title: Differential gene expression in ADGRF5 overexpression versus control in neonatal rat ventricular myocytes

city: Philadelphia

first: Jeanette

middle: Marie

last: Einspahr

state: Pa

address: 3500 N Broad Street

department: Aging & Cardiovascular Discovery Center

institute: Temple University School of Medicine

summary: G-Protein Coupled Receptors (GPCRs) represent one of the most targeted drug classes in the human genome, accounting for greater than 40% of all FDA-approved drugs, including gold-standard heart failure (HF) therapeutics. However, the second-largest family of GPCRs, known as Adhesion GPCRs (aGPCR), have yet to serve as a clinical target despite increasing evidence of their pathophysiological contributions, representing an area of highly unexplored druggable potential. We identified an aGPCR, known as ADGRF5, to be highly expressed in the left ventricle (LV) of C57BL/6, which we also found increases in response to transaortic constriction (TAC) surgery. To determine the impact of ADGRF5 on cardiac parameters normally or during HF, we generated constitutive cardiomyocyte-specific (αMHC-Cre-driven) ADGRF5 knockout mice (cmF5KO), which displayed normal cardiac structure and function at 12 weeks of age, as assessed via echocardiography, gravimetrics and immunohistochemistry. However, over time cmF5KO mice developed worsened cardiac function and remodeling with increased mortality, even in the absence of pathologic insult, suggesting ADGRF5 provides a homeostatic regulatory function in the heart. To determine the impact of the receptor in vitro, we overexpressed ADGRF5 in neonatal rat ventricular myocytes (NRVMs) versus vehicle controls, and subsequently performed RNA-sequencing to determine differential gene expression. We identified 2039 differentially expressed genes (815 upregulated, 1225 downregulated).

overall_design: Four biological replicates were analyzed for each condition (two conditions; 4 replicates per condition).

first: Douglas

last: Tilley


title: A Role for Rapid Transcription Dynamics in Cytarabine Resistance in Acute Myeloid Leukemia [RNA-seq]

status: Public on Mar 09 2026

city: Bronx

first: Russell

last: Scharf

state: New York

address: 1945 Eastchester Rd

country: USA

institute: Albert Einstein College of Medicine

type: Expression profiling by high throughput sequencing

summary: Chemotherapy resistance remains a critical challenge in the treatment of patients with cancer including acute myeloid leukemia (AML). While genetic alterations contribute to chemotherapy resistance, rapid-adaptive non-genetic mechanisms, particularly transcription dynamics, remain poorly understood. Given the role of cell-intrinsic transcriptional variability in cell fate decisions, understanding and targeting chemotherapy-induced changes in transcription dynamics could offer new strategies to prevent chemotherapy resistance. In this study, we demonstrate that short-term treatment with the widely used chemotherapeutic cytarabine (AraC) leads to the rapid emergence of RNA-induced AML cells with increased AraC resistance in both cell lines as well as primary patient samples. Mechanistically, through global transcriptomic analyses and targeted high-resolution analysis of transcription dynamics using single-molecule RNA FISH, we found rapid induction of transcriptional dynamics and upregulation of key transcription factors (TFs) - which we term “AraC rapid response TFs (ARR-TFs)” and which include PU.1 and GATA1 – following chemotherapy treatment. At a functional level, short-term pre- and co-treatment with RNA transcription inhibitors effectively suppressed chemotherapy-induced RNA induction and prevented resistance acquisition, in both in vitro and in vivo models. Furthermore, we found that CRISPR-mediated suppression of PU.1 and GATA1 induction significantly attenuated AraC resistance. In summary, our findings reveal a previously unrecognized role of rapid and early adaptive transcriptional dynamics in AML chemotherapy resistance, highlighting master TFs as key regulators of drug resistance. These insights offer a novel, pharmacologically targetable and accessible approach to alleviate chemotherapy resistance, and encourage further testing with the ultimate goal to improve AML treatment outcomes.

overall_design: HL60 cells were treated with 250nM AraC for 1 day. Controls were treated with the AraC solvent DMSO. Cells were stained for Pyronin Y and Hoechst and G0/G1 cells were sorted using flow cytometry for RNA seq analysis

first: Goichi

last: Tatsumi

first: Rajni

last: Kumari

first: Yutaro

last: Suzuki

first: Shuting

last: Li

first: Taylor

last: Samuel

first: Sriram

last: Sundaravel

first: Amit

last: Verma

first: Justin

last: Wheat

first: Ulrich

last: Steidl


title: GADD45A links Hippo-TEAD signaling to estrogen response and luminal identity in HR-positive breast cancer

city: Taipei

first: Yi-Ru

last: Tseng

address: No. 201, Sec. 2, Sh ipai Rd.

country: Taiwan

institute: Taipei Veterans General Hospita

summary: Growth arrest and DNA damage-inducible alpha (GADD45A) has context-dependent roles in cancer, yet its function in hormone receptor-positive (HR+) breast cancer remains poorly defined. We characterized GADD45A across breast cancer subtypes and investigated its prognostic significance and connection to estrogen and Hippo-TEAD signaling in HR+HER2− breast cancer. GADD45A protein expression was evaluated by immunohistochemistry in 100 tissue microarray samples and 16 CDK4/6 inhibitor-treated metastatic cases. Prognostic associations were assessed in the METABRIC (n = 1,904) and TCGA-BRCA cohorts. siRNA-mediated GADD45A knockdown was performed in MCF7 and T47D cells followed by RNA sequencing and integration with TEAD4 ChIP-seq data. GADD45A protein was preferentially expressed in HR+HER2− tumors and correlated with estrogen receptor levels. High GADD45A expression was associated with improved overall and relapse-free survival in HR+HER2− breast cancer but not in other subtypes. GADD45A knockdown led to downregulation of estrogen response and Hippo-TEAD positive effector programs with concurrent upregulation of E2F targets and cell cycle pathways. Integration with TEAD4 ChIP-seq data demonstrated significant enrichment of TEAD4 promoter binding among GADD45A-regulated genes, including Hippo-TEAD components, chromatin remodeling machinery, and luminal identity regulators. GADD45A is a subtype-specific prognostic marker in HR+HER2− breast cancer that sustains estrogen-responsive and TEAD-mediated transcriptional programs, identifying it as a previously unrecognized link between Hippo-TEAD signaling and luminal gene maintenance.

overall_design: siRNA-mediated knockdown of GADD45A was performed using siGADD45A2 (MCF7) or siGADD45A1 (T47D) with scramble siRNA controls, in biological duplicate per condition (8 samples total).

first: Chih-Yi

last: Lin

first: Chun-Yu

last: Liu

first: Ta-Chung

last: Chao

first: Chi-Cheng

last: Huang

first: Yi-Fang

last: Tsai

first: Ling-Ming

first: Jiun-I

last: Lai


title: Regulation of neuronal invasion of small cell lung cancer by STMN2/β-alanine-controlled metabolic reprogramming [beta-alanine-RNAseq]

city: Hangzhou

first: Yongjie

last: Wang

address: 2318 Yuhangtang Rd

country: China

institute: Hangzhou Normal University

summary: Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine malignancy characterized by rapid progression and a poor prognosis. The perineural invasion (PNI) phenomenon is prevalent in SCLC and represents a critical clinical-pathological feature; nevertheless, the mechanisms and its influence on disease progression and patient survival remain unclear. In this study, we established PNI as an independent adverse prognostic factor of SCLC based on surgical cohort evidