repair

country: United Kingdom

institute: University of Edinburgh

summary: Pioneer transcription factors (TFs) such as SOX2 play critical roles in control of stem cell identity and are dysregulated in many human cancers. For example, SOX2 regulates the self-renewal of neural stem cells (NSCs), and is typically highly expressed in glioblastoma stem cells, where it is known to induce an immature NSC-like state. Here, we explored the regulation of SOX2 by phosphorylation during cell division and identify an unexpected role for mitotic SOX2 as an inducer of genomic damage. We find that SOX2 adopts a distinct phosphorylated state in NSCs during mitosis, regulated by the mitotic kinase CDK1. Mapping of SOX2 genome occupancy and chromatin accessibility shows that a subset of SOX2 remains bound to the interphase targets, consistent with mitotic bookmarking, but phosphorylated SOX2 is redistributed to constitutive heterochromatin, including telomeres. Ablation of SOX2 phosphorylation leads to promiscuous chromatin binding across the genome and triggers prolonged mitotic transit times and increased susceptibility to DNA damage. These findings show that excessive levels of SOX2 protein in mitosis trigger inappropriate opening of chromatin and downstream chromosomal damage. Elevated levels of SOX2 in cancers may therefore have dual oncogenic roles: imposing stemness during interphase via their well-known transcriptional roles, but simultaneously inducing genomic damage during mitosis due to their unconstrained pioneer factor activity.

overall_design: Genome binding/occupancy profiling and transcriptional profiling were performed using ChIP-seq, RNA-seq and ATACseq). This dataset refers to the ChIPseq analysis of SOX2 phsophsorylated at S253 and total SOX2 during mitosis and interphase. Experimental groups include control mNSCs or those arrested in mitosis .

first: Dounia

last: Djeghloul

first: Nicolas

last: Veland

first: Alhafidz

last: Hamdan

first: Maria

last: Kalantzaki

first: Erika

last: Lo

first: Robert

last: Illingworth

first: Alex

last: Von Kriegsheim

first: Amanda

middle: G

last: Fisher

first: Abdenour

last: Soufi

first: Steven

last: Pollard


title: Genome-wide screening of FOXM1 expression  in multiple myeloma cells

city: Shimotsuke

first: Sae

last: Matsuoka

state: Tochigi

department: Center for Molecular Medicine

institute: Jichi  Medical  University

summary: We analyzed expression of FOXM1 gene  in multiple myeloma using ChIP-seq.

overall_design: We isolated fixed chromatin from KMM1 cells and performed immunoprecipitation with anti-FOXM1 antibody, followed by deep sequencing.

first: Jiro

last: Kikuchi


title: Disordered DNA methylation leads to targetable transcriptional plasticity in ATRT [WGBS cell line]

last: Koldobskiy

state: MD

address: 1800 Orleans St., Bloomberg 11379

department: Oncology

institute: Johns Hopkins University

type: Methylation profiling by high throughput sequencing

summary: Atypical teratoid rhabdoid tumor (ATRT) is a highly aggressive but genetically simple pediatric central nervous system tumor, defined by biallelic inactivation of the chromatin regulator SMARCB1 with remarkably few other cooperating mutations. Despite its genetic homogeneity, ATRT exhibits profound clinical and epigenetic heterogeneity, with three major subgroups (ATRT-TYR, ATRT-MYC, and ATRT-SHH) defined by DNA methylation and transcriptional signatures. Beyond these subgroup-defining features, we aimed to investigate epigenetic variability within tumors by applying whole-genome bisulfite sequencing and probabilistic modeling to quantify stochastic DNA methylation in primary ATRT samples encompassing all three subgroups. We show that ATRT exhibits a destabilized and increasingly stochastic methylome. While ATRT global methylation patterns diverge according to subgroup, some methylation perturbations, such as hypermethylation and increased methylation entropy over bivalent promoters, are consistent across subgroups. We find that methylation stochasticity alterations map onto potential drivers of ATRT, such as LIN28a, the HOXD cluster for ATRT-MYC, and OTX2 for ATRT-TYR, and identify actionable targets, such as hypermethylation of the tumor suppressor CDKN2a across all subgroups. We investigate the sensitivity of the aberrant DNA methylation landscape of ATRT to pharmacologic DNA methyltransferase inhibition and histone deacetylase inhibition (HDACi). We show that decitabine leads to profound demethylation of patient-derived ATRT cell lines, including reversal of hypermethylation at bivalent promoters and the CDKN2a locus. The addition of HDACi leads to dramatic gene expression changes, including upregulation of innate immune signaling pathways, such as STING/interferon signaling, genes under the regulation of bivalent promoters, and reactivation of the tumor suppressor CDKN2A. The combination of DNMTi and HDACi synergistically reduces cell viability. Taken together, we show that ATRT has a highly stochastic methylome sensitive to epigenetic manipulation.

overall_design: WGBS on 22 primary patient samples of ATRT and 4 normal fetal brain controls. Additional files include WGBS output from 4 ATRT cell lines, CHLA02, CHLA05, CHLA06, and CHLA266, treated with 5 days of 100 nM decitabine +/- RG2833.

first: Michael A.

first: Ashley R.

last: Tetens


title: Disordered DNA methylation leads to targetable transcriptional plasticity in ATRT [WGBS]

first: Michael

overall_design:
WGBS on 22 primary patient samples of ATRT and 4 normal fetal brain controls. Additional files include WGBS output from 4 ATRT cell lines, CHLA02, CHLA05, CHLA06, and CHLA266, treated with 5 days of 100 nM decitabine +/- RG2833.

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


title: Investigating milk-derived extracellular vesicles as mediators of maternal stress and environmental intervention [miRNA-seqMilk]

city: New York

first: Jordan

last: Marrocco

state: NY

address: 3 Times Sq, Office 752

department: Biology

institute: Touro University

type: Non-coding RNA profiling by high throughput sequencing

summary: It was our goal to identify changes in milk-derived extracellular vesicle miRNA cargos after maternal immune activation in lactating Sprague-Dawley rat mothers.

overall_design: Sprague-Dawley rats mothers were housed in either standard laboratory cages or in environmental enrichment. Rat dams were injected with either lipolysaccharide (LPS) to induce an immune response or a saline vehicle on postnatal day (P)10. Maternal milk samples were collected 2 hours later on P10 and again 24-hours post-injection on P11. Milk-derived extracellular vesicles were isolated from the milk samples and miRNA cargo evaluated.

first: Julia

last: Martz

first: Baila

last: Hammer

first: Tristen

last: Langen

first: Benjamin

last: Berkowitz

first: Benzion

first: Jasmyn

last: Storm

first: Jueqin

last: Lu

first: Deepali

last: Lehri

first: Sanoji

last: Wijenayake

last: Kentner


title: Investigating milk-derived extracellular vesicles as mediators of maternal stress and environmental intervention [miRNA-seqHippo]

summary: It was our goal to identify changes in miRNA expression in the neonatal offspring hippocampus (on postnatal [P]10), after maternal immune activation in lactating Sprague-Dawley rat mothers.

overall_design: Sprague-Dawley rats mothers were housed in either standard laboratory cages or in environmental enrichment. Rat dams were injected with either lipolysaccharide (LPS) to induce an immune response or a saline vehicle on postnatal day (P)10. Offspring hippocampus was collected on postnatal day (P)12 and miRNA cargo evaluated.

first: Amanda


title: Liver-specific LXR inverse agonist restores lipid homeostasis in animal models and humans

city: Lausanne

first: Xiaoxu

last: Li

address: Station 15 - CH

country: Switzerland

institute: EPFL

summary: Despite advances in lipid-lowering treatment, atherosclerotic cardiovascular disease (ASCVD) remains the leading cause of mortality, underscoring the need for treatments that address residual risk. Targeting both the synthesis and clearance of triglyceride (TG)-rich lipoproteins is a promising approach. Liver X receptor (LXR) inhibition suppresses de novo lipogenesis and enhances clearance of TG-rich lipoproteins, but its clinical utility remains unexplored. Here, we demonstrate the pivotal role of LXR in lipid metabolism and metabolic diseases. In dysmetabolic animal models, LXR inverse agonists reduced plasma TG and cholesterol and improved insulin sensitivity by suppressing LXR activity and intestinal lipid absorption. Given concerns that systemic LXR inhibition may impair reverse cholesterol transport, we developed TLC-2716—an orally-administered, gut- and liver-restricted LXR inverse agonist. In human liver organoids modeling steatohepatitis, TLC-2716 reduced lipid accumulation and downregulated inflammation and fibrotic gene expression. In a Phase 1 clinical trial (NCT05483998), 14-day treatment with TLC-2716 was safe and resulted in placebo-adjusted reductions up to 38.5% in plasma TG and 60.8% in postprandial remnant cholesterol. These results highlight the safety and therapeutic potential of TLC-2716 as a novel treatment for managing dyslipidemia and reducing residual ASCVD risk.

overall_design: The effect of TLC-2716 on lipid accumulation was also evaluated in an induced pluripotent stem cell (iPSC)-derived human liver organoid (HLO) model established from human donors with different genetic backgrounds, including those with a known risk variant for MASH (the glucokinase regulatory protein [GCKR] rs1260326:C>T variant). Mutations in GCKR (GCKRTT) have been associated with increased hepatic DNL and plasma lipids, as well as metabolic diseases including diabetes and MASH. Briefly, the wildtype and GCKR mutation carried HLOs were exposed to high concentrations of oleate for three days to induce a steatotic MASH phenotype (sHLOs) and then treated with TLC-2716 or vehicle.

first: Masaki

last: Kimura

first: Archana

last: Vijayakumar

first: Robert

last: Myers

first: Takanori

last: Takebe

first: G. Mani

last: Subramanian

first: Johan

last: Auwerx


title: Disordered DNA methylation leads to targetable transcriptional plasticity in ATRT [RNA-seq]

first: Michael

last: Koldobskiy

overall_design: RNA-Seq was performed on 4 ATRT cell lines, CHLA02, CHLA05, CHLA06, and CHLA266, after 5 day treatment with low dose decitabine (100 nM) plus one day of 2.5 uM RG2833 on the 5th day. Cells were harvested 72 hours after the final treatment dose.

first: Michael A.

first: Ashley R.

last: Tetens


title: Pathogenetic mechanisms of muscle-specific ribosomes in dilated cardiomyopathy (AC16 RNA-seq)

first: Xuebing

last: Wu

address: 630 West 168th Street, P&S 10-401

institute: Columbia University

summary: The heart employs a specialized ribosome in its muscle cells to translate genetic information into proteins, a fundamental adaptation with an elusive physiological role. Its significance is underscored by the discovery of neonatal patients suffering from often fatal heart failure caused by rare biallelic variants in RPL3L, a muscle-specific ribosomal protein that replaces the ubiquitous RPL3 in cardiac ribosomes. RPL3L-linked heart failure represents the only known human disease arising from mutations in tissue-specific ribosomes, yet the underlying pathogenetic mechanisms remain poorly understood despite an increasing number of reported cases. While the autosomal recessive inheritance pattern suggests a loss-of-function mechanism, Rpl3l-knockout mice display only mild phenotypes, attributed to up-regulation of the ubiquitous Rpl3. Interestingly, living human knockouts of RPL3L have been identified. Here, we report two new cases of RPL3L-linked severe neonatal heart failure and uncover an unusual pathogenetic mechanism through integrated analyses of population genetic data, patient cardiac tissue, and isogenic cells expressing RPL3L variants. Our findings demonstrate that patient hearts lack sufficient RPL3 compensation. Moreover, contrary to a simple loss-of-function mechanism often associated with autosomal recessive diseases, RPL3L-linked disease is driven by a combination of gain-of-toxicity and loss-of-function. Most patients harbor a recurrent toxic missense variant alongside a non-recurrent variant. The non-recurrent variant is often loss-of-function and enables partial compensation through RPL3, similar to Rpl3l-knockout mice. However, the recurrent missense variant exhibits increased affinity for the RPL3/RPL3L chaperone GRWD1 and 60S biogenesis factors, retains 28S rRNA in the nucleus, disrupts ribosome biogenesis, and induces severe cellular toxicity that extends beyond the loss of ribosomes. These findings elucidate the pathogenetic mechanisms underlying muscle-specific ribosome dysfunction in neonatal heart failure, providing critical insights for genetic screening and therapeutic development. Our findings also suggest that gain-of-toxicity mechanisms may be more widespread in autosomal recessive diseases, especially for those involving genes with paralogs.

overall_design: whole cell polyA RNA-seq and nuclear RNA-seq of human AC16 cells expressing RPL3L variants

last: Murphy


title: Transcriptional differences between freshly isolated and long-lived naïve P14 CD8 T cells prior to infection

city: Cincinnati

first: Yamato

last: Sajiki

state: OH

address: 3333 Burnet Avenue

institute: Cincinnati Children's Hospital Medical Center

summary: The heterogeneity of antigen-specific naïve CD8 T cells has not yet been fully elucidated. We found that long-lived naïve CD8 T cells, incubated in naïve B6 mice for more than 30 days, generate significantly higher numbers of effector and memory CD8 T cells after lymphocytic choriomeningitis virus (LCMV)-Armstrong infection compared to freshly isolated naive CD8 T cells. To examine differences between long-lived and freshly isolated naive CD8 T cells prior to infection, a bulk RNA-seq analysis was performed.

overall_design: RNA-seq profiles of P14 T cell receptor transgenic CD8 T cells incubated in naive B6 mice for 1.5 days (fresh) and 35 days (long-lived).

first: Koichi

last: Araki


title: Transcriptional differences between effector cells derived from freshly isolated and long-lived naïve P14 CD8 T cells on day 5 post-infection.

summary: The heterogeneity of antigen-specific naïve CD8 T cells has not yet been fully elucidated. We found that long-lived naïve CD8 T cells, incubated in naïve B6 mice for more than 30 days, generate significantly higher numbers of effector and memory CD8 T cells after lymphocytic choriomeningitis virus (LCMV)-Armstrong infection compared to freshly isolated naive CD8 T cells. To examine the mechanism of why long-lived naive CD8 T cells exhibit superior responses to infection, we compared transcriptional profiles between effector CD8 T cells derived from long-lived and freshly isolated naive CD8 T cells.

overall_design: RNA-seq profiles of effector P14 T cell receptor transgenic CD8 T cells derived from freshly isolated and long-lived P14 CD8 T cells on Day 5 post-LCMV-Armstrong infection.


title: RNAseq of conventional dendritic cells generated from mouse bone marrow cultures under different conditions

city: Parkville

first: Gordon

middle: K

last: Smyth

state: Vic

address: 1G Royal Pde

department: Bioinformatics

country: Australia

institute: Walter and Eliza Hall Institute of Medical Research

summary: Type 1 conventional dendritic cells (cDC1s) are infrequent immune cells with an essential role in orchestrating immune responses to malignancies or infections. Despite their significance in regulating adaptive immunity, the absence of efficient manufacturing techniques to produce adequate yields of cDC1s has hindered their application in therapeutic settings. Here, we find that Interleukin-4 (IL-4) enhances the yield of cDC1 cells produced from Flt3 ligand (Flt3L) cultures of hematopoietic progenitors from either mice or humans, while inhibiting the generation of plasmacytoid DCs. IL-4 acted predominantly on DC progenitors and its activity relied on the cell intrinsic expression of IL4-RA or STAT6. Both in vitro and in vivo, cDC1s that had been stimulated by IL-4 were effective in activating and promoting the expansion of cytotoxic CD8 T cells. Gene expression analysis pointed to a critical role for IL-4 in promoting cDC1 proliferation in vitro. Overall, our research reveals an unanticipated role for IL-4 in fostering the development and manufacture of bona-fide cDC1s, making it easier to employ them for further mechanistic studies, and opening the door to leveraging their potential use in human diseases.

overall_design: RNA-seq profiling of sorted cDC1 and cDC2s generated from mouse bone marrow cultures with different conditions:  Flt3L, Flt3L + IL-4, Flt3L + IL-4+ OP9, Flt3L + OP9, Flt3L + IL-4 + OP9DL-1, and Flt3L + OP9DL-1. Three biological replicates per group except the cDC2 Flt3L + IL-4 + OP9DL-1 group, which has two replicates. Samples were sequenced with an Illumina NextSeq 500 sequencing system, producing between 18-93 million single end 86 bp reads per sample.

first: Hannah

middle: D

last: Coughlan

first: Stephen

middle: L

last: Nutt

last: Chopin


title: Genetic diversity of Collaborative Cross mice reveals FFAR3 as a target for ILC2 anti-inflammatory reprogramming

city: Nashville

first: Mark

last: Rusznak

state: Tennessee

address: 1161 21st Ave S

department: Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine

institute: Vanderbilt University Medical Center

summary: Pulmonary group 2 innate lymphoid cells (ILC2s) are key drivers of Type 2 inflammation in diseases like asthma, yet the molecular mechanisms regulating their function are incompletely understood. Using the genetically diverse Collaborative Cross (CC) mouse panel, we mapped a quantitative trait locus (QTL) that governs ILC2 prevalence in the lung after aeroallergen exposure. This QTL creates a large population of ILC2s in the lung that are resistant to activation and have diminished Type 2 effector function. We identified free-fatty acid receptor 3 (FFAR3) as a gene responsible for this effect and demonstrated that FFAR3 signaling reprograms ILC2s to an anti-inflammatory state by promoting their survival, reducing Type 2 cytokine production, and enhancing IL-10 expression. This reprogramming is mediated by epidermal growth factor receptor (EGFR) upregulation. We showed that FFAR3's anti-inflammatory effect is conserved in human ILC2s, making it a potential therapeutic target for Type 2 inflammation.

overall_design: Naive group 2 innate lymphoid cells (ILC2s) were isolated from the unchallenged lungs of mice by flow cytometry. We obtained ILC2s from both female C57BL/6J mice and female CC030/GeniUnc (Collaborative Cross) mice. Experiment 1: ILC2s were sorted from unchallenged C57BL/6J and CC030/GeniUnc lungs directly into buffer for analysis, allowing for comparison of C57BL/6J and CC030/GeniUnc ILC2s in the naive state. Cells were sorted separately from 3 mice of each strain. Experiment 2: ILC2s were flow sorted from CC030/GeniUnc, and all ILC2s were cultured with IL-33, TSLP, and IL-2. ILC2s received either DMSO vehicle or AR420626 treatment (FFAR3 agonist, 10uM). ILC2s were isolated from 5 CC030/GeniUnc, and cells from each biological replicate were split between vehicle and treatment condition to allow for paired samples. Cells were harvested at Day 2 and Day 6 of culture for analysis.

first: Shinji

last: Toki

first: Yajing

last: Hao

first: Marc

last: Todd

first: Liddy

last: Malone

first: Julia

last: Goodhead

first: Catherine

last: DuPuy

first: Dominique

last: Babin

first: Christian

last: Warren

first: Masako

last: Abney

first: Matthew

middle: T

last: Stier

first: Christopher

last: Thomas

first: Jing

last: Li

first: Justin

last: Jacobse

last: Petrovic

first: Jacqueline-Yvonne

last: Cephus

first: Shelby

middle: N

last: Kuehnle

first: Allison

last: Norlander

first: Fang

last: Yan

first: Jeremy

last: Goettel

first: Darla

last: Miller

first: Rachel

last: Lynch

first: Daniel

middle: P

last: Cook

first: Dawn

last: Newcomb

first: Fei

last: Zou

first: R Stokes

last: Peebles Jr


title: STING agonism overcomes STAT3-mediated immunosuppression and adaptive resistance to PARP inhibition in ovarian cancer

first: Jean

last: Zhao

state: MA

address: 450 Brookline Ave

institute: Dana Farber Cancer Institute

summary: Background: PARP inhibition (PARPi) has demonstrated potent therapeutic efficacy in patients with BRCA-mutant ovarian cancer. However, acquired resistance to PARPi remains a major challenge in the clinic.   Methods: PARPi-resistant ovarian cancer mouse models were generated by long-term treatment of olaparib in syngeneic Brca1-deficient ovarian tumors. STAT3-mediated immunosuppression was investigated in vitro by co-culture experiments and in vivo by analysis of immune cells in the TME of human and mouse PARPi-resistant tumors. Whole genome transcriptome analysis was performed to assess the anti-tumor immunomodulatory effect of STING (stimulator of interferon genes) agonists on myeloid cells in the TME of PARPi-resistant ovarian tumors. A STING agonist was used to overcome STAT3-mediated immunosuppression and acquired PARPi resistance in syngeneic and PDX models of ovarian cancer.  Results: In this study, we uncover an adaptive resistance mechanism to PARP inhibition mediated by tumor associated macrophages (TAMs) in the tumor microenvironment (TME). Markedly increased populations of pro-tumor macrophages are found in BRCA-deficient ovarian tumors that rendered resistance to PARPi in both murine models and patients. Mechanistically, PARP inhibition elevates the STAT3 signaling pathway in tumor cells, which in turn promotes pro-tumor polarization of TAMs. STAT3 ablation in tumor cells mitigates polarization of pro-tumor macrophages and increases tumor-infiltrating T-cells upon PARP inhibition. These findings are corroborated in patient-derived, PARPi-resistant BRCA1-mutant ovarian tumors. Importantly, STING agonists reshape the immunosuppressive TME by reprogramming myeloid cells and overcome the TME-dependent adaptive resistance to PARPi in ovarian cancer. This effect is further enhanced by addition of PD-1 blockade.  Conclusions: We elucidate an adaptive immunosuppression mechanism rendering resistance to PARPi in BRCA1-mutant ovarian tumors. This is mediated by enrichment of pro-tumor TAMs propelled by PARPi-induced STAT3 activation in tumor cells. We also provide a new strategy to reshape the immunosuppressive TME with STING agonist and overcome PARPi resistance in ovarian cancer.

overall_design: PBM and PBM-R tumors: transcriptome analysis were performed on bulk tumors from PBM and PBM-R tumor-bearing mice. PBM tumors were generated by orthotopic injection of PBM tumor cells into FVB/NJ mice. PBM-R are refactory tumors generated in PBM tumor-bearing mice after long-term treatment with olaparib (50 mg/kg/day).   Myeloid cells: about 1 x10^6 PBM-R tumor cells were intraperitoneally injected to FVB/NJ mice. About 2-3 weeks after injection, mice were grouped and treated with control, olaparib, MSA-2 and MSA-2 in combination with MSA-2 for 24 hours. After treatment, myeloid cells (CD45+CD11b+) were isolated from the ascites of each mouse (n=3 per group) by EasySep Mouse CD11b positive Selection Kit II (STEMCELL Technology, #18970) and subjected for transcriptome analysis.

first: Liya

last: Ding

first: Qiwei

last: Wang

first: Shaozhen

last: Xie

first: Ziying

last: Lin


title: SOX2 phosphorylation during mitosis limits genomic damage [RNA-Seq]

first: Charles

last: Williams

overall_design: Genome binding/occupancy profiling/transcriptional profiling by high throughput sequencing

first: Dounia

last: Djeghloul

first: Nicolas

last: Veland

first: Alhafidz

last: Hamdan

first: Maria

last: Kalantzaki

first: Erika

last: Lo

first: Robert

last: Illingworth

first: Alex

last: Von Kriegsheim

first: Amanda

last: Fisher

first: Abdenour

last: Soufi

first: Steven

last: Pollard


title: Antioxidant program of islet-resident macrophages preserves β cell physiology

city: Paris

last: Venteclef

address: 156 rue de Vaugirard

country: France

institute: Institut Necker Enfants Malades, INSERM U1151

summary: Macrophages residing within mouse pancreatic islets display an activated phenotype at steady state, yet the molecular signatures underlying their polarization remain unclear. To investigate islet macrophage heterogeneity under physiological conditions, we isolated islet-resident immune cells from chow diet-fed adult male mice and performed single-cell RNA sequencing.

overall_design: Islets were isolated and pooled from 6–8 mice, dissociated into single cells, and CD45+ immune cells were FACS-sorted. These cells were then subjected to single-cell RNA sequencing using the 10X Genomics platform.

first: A

last: Grosjean

first: M

last: Diedisheim

first: E

last: Dalmas

first: LG

last: Baudrin

first: S

last: Baulande


title: SOX2 phosphorylation during mitosis limits genomic damage [ChIP-Seq]

last: Williams

overall_design: Genome binding/occupancy profiling and transcriptional profiling were performed using ChIP-seq, RNA-seq and ATACseq). This dataset refers to the ChIPseq analysis of SOX2 during mitosis. Mouse Neural Stem Cells (mNSCs) engineered with a PiggyBac transposon-based doxycycline-inducible SOX2 expression system (Martella et al., ACS Synthetic Biology, 2017) were used. Sample Preparation and Treatments: SOX2 Induction: mNSCs were treated with 1 μg/ml doxycycline for 72 hours to induce SOX2 expression. Mitotic Arrest: Cells were treated with 50 ng/ml Nocodazole to induce mitotic arrest, with or without concurrent SOX2 induction. Experimental groups include mNSCs with induced SOX2 expression, and cells arrested in mitosis with/without SOX2 induction.


title: The role of EHMT2 in Uveal melanoma progression [EHMT2_Inhibitors]

city: Birmingham

first: Narendra

last: Wajapeyee

state: AL

address: 720 20th Street South, Kaul 540A

department: Department of Biochemistry and Molecular Genetics

institute: University of Alabama at Birmingham

summary: EHMT2/G9a expression is elevated in many cancer types including uveal melanoma. Here in the present study, we have identified pharmacological inhibition of EHMT2/G9a in MP41 cells and Mel92.1 cells promote the sensitivity to NK cells in vitro. By keeping this in mind, we have performed RNA-seq of inhibition of EHMT2/G9a validated cells to identify the differentially regulated genes from various pathways that are responsible for promoting resistance to NK cell mediated cytotoxicity.

overall_design: MP-41 and Mel 92.1 cells were treated with EHMT2/G9a inhibitor UNC0642 & A366, samples were harvested and were used to prepare the total RNA, followed by RiboRNA depletion with random priming and RNASeq Illumina NextSeq 500 platform, with three biological replicates for each sample.


title: HTGTS-TCR-seq for profiling of mouse and human T-cell receptor α and β gene rearrangements and diversity [HTGTS_TCR-seq]

status: Public on Nov 13 2025

city: Shanghai

first: Haiqiang

last: Dai

address: 320 Yue Yang Road

country: China

institute: Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell  Science

summary: Developing αβ T lymphocytes generate T-cell receptor (TCR) diversity through V(D)J recombination, which assembles Tcra and Tcrb genes from germline variable (V), diversity (D), and joining (J) segments. Approaches to characterize TCR rearrangements and diversity are critical for studying T-cell development and immune function. Several existing methods, such as multiplex PCR and 5′RACE, have advanced the field; however, each carries inherent technical limitations. Here, we present High-Throughput Genome-Wide Translocation Sequencing–based TCR sequencing (HTGTS-TCR-seq), a complementary and cost-effective strategy for quantitative profiling of Tcra and Tcrb gene rearrangements. HTGTS-TCR-seq employs a limited set of 3-5 J or V region primers to enrich for V(D)J recombination products, allowing detection of both productive and nonproductive rearrangements. Application to wild-type murine thymocytes at defined developmental stages, as well as young and aged T cells, reveals stage-specific V and J usage and age-associated repertoire alterations. Analysis of Wapl-knockout preselection double-positive thymocytes uncovers a cell division-independent role for the cohesin-unloading factor WAPL in Tcra rearrangement. Moreover, analysis of human peripheral T cells demonstrates conserved complementarity-determining region 3 (CDR3) features and subset-specific Vβ usage across species. Collectively, HTGTS-TCR-seq provides an efficient and accessible approach for quantifying TCR rearrangements and diversity across development, aging, and immune-related conditions.

overall_design: We employed HTGTS-TCR-seq to asess rearrangement events and TCR repertoire changes across developmental stages and T cell subsets at DNA level. DN3 thymocytes, preselection DP thymocytes from mice and CD4+ and CD8+ T cells from human PBMCs were FACS sorted. Total thymocytes and splenic CD4+ and CD8+ T cells from young and aged mice were collected. Genome DNA were extracted, sonicated and utilized to conduct HTGTS-TCR-seq.

first: Rui

last: Luo

first: Yawei

last: Song

first: Meichen

last: Wang

first: Longhao

last: Zou

first: Fangtai

last: Jiao

first: Tiange

last: Yang

first: Guangchuan

first: Wei

last: Wu

first: Hai-Qiang


title: HTGTS-TCR-seq for profiling of mouse and human T-cell receptor α and β gene rearrangements and diversity [RNA-seq]

overall_design: We employed RNA-seq to asess the deletion efficiency of Wapl in preselection DP thymocytes and to explore whether deletion of Wapl led to dysregulation of certain development-related genes.


title: Microbiota-derived bile acid ameliorates lung inflammation through metabolic rewiring of alveolar macrophages

city: Warsaw

first: Bartosz

last: Wojtas

address: Pasteura 3

country: Poland

institute: Nencki Institute of Experimental Biology

summary: Using mouse models of airway inflammation, we identified a causal link between the microbiota-dependent bile acid isolithocholic acid and protection against lung immunopathology. We further found that this protection involves Sphingosine-1-phosphate receptor 2–dependent metabolic rewiring of alveolar macrophages as a key mechanism. Collectively, our findings demonstrate that isolithocholic acid, a microbiota-derived bile acid, can ameliorate lung inflammation through previously unrecognized pathways, highlighting its therapeutic potential—and that of its derivatives—in lung disease.

overall_design: Acute lung injury was induced in six wild-type C57BL/6J mice by intranasal instillation of LPS. Following induction, half of the mice were treated with isolithocholic acid for three days, while the other half received vehicle. A control group of mice that did not receive LPS underwent the same treatment regimen. After four days, lungs were collected, dissociated into single-cell suspensions, and analyzed using scRNA-seq.


title: Pathogenetic mechanism of muscle-specific ribosome in heart failure

first: Xuebing

overall_design: RNA-seq from ventricular tissue of explanted human heart

first: Michael

last: Murphy

first: Mythily

last: Ganapathi

first: Teresa

last: Lee

first: Wendy

last: Chung


title: HTGTS-TCR-seq for profiling of mouse and human T-cell receptor α and β gene rearrangements and diversity [RACE]

overall_design: We employed 5'RACE to validate HTGTS-TCR-seq assay by extracting DNA and RNA from preselection DP and total thymocytes.


title: Transcription factor ZNF263 primes human embryonic stem cells for pluripotency dissolution and early differentiation [RNA-Seq 2]

city: shanghai

first: Liu

last: Xin

address: 320 Yueyang Road

institute: Shanghai Institute of Biochemistry and Cell Biology, CAS

summary: Conventional human embryonic stem cells (hESCs) are capable of self-renewal and simultaneously poised for differentiation, and thus have been considered to resemble a primed pluripotent state. But the underlying regulatory mechanisms remain largely underexplored. Especially, little was known about the pivotal transcription factors (TFs) driving hESCs through the naïve-to-primed transition to prepare for differentiation. Here, we conduct a large-scale in-silico screening and identify ZNF263 as a new key TF in primed hESCs, as evidenced by its extensive binding capacity at the active and poised genes. Genetic and functional assays reveal that ZNF263 directly initiates the incipient expression of early differentiation genes and concurrently dampens the core pluripotency circuitry in hESCs, greatly tilting the balance from pluripotency maintenance to lineage priming. Importantly, ZNF263 deficiency in hESCs further led to clear defects in pluripotency dissolution and lineage commitment, suggesting it’s indispensably required for the whole process. Together, our results demonstrate a pivotal role of ZNF263 in coordinating the establishment of primed pluripotency state in hESCs and facilitating the differentiation into primary germ layer lineages, which opens a new gate for understanding the central regulatory network governing human early development.

overall_design: To investigate the role of ZNF263 in the process of pluripotency regulation, we conducted ZNF263 overexpression and cofactor(ZIC2) overexpression experiments to demonstrate corresponding molecular mechanism in hESCs, and then performed gene expression profiling using RNA-seq.

first: Zhen

last: Shao


title: Uncovering pre-sensitizing agents to FLT3 inhibitors in acute myeloid leukemia with ReSisTrace lineage tracing

city: Helsinki

first: Li

last: Shen

address: Haartmaninkatu 8

country: Finland

institute: University of Helsinki

summary: FLT3 inhibitors have significantly advanced the treatment of aggressive FLT3-mutated acute myeloid leukemia (AML), yet the development of resistance remains a major therapeutic challenge. Using the single-cell lineage-tracing method ReSisTrace, we identified transcriptional signatures distinguishing pre-resistant from pre-sensitive AML cells exposed to midostaurin or quizartinib. Targeting the pre-resistance gene GSPT1 with a CC-90009 degrader synergized with FLT3 inhibitors in AML cell lines, patient samples, and a PDX model, significantly improving survival. W further identified small molecules, including vistusertib (mTOR inhibitor), linsitinib (IGF1R/INSR inhibitor), and meisoindigo (IGF1R/Src inhibitor), capable of reversing pre-resistance transcriptional programs and shifting cells toward a FLT3 inhibitor-sensitive state. In summary, ReSisTrace unveils pre-existing transcriptional features of treatment vulnerability in hematological cancers, and elucidates novel strategies for enhancing FLT3 inhibitor treatment efficacy in FLT3-ITD-positive AML.

overall_design: We applied ReSisTrace to reveal cell states that are primed to FLT3 inhibitor resistance in an FLT3-ITD-mutated MOLM-13 AML cell line. Uniquely labeled, synchronized cells were allowed to divide once, after which half of the cells were analyzed by scRNA-seq (pre-treatment samples), while the other half underwent DMSO (control), midostaurin, or quizartinib treatment. The lineage labels of cells that survived the treatment were determined by scRNA-sequencing (post-treatment sample) and were used to identify pre-resistant cells in the pre-treatment sample.

first: Johanna

last: Eriksson

first: Shuyu

last: Zheng

first: Jing

last: Tang


title: Perimenopausal State Oestradiol to Progesterone Imbalance Drives Alzheimer’s Risk via ERRα Dysregulation and Energy Dyshomeostasis [EnrichedNeuron_RNASeq]

city: Sha Tin

first: Jacquelyne

last: Sun

state: New Territories

address: Room 612A, Mong Man Wai Building,

department: School of Life Sciences

country: Hong Kong

institute: The Chinese University of Hong Kong

summary: Sex-biased differences in Alzheimer’s disease (AD) are well documented, but the mechanisms underlying increased vulnerability in postmenopausal women remain unclear. This study aimed to model the effects of perimenopausal hormonal fluctuations on AD pathophysiology. Using a VCD-induced accelerated ovarian failure model in young female C57BL/6J and 3xTg mice, we simulated a perimenopausal state with hormonal changes characterized by elevated oestradiol levels and reduced progesterone levels. Supporting human brain transcriptomic and metabolomic data from the ROSMAP study revealed that impaired oestrogen-related receptor alpha (ERRα) function was a key driver of female sex-biased vulnerability. In female mice, progesterone-guided oestrogen receptor signalling maintained ERRα activity by regulating neuronal cholesterol homeostasis and the TCA cycle. Hormonal imbalances disrupted this mechanism, triggering an aspartate-driven “minicycle,” which increased glutamate release, neuronal excitability, ATP depletion, and energy crisis susceptibility. This study demonstrates how perimenopausal hormonal imbalances exacerbate AD risk via ERRα dysfunction, linking neuronal cholesterol and energy homeostasis to disease vulnerability.

overall_design: To investigate the overall effects of Esrra knockdown in neurons, stereotaxic injections was performed and neuronal cells from the mouse brains were enriched using Miltenyi Biotech's enrichment protocol for neurons before the enriched neuronal fraction was sent for transcriptomic profiling.

first: Kim Hei-Man

last: Chow


title: Transcriptional profiling of Hutchinson-Gilford Progeria patients identifies primary target pathways of the disease-causing progerin protein

city: Bethesda

first: Sandra

last: Vidak

address: 9000 Rockville Pike

institute: NIH

summary: Hutchinson Gilford Progeria Syndrome (HGPS) is an ultra-rare pediatric premature aging disorder. The disease is caused by a point mutation in the LMNA gene leading to the production of the dominant-negative progerin isoform of the nuclear envelope protein lamin A. Disease severity and progression amongst the population of ~140 known patients is variable. Numerous cellular pathways have been implicated in the disease mechanism, including mTOR signaling, stem cell differentiation and the oxidative stress response. Most of mechanistic insights into the disease have come from studies using cellular or mouse models of HGPS. To probe the clinical relevance of previously implicated cellular pathways and to address the extent of gene expression heterogeneity between patients, we have performed transcriptomic analysis of a comprehensive set of HGPS patients. We find misexpression of several of the major HGPS-associated cellular pathways across the patient population, particularly of multiple signaling pathways as well as the Unfolded Protein Response (UPR) and mesodermal cell fate specification. Variability amongst individual patients was limited with misregulation of the major pathways observed in most patients. Comparing the transcriptome of patients with an inducible HGPS cell model, we distinguished immediate-early cellular response pathways from secondary adaptive pathways and identify mTOR, the UPR, UV response, apoptosis and TNFa signaling via NFkB as primary targets of the disease-causing progerin protein.

overall_design: RNA-seq profiling of primary human HGPS fibroblasts and primary human wild type control fibroblats, as well as hTERT-immortalized GFP-progerin doxycycline inducible dermal fibroblast cell line where progerin was expressed for 0 or 6 days.

first: Sohyoung

last: Kim

first: Tom

last: Misteli


title: SOX2 phosphorylation during mitosis limits genomic damage [ATAC-Seq]

first: Charles

last: Williams

address: 5 Little France Dr, Bioquarter, Scottish Ctr For Regenerative Medicine, Scottish Ctr For Regenerative Medicine

institute: University of Edinburgh

summary: Pioneer transcription factors (TFs) such as SOX2 play critical roles in control of stem cell identity and are dysregulated in many human cancers. For example, SOX2 regulates the self-renewal of neural stem cells (NSCs), and is typically highly expressed in glioblastoma stem cells, where it is known to induce an immature NSC-like state. Here, we explored the regulation of SOX2 by phosphorylation during cell division and identify an unexpected role for mitotic SOX2 as an inducer of genomic damage. We find that SOX2 adopts a distinct phosphorylated state in NSCs during mitosis, regulated by the mitotic kinase CDK1. Mapping of SOX2 genome occupancy and chromatin accessibility shows that a subset of SOX2 remains bound to the interphase targets, consistent with mitotic bookmarking, but phosphorylated SOX2 is redistributed to constitutive heterochromatin, including telomeres. Ablation of SOX2 phosphorylation leads to promiscuous chromatin binding across the genome and triggers prolonged mitotic transit times and increased susceptibility to DNA damage. These findings show that excessive levels of SOX2 protein in mitosis trigger inappropriate opening of chromatin and downstream chromosomal damage. Elevated levels of SOX2 in cancers may therefore have dual oncogenic roles: imposing stemness during interphase via their well-known transcriptional roles, but simultaneously inducing genomic damage during mitosis due to their unconstrained pioneer factor activity.

first: Dounia

last: Djeghloul

first: Nicolas

last: Veland

first: Alhafidz

last: Hamdan

first: Maria

last: Kalantzaki

first: Erika

last: Lo

first: Robert

last: Illingworth

first: Alex

last: Von Kriegsheim

first: Amanda

last: Fisher

first: Abdenour

last: Soufi

first: Steven

last: Pollard


title: Genome-wide profile of Mpp8- and Tasor-DNA binding in mouse embryonic stem cells (mESCs) [CUT&Tag]

city: Nanjing

first: Bowen

last: Wang

address: 12 Xue-fu Road, Pukou District

institute: Nanjing University

summary: Purpose: To determine the genome-wide binding sites of Mpp8 and Tasor in mESCs

overall_design: CUT&Tag of Mpp8 and Tasor in mESCs


title: Widespread low-affinity motifs enhance chromatin accessibility and regulatory potential in mESCs [ATAC-seq]

city: Kansas City

first: Melanie

last: Weilert

state: MO

institute: Stowers Institute for Medical Research

summary: Low-affinity transcription factor motifs are an important element of the cis-regulatory code, yet they cannot be mapped by local sequence scanning without knowledge of the sequence context in which they are functional. Here we leverage sequence-to-profile models of chromatin accessibility and binding in mouse embryonic stem cells to reliably map low-affinity motifs across the genome and deduce the mechanisms by which they become functional. We show that low-affinity motifs are used by pioneer transcription factors and that their effect is disproportionately enhanced by intra-nucleosomal cooperativity with stronger pioneer motifs. By exploring the underlying nucleosome-mediated mechanism with a kinetic model, we discover that pioneer cooperativity, and not low-affinity motifs per se, renders enhancers more responsive to changes in TF concentrations by increasing their regulatory potential. These results show that low-affinity motifs can be accurately mapped and suggest that their inherently strong cooperativity is an important property of developmental enhancers.

overall_design: Assay for transposase-accessible chromatin with sequencing (ATAC-seq) experiments were conducted in mouse embryonic stem cell lines (R1) across wildtype lines and two suites of CRISPR/Cas9 clones at an Akr1cl and Btbd11 locus.

first: Kaelan

last: Brennan

first: Khyati

last: Dalal

first: Sabrina

last: Krueger

first: Haining

last: Jiang

first: Rosa

last: Martinez-Corral

first: Julia

last: Zeitlinger


title: Inhibition of heme biosynthesis triggers cuproptosis in acute myeloid leukemia [RNA-Seq OCIAML3_ALAS1_KO_and_SA]

city: Melbourne

first: Emily

last: Gruber

state: VIC

address: 305 Grattan St

institute: Peter MacCallum Cancer Centre

summary: The ubiquitous metabolite heme has diverse enzymatic and signaling functions in most mammalian cells. Through integrated analyses of mouse models, human cell lines and primary patient samples, we identify de novo heme biosynthesis as a selective dependency in acute myeloid leukemia (AML). The dependency is underpinned by a propensity of AML cells, and especially leukemic stem cells (LSCs), to downregulate heme biosynthesis enzymes (HBEs) which promotes their self-renewal. Inhibition of HBEs causes collapse of mitochondrial Complex IV (CIV) and dysregulates the copper-chaperone system inducing cuproptosis, a form of programmed cell death brought about by the oligomerization of lipoylated proteins by copper. Moreover, we identify pathways that are synthetic lethal with heme biosynthesis, including glycolysis, which can be leveraged for combination strategies. Altogether, our work uncovers a heme rheostat that controls gene expression and drug sensitivity in AML and implicates HBE inhibition as a novel cuproptosis trigger.

overall_design: OCI-AML3 cells containing sgRNAs targeting ALAS1 (or scrambled) were treated with doxycyline to induce sgRNA expression and knockout ALAS1 for 7 days. Additionally, OCI-AML3 cells were treated with SA (500uM) or vehicle for 6 days and RNA isolated for RNAseq.

first: Lev

last: Kats


title: Phase 2 Trial (COTC-030) of adjuvant inhaled recombinant human IL-15 combined with amputation and adjuvant carboplatin chemotherapy in dogs with appendicular osteosarcoma

first: Anjali

last: Garg

address: Building 10, Room 1B53

department: Comparative Oncology Program,NCI

institute: National Institute of Health

summary: We have previously shown that inhaled IL-15 is associated with anti-tumor responses in dogs with pulmonary metastatic osteosarcoma (OSA) and melanoma. Here, we evaluated inhaled IL-15 combined with amputation and chemotherapy for dogs with localized OSA eligible for treatment with curative intent. In a multicenter COTC phase II trial for dogs with limb OSA, we hypothesized that 2 weeks of inhaled rhIL-15 after amputation and prior to chemotherapy would reduce the risk of metastatic failure at the completion of chemotherapy from a historical rate of 40% to 20%. Using a 2-sided alpha of 0.05, we planned an accrual of 40 dogs to test this hypothesis with 80% power. We also performed immune correlative assays and sequencing of blood immune cells (PBMCs) and primary amputation specimens. Unexpectedly, disease-free and survival outcomes for dogs in the intent-to-treat population were statistically inferior to a well-validated historical control cohort (P=0.03, P=0.003), so the trial was halted for futility. Cytotoxicity assays of PBMCs showed significant decreases after both surgery and chemotherapy with an overall decrease from the start to the end of therapy (-18.2±16.1%, P<0.001). Some dogs demonstrated positive fold change in PBMC cytotoxicity, and this correlated significantly with improved dog survival (P=0.004, r=0.62). Although plasma concentrations of key cytokines varied markedly with no significant differences between disease-free and metastatic-failure patients, inflammatory cytokines such as IL-6 showed absolute increases post-amputation and post-chemotherapy correlating with decreases in cytotoxicity. Tumor sequencing data reproduced immune signatures as observed in both human and canine cohorts, and PBMC sequencing data from 4 dogs showed upregulation of IL-15 receptor alpha on myeloid derived suppressor cells post-surgery. Inhaled IL-15 as part of multimodality approach with amputation and chemotherapy appears to be associated with worse outcomes in dogs with appendicular OSA. Correlative assays suggest significant immunological effects of amputation and chemotherapy on immune responses. These data have important implications for the design and testing of novel immunotherapy strategies involving multimodality approaches including surgery and chemotherapy.

overall_design: RNA for mRNAseq was isolated from canine frozen tumor tissue in RNAlater

middle: B

last: Rebhun

first: Sylvia

last: Cruz

first: Daniel

last: York

first: Christina

last: Mazcko

first: Aryana

last: Razmara

first: Sushant

last: Patkar

first: Sean

last: Judge

first: Cyrus

last: Sholevar

first: Ravi

last: Shah

first: Anthony

last: Zamora

first: Sami

last: Al-Nadaf

first: David

last: Vail

first: Timothy

last: Fan

first: Jenna

middle: H

last: Burton

first: Madison

last: Luker

first: Katherine

middle: A

last: Skorupski

first: Amandine

last: LeJeune

first: Kevin

last: Woolard

first: Susan

last: Stewart

first: Ellen

last: Sparger

first: Jacque

last: Young

first: Tamar

last: Davidyan

first: Erich

last: Huang

first: Jessica

last: Beck

first: William

last: Murphy

first: Michael

middle: S

last: Kent

last: Culp

first: Amy

last: LeBlanc

last: Canter


title: Pan-modification profiling facilitates a cross-evolutionary dissection of the thermoregulated ribosomal epitranscriptome - extradata2

city: Rehovot

first: Miguel

middle: A.

last: Garcia-Campos

state: Center District

address: Herzl 234, Meyer Bldg., Room 1410

department: Molecular Genetics

country: Israel

institute: Weizmann Insitute of Science

summary: Over 170 modifications are deposited on RNA, across the tree of life. A major bottleneck to systematically dissecting RNA modifications has been the lack of methods allowing to acquire systematic, high-confidence maps of many RNA modifications and at scale. Here we have developed Pan-Mod-seq, which permits de-novo, sensitive and specific identification of 16 distinct modifications in dozens of samples in parallel. We applied Pan-Mod-seq to RNA from 14 different species from all 3 domains of life, most of which sampled under highly variable chemical/physical/biological gradients, aiming to systematically explore the plasticity of rRNA modifications. While dynamically modified sites are relatively rare in mesophiles, we find that these are widespread in hyperthermophiles, where ~50% of identified modifications were induced with temperature. We focused on dissection of three key dynamically induced modifications: m5C, ac4C and Ψ. We uncover an m5C program encompassing dozens of targets across both rRNA subunits, all of which are dramatically induced with growth temperature in two of the sampled hyperthermophiles. In both cases m5C is introduced at a single consensus motif, via a single enzyme which is essential for growth at higher temperatures. ac4C in turn is dynamically induced among all three sampled hyperthermophiles at hundreds of sites, also at a single consensus motif. Remarkably, the methylation and acetylation overlap, and together form a tandemly modified temperature induced G-m5C-ac4C-G sequence motif.  Temperature-dependent induction of both enzymes can be recapitulated in vitro, establishing that it is an intrinsic property of the two enzymes rather than externally regulated. We obtain high-resolution cryoEM structures of ribosomes from WT strains at low and high temperatures and ones deleted of the methylating enzyme, and identify numerous mechanisms via which m5C confers structural stability. Finally, we also uncover a systematic induction in Ψ levels in T. kodakarensis, and reveal that it is largely coordinated by a single temperature-induced sRNA guiding pseudouridine formation at an unprecedented number of targets, whose loss results in growth deficiency at higher temperatures. Our findings offer a wide and systematic view of rRNA modification plasticity across representative species sampled from the tree of life, dissect the ability of different modifications to contribute to the stability of ribosomes at higher temperatures, and present a methodology allowing an unprecedented view on the the rRNA epitranscriptome in health and in disease.

sample_organism:
Pyrococcus furiosus
Saccharomyces cerevisiae

overall_design: In-vitro methylation followed by bisulfite sequencing. A mixture of total RNA of Saccharomyces cerevisiae (WT) and Pyrococcus furiosus (ΔRsmB) were in-vitro methylated with either Pfu-RsmB or TK1935under different temperatures (37 °C, 55 °C, 75 °C, 85 °C, 95 °C, and 105 °C), and a no enzyme control kept at 4 °C. Samples were subjected to bisulfite sequencing. One replicate per condition.

first: Schraga

last: Schwartz

first: Joe

last: Georgeson

first: Ronit

last: Nir

first: Vinithra

last: Iyer

first: Anatoly

last: Kustanovich


title: Inhibition of heme biosynthesis triggers cuproptosis in acute myeloid leukemia [RNA-Seq DIN]

first: Emily

last: Gruber

overall_design: The DIN model was generated by co-transducing fetal liver cells with MSCV-TRI-DNMT3AR882H-dsRed (TRI-DNMT3AR882H), MSCV-GFP-IDH2R140Q (MIG-IDH2R140Q) and MSCV-NRASG12D-TTA (MIT- NRASG12D) into sub-lethally irradiated (5.5Gy whole body irradiation delivered by an X-RAD 320 (Precision) female Ptprca recipients. Cryopreserved cells from moribund mice were transplanted into Ptprca recipients and mice were given either normal or doxycyline chow and water for 5 or 14 days. Doxycyline de-induces the expression of DNMT3AR882H. cKit+/Cd11b- DIN progenitor cells from the bone marrow and spleens of mice were isolated by FACS and subjected to RNA sequencing.

first: Lev

last: Kats


title: Inhibition of heme biosynthesis triggers cuproptosis in acute myeloid leukemia [ChIP-Seq]

overall_design: Hoxb8 cells expressing empty vector (EV), DNMT3AR882H, or DNMT3AR882H and treated with Hemin were isolated for ChIPseq.


title: Inhibition of heme biosynthesis triggers cuproptosis in acute myeloid leukemia [RNA-Seq Hoxb8_R882H_v_EV]

overall_design: RNA from Hoxb8 cells expressing empty vector (EV) or DNMT3AR882H was isolated for RNAseq.


title: Pan-modification profiling facilitates a cross-evolutionary dissection of the thermoregulated ribosomal epitranscriptome - thirdBatch1

overall_design: In-vitro treatment of WT and TkNat10 KO T. kodakarensis strains with Pfu-RsmB at at 85°C, followed by bisulfite treatment, in triplicates


title: Pan-modification profiling facilitates a cross-evolutionary dissection of the thermoregulated ribosomal epitranscriptome - extradata4

overall_design: Total RNA from Thermococcus kodakarensis, WT, Tko105 KO, or Tko35 KO was subjected to BID-seq treatment. WT RNA was also left untreated (input). The WT strain was grown at 65 °C, 75 °C, 85 °C and 95 °C, while the Tko105 KO strain and Tko35 KO strain were grown at  75°C and 95°C. three replicates


title: Pan-modification profiling facilitates a cross-evolutionary dissection of the thermoregulated ribosomal epitranscriptome - extradata3

overall_design: total RNA extracted from a ΔTkNat10 Thermococcus kodakarensis strain was subjected to in-vitro acetylation using purified TkNat10, grown at 37 °C, 55 °C, 65 °C, 75 °C, 85 °C, or 95 °C; RNA was subjected to ac4C-seq using NACNBH3 or was left untreated (control), one replicate


title: A Split Luciferase Biosensing Platform for Detection and Imaging of Chromatin Loops in Individual Live Cells

city: Davis

first: Henriette

last: O'Geen

state: CA

address: 451 Health Sciences Drive, GBSF 4617A

department: Genome Center

institute: UC Davis

summary: Eukaryotic cells regulate higher-order chromatin architecture, gene expression, and gene recombination via compaction of the genome into chromatin loops and topologically associating domains (TADs). While chromatin architecture has been thoroughly characterized for many eukaryotic genomes using cell-destructive techniques such as 3C-based methods, live-cell biosensing tools that can probe three-dimensional chromatin contacts in real-time are lacking. Using a dual dCas9 DNA biosensor based on a split NanoLuc luciferase reporter, we directly detected chromatin loops in live cells using luminescence quantification in a luminometer. We were able to see signal above background ratios of up to 10-fold. In addition, we directly visualized chromatin looping at the MYC TAD in live cells using high-resolution, low light live-cell imaging. Our biosensing platform therefore provides a useful methodology for live-cell, real-time detection of known or novel loops and for monitoring looping dynamics upon alterations in cell state.

overall_design: 4C-seq of cell-type specific chromatin loops in WT HCT116 cells, WT K562 cells, HCT116 E7 enhancer deletion cells, and HCT116 RAD21-mAC cells treated with either 1 uM DMSO or 1 uM auxin for 120 min using a non-blind viewpoint centered on the conserved CTCF binding site upstream of the MYC promoter with three replicates per cell type.

first: Nicholas

last: Heath

first: Antonio

last: Gomez

last: O’Geen

first: David

middle: J

last: Segal


title: Senataxin Promotes RNA Polymerase II Elongation [Hap1_TTseq]

city: Copenhagen

first: Zhong

last: Han

address: Blegdamsvej 3B

department: ICMM, Institut for Cellulær og Molekylær Medicin

country: Denmark

institute: University of Copenhagen

summary: Senataxin (SETX) is recognized as a critical regulator of RNA Polymerase II (RNAPII) transcription and plays a pivotal role in maintaining genomic stability. Despite its importance, the direct in vivo functions of SETX remain inadequately characterized. Employing the degradation tag (dTAG) system for acute protein depletion, we demonstrate that SETX does not influence transcription termination but significantly enhances RNAPII elongation. Through in vitro reconstitution assays, we show that SETX actively drives RNAPII forward across complex transcriptional landscapes, akin to the way bacterial ribosomes mitigate transcriptional pauses. In vivo analyses further reveal an accumulation of pausing RNAPII at early transcription stages and a corresponding increase in R-loop formation at these sites following SETX depletion. These insights not only redefine our understanding of SETX's role in transcriptional regulation but also highlight its potential as a therapeutic target in disorders linked to transcriptional dysregulation.

overall_design: Determination of nascent RNA synthesis by TT-seq under DMSO vs dTAGV1 treatment (4h)

first: Jesper

middle: Q

last: Svejstrup


title: Pan-modification profiling facilitates a cross-evolutionary dissection of the thermoregulated ribosomal epitranscriptome - extradata1

overall_design: Total RNA from Pyrococcus furiosus strain VC1, either WT or knockout (ΔRsmB), grown at 95 °C, was subjected to bisulfite sequencing, three replicates


title: Enhanced phase separation of BRD4S derived from CDK12 inactivation-induced IPA drives cancer metastasis

city: Rochester

first: LIGUO

last: WANG

state: MN

address: 200 1st St SW

department: Division of Computational Biology

institute: Mayo Clinic

summary: CDK12 gene is frequently deleted/mutated in human cancers and CDK12 loss associates with intronic polyadenylation (IPA). Here, we show that CDK12 deficiency induces IPA usage in BRD4 gene locus and expression of the BRD4 short form (BRD4S). Different from the full-length BRD4 (BRD4L) that possesses a C-terminal intrinsically disordered region (IDR), surprisingly, BRD4S has a greater capacity to form phase separation in a manner dependent on oligomerization mediated by a base-interacting structural domain (BID). Through CUT&Tag profiling, we identify BRD4S specifically binds to the promoters of and transcriptionally upregulates TGF-β signaling genes including TGFB2 and LTBP1. Our study identifies IPA usage at the BRD4 gene locus as a previously unrecognized mechanism responsible for BRD4S genesis. Our findings also reveal an essential role of BRD4S condensation formation and a unique ability of BRD4S to turn on TGF-β signaling gene expression.

overall_design: Genome-wide binding profiling of BRD4L and BRD4S by CUT&Tag in OVCAR8 cells ectopically expressing FLAG-tagged BRD4L or BRD4S individually.

first: Rui

last: Sun

first: Liguo

last: Wang

first: Haojie

last: Huang


title: Genome structure in single nucleoli by sequencing of laser capture nucleolar microdissection (scRNA-Seq)

city: Zurich

first: Raffaella

last: Santoro

address: Winterthurerstrasse 190

department: Dep. of Molecular Mechanisms  of Disease

institute: University of Zurich

summary: The three-dimensional architecture of the eukaryotic genome is a critical determinant in the regulation of gene expression programs. Repressive chromatin regions are localized either at the nuclear lamina or the nucleolus. While the role of the nuclear lamina in genome organization has been widely studied, the role of the nucleolus has only recently come under investigation. Here, we present a new method called nucleolus architecture mapping (NAM) for identifying nucleolus-associated genomic domains (NADs) at a single nucleolus resolution. NAM combines laser capture microdissection and DNA sequencing. We applied NAM to embryonic stem cells (ESCs) and neural progenitor cells and observed a distinct pattern of nucleolus-to-nucleolus heterogeneity for NADs. NAM not only identified characteristic features of NADs, such as high levels of H3K9me2, depletion of H3K27me3, low active histone marks, and repressed gene expression but also revealed that the genomic domains seen in cell population studies to contact both nucleoli and nuclear lamina (NAD/LAD regions) can contact the nucleolus in one cell and the NL in another cell, but rarely both nucleolus and NL in the same cell. Additionally, we found that ribosomal protein (RP) genes are frequently associated with the nucleolus, suggesting a potential direct crosstalk between the nucleolus and the regulation of RP genes, and hence ribosome biogenesis. We also performed NAM upon perturbation of the nucleolus structure by inhibiting PolI transcription with Actinomycin D. This showed a loss of mostly NADs from the nucleolus other than the chromosomes containing rRNA genes, demonstrating that nucleolar integrity is required for genomic contacts with the nucleoli. Additionally, NAM applied to hybrid ESCs demonstrates a predominantly monoallelic distribution of NADs in single cells. We identify parental-specific NADs exhibiting a non-uniform distribution across different chromosomes. Our findings underscore NAM as an invaluable tool for investigating nucleolar organization in individual cells and, potentially, genome organization in other large phase-separated organelles in the future.

overall_design: scRNAseq F123 ESCs

first: Kaivalya

last: Walavalkar


title: Widespread low-affinity motifs enhance chromatin accessibility and regulatory potential in mESCs [ChIP-nexus]

first: Melanie

last: Weilert

overall_design: Chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode and single ligation (ChIP-nexus) experiments were conducted in mouse embryonic stem cell lines (R1) across wildtype lines and one set of CRISPR/Cas9 clones at an upstream Akr1cl enhancer.

first: Kaelan

last: Brennan

first: Khyati

last: Dalal

first: Sabrina

last: Krueger

first: Haining

last: Jiang

first: Rosa

last: Martinez-Corral

first: Julia

last: Zeitlinger


title: Allelic topological centering by transcription factors drives oncogenic multi-enhancer transcriptional regulation [Micro-C]

city: Philadelphia

first: YEQIAO

last: ZHOU

state: PA

address: 3600 Civic Center Blvd 14 114

department: Pathology

institute: University of Pennsylvania

summary: Multiple enhancers, often located across vast genomic distances, regulate key genes in cancer. However, how organization of chromatin topology at individual allele enables cancer type-restricted multi-enhancer gene regulation remains unclear. Using acute protein degradation and time-course population-average chromatin conformation capture in lymphoma, we found that the B-cell lineage-determining transcription factor EBF1 preferentially positions multiple enhancers at loci containing sparsely distributed genes essential for B-cell identity and oncogenesis. Our time-resolved sub-diffraction optical tracing of chromatin architecture of over 100,000 alleles further revealed diverse topological conformations that facilitate multi-enhancer interactions in individual lymphoma cells. Mechanistically, we found that positioning of enhancers at the topological center of the allele is required for their interactions with the target promoter, with EBF1 serving as a barrier to the loop extruding cohesin on the enhancers. These findings, which we demonstrate their generalizability to the T-cell lineage-determining transcription factor TCF1 in T-cell leukemia, suggest that lineage-determining transcription factors establish local radial positioning of enhancers and promoters to enable multi-enhancer regulation of key genes in cancer.

overall_design: Micro-C was performed in Granta519-EBF1-FKBP-KI cells to assess the impact of EBF1 on chromatin folding

first: Robert

last: Faryabi

first: Yeqiao

last: Zhou


title: Allelic topological centering by transcription factors drives oncogenic multi-enhancer transcriptional regulation [ChIP-seq]

overall_design: ChIPseq was performed to assess protein binding in Granta519 cells with or without EBF1


title: Transcriptional profiles of castration resistant prostate cancer cells treated with ITRI-148 PROTAC targeting AR and AR-V7

city: Taoyuan

first: Ling-Yu

address: No.259, Wenhua 1st Rd., Guishan Dist.,

department: Biochemistry and Molecular Biology

institute: Chang Gung University

summary: Androgen receptor (AR) signaling remains a key driver of castration-resistant prostate cancer, with AR splice variants like AR-V7 mediating resistance to second-generation antiandrogens. Targeting the AR N-terminal domain (NTD) offers a strategy to bypass ligand-binding domain-mediated resistance. We developed a VHL-based (ITRI-90) and a CRBN-based (ITRI-148) PROTAC degrader capable of targeting AR and AR-V7 via NTD. These compounds demonstrate potent antitumor activity towards castration-resistant and enzalutamide resistant xenograft models. Transcriptomic analyses confirm robust suppression of AR and AR-V7 target genes by ITRI-148 in VCaP cells. The transcriptomic effects of these AR-NTD degraders in enzalutamide-resistant C4-2B/MDVR cells closely mirror that of AR knockdown.

overall_design: RNA-seq profiling of VCaP cells treated with DMSO or 1 μM ITRI-148 for 48 hours; and the profiling of enzalutamide-resistant C4-2B/MDVR cells treated with DMSO, 5 μM ITRI-90, 5 μM ITRI-148 or infected with Lentivirus carrying pLKO1 control vector or AR-knockdown shRNA.

first: Chiu-Lien

last: Hung


title: TGF-beta dynamically controls epithelial identity in a 3D model of human epiblast [RNAseq_CRISPRi_ZNF398]

city: Turin

first: Andrea

last: Lauria

address: Via Accademia Albertina, 13

department: Life Sciences and Systems Biology

country: Italy

institute: University of Turin

summary: Pluripotency is the ability to give rise to all cell types of the body and is first observed in a mass of disorganised cells of the embryo. Upon implantation, pluripotent cells form a columnar epithelium and undergo lumenogenesis. At gastrulation, a portion of the pluripotent epiblast will undergo epithelial to mesenchymal transition (EMT), forming the primitive streak (PS). It still remains unclear what molecular mechanism supports the epithelial identity of the pluripotent epiblast before gastrulation. Here we developed an optimised, chemically defined 3D model of human pluripotent epiblast formation in which conventional pluripotent stem cells (PSCs) self-organise into a columnar epithelium with a lumen in 48 hours. From 72 hours we observed spontaneous symmetry breaking and specification of PS-like cells, as confirmed by single-cell RNA sequencing. We found that Insulin and FGF signalling are both required for the proliferation and survival of the pluripotent epiblast model. Conversely, TGF-beta signalling maintains epithelial identity. Epithelial identity appears uncoupled from the expression of canonical pluripotency markers OCT4, NANOG and PRDM14, but under the control of ZNF398. Once the pluripotent epithelium is established, TGF-beta inhibition is inconsequential, and stimulation with Activin A leads to highly efficient PS induction. We conclude that TGF-beta dynamically orchestrates epithelial identity of human pluripotent cells.

overall_design: Bulk RNA-seq profiling of gRNA-ZNF398 PSCs in 2D and 3D-hE-gastruloids, with (ZNF398 Knock-Down) or without (Control) Dox+TMP induction. 8 samples, 2 replicates per condition.

first: Irene

last: Zorzan

first: Elena

last: Carbognin

first: Valentina

last: Proserpio

first: Davide

last: Benvegnù

first: Susana

last: De Juambelz Urias

first: Caterina

last: Dalrio

first: Giorgia

last: Panebianco

first: Rebecca

last: Scarfò

first: Eleonora

last: Pensabene

first: Mattia

last: Arboit

last: Paolucci

last: Drusin

first: Monika

last: Sledziowska

first: Paola

last: Braghetta

last: Ditadi

first: Gianluca

last: Amadei

first: Salvatore

last: Oliviero

first: Graziano

last: Martello


title: TGF-beta dynamically controls epithelial identity in a 3D model of human epiblast [RNAseq_H9_KiPS]

last: Lauria

overall_design: Bulk RNA-seq profiling of 3D-hE-gastruloids generated from H9 and KiPS cell lines, collected at 0, 4 and 8 days of gastruloid formation. 12 samples, 2 replicates per condition.

last: Zorzan

last: Carbognin


title: Senataxin Promotes RNA Polymerase II Elongation [Hap1_DRBrelease_TTseq]

first: Zhong

last: Han

overall_design: Determination of RNAPII elongation speed by DRBrelease_TTseq under DMSO vs dTAGV1

first: Jesper

last: Svejstrup


title: The Function of Tetrameric MADS-box Transcription Factors in Floral Fate Determination [Mnase-seq]

city: Berlin

first: Manuel

last: Neumann

address: Philippstr. 13

country: Germany

institute: HU Berlin

summary: MADS-domain transcription factors (TF) are critical in determining floral cell fate and floral organ identities. However, their function molecular mechanisms are still poorly understood. Here, we show that effective DNA-binding in closed chromatin and target gene regulation of APETALA1 (AP1), a  key floral MADS-box TF requires tetramerisation, indicating the fundamental role of tetrameric MADS-box TFs in floral cell fate programming.

overall_design: MNase-seq were generated with seedling tissues induced by dexamethasone for 24 hours for AP1 expression. In addition,same amount of mock-treated seedlings were collected as a control. Three biological replicates were generated.

first: Kerstin

last: Kaufmann

first: Xiaocai

last: Xu


title: HTGTS-TCR-seq for profiling of mouse and human T-cell receptor α and β gene rearrangements and diversity [HTGTS-TCRseq]

city: Shanghai

first: Haiqiang

last: Dai

address: 320 Yue Yang Road

institute: Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell  Science

summary: T-cell receptor (TCR) diversity is generated through V(D)J recombination, which are essential for adaptive immunity. Although many methods have been applied, a simple method to detect TCR rearrangement at the DNA level remains lacking. Here we introduce HTGTS-TCR-seq, a novel approach derived from high-throughput genome-wide translocation sequencing (HTGTS), to comprehensively and unbiasedly analyze TCR recombination. Unlike multiplex PCR requiring extensive V-J segment primers, HTGTS-TCR-seq enables unbiased TCR profiling through targeted enrichment and deep sequencing by a single primer. This method identifies biased V(D)J segment usage in developing T cells through V/J primer-based amplification and provides unique complementarity determining region 3 (CDR3) profiles in mature T cells via J primers. HTGTS-TCR-seq detects rearrangement at both alleles, enabling quantification of DJβ and VβDJβ rearrangement products with productive and nonproductive profile. Applied to human peripheral blood mononuclear cells (PBMCs), HTGTS-TCR-seq reveals a shared CDR3 motif between humans and mice. HTGTS-TCR-seq is a robust tool for T cell immunity research, with potential application in immune responses analysis, cancer research, and autoimmune diseases investigation.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: We employed HTGTS-TCR-seq to asess rearrangement events and TCR repertoire changes across developmental stages and T cell subsets at DNA level. DN3 thymocytes, preselection DP thymocytes, splenic CD4+ and CD8+ T cells from mice and CD4+ and CD8+ T cells from human PBMCs were FACS sorted. Total thymocytes from young and aged mice were collected. Genome DNA were extracted, sonicated and utilized to conduct HTGTS-TCR-seq.

first: Rui

last: Luo

first: Yawei

last: Song

first: Meichen

last: Wang

first: Longhao

last: Zou

first: Tiange

last: Yang

first: Guangchuan

first: Wei

last: Wu

first: Hai-Qiang


title: Multiomics profiling of molecular and cellular dynamics of spinal cord injury in a rat [mRNA-seq]

city: Vestec

first: Ruslan

last: Klassen

address: Průmyslová 595

department: Institute of Biotechnology

country: Czech Republic

institute: Czech Academy of Science

summary: Spinal cord injury (SCI) is a debilitating condition with no effective treatment. The injury triggers a complex cascade of molecular and cellular events that drive both damage and repair processes. To explore these mechanisms, we performed a comprehensive multiomics analysis in a rat compression model of SCI, focusing on the acute phase. Transcriptomic profiling revealed extensive gene dysregulation, highlighting early inflammation, neuronal death, and synaptic dysfunction, followed by the initiation of reparative processes. Cell type composition analysis showed a rapid infiltration of peripheral immune cells, activation of microglia, and loss of neurons, astrocytes, and oligodendrocytes. miRNA profiling uncovered a highly dysregulated miRNA landscape, with the miR-17~92 cluster emerging as a key regulator of neurogenesis, synaptic activity, and cell survival. Integrative miRNA-mRNA-protein analysis identified potential therapeutic targets, including miR-20a, whose inhibition in vitro supported neurogenesis and reduced apoptosis under oxidative stress. Our findings provide new insights into the molecular mechanisms of SCI and highlight miRNAs as potential targets for therapeutic intervention.

overall_design: Adult male Wistar rats (n=64) were used as an experimental model. The rats were obtained from the facility's breeding center (Physiological Institute of the Academy of Sciences of the Czech Republic, Prague, Czech Republic). Animals undergoing surgery weighed 300 ± 30 g and were 10 weeks old. Animals were maintained on a 12-h light/dark cycle with ad libitum access to food and water. Rats were randomly divided into experimental groups: SCI 3 hpi (n=4), 12 hpi (n=4), 24 hpi (n=4), 3 dpi (n=4), 7 dpi (n=4), time- corresponding shame-operated groups (n=4 for each group) and an intact control group (n=4), also 8 animals were used for behavioral tests and 12 animals were used for immunohistochemical analysis.

first: Sarka

last: Chytilova

first: Ivan

last: Arzhanov

first: Daniel

last: Zucha

first: Eva

last: Rohlova

first: Peter

last: Androvic

first: Pavel

last: Abaffy

first: Lucia

last: Urdzikova-Machova

first: Mikael

last: Kubista

first: Nataliya

last: Romanyuk

first: Lukas

last: Valihrach


title: Allelic topological centering by transcription factors drives oncogenic multi-enhancer transcriptional regulation [Hi-C]

first: YEQIAO

last: ZHOU

overall_design: Hi-C was performed to assess genome folding in Granta519 Cas9 cells with or without EBF1

first: Robert

last: Faryabi

first: Yeqiao

last: Zhou


title: TGF-beta dynamically controls epithelial identity in a 3D model of human epiblast [RNAseq_ZNF398_OE]

first: Andrea

last: Lauria

overall_design: Bulk RNAseq profiling of KiPS stably expressing the empty vector control without or with SB43 (TGF-beta inhibition), or expressing ZNF398 without or with SB43, in 2D and 3D after 4 days of self-organisation. 12 samples, 2 replicates per condition.

first: Irene

last: Zorzan

first: Elena

last: Carbognin

first: Valentina

last: Proserpio

first: Davide

last: Benvegnù

first: Susana

last: De Juambelz Urias

first: Caterina

last: Dalrio

first: Giorgia

last: Panebianco

first: Rebecca

last: Scarfò

first: Eleonora

last: Pensabene

first: Mattia

last: Arboit

last: Paolucci

last: Drusin

first: Monika

last: Sledziowska

first: Paola

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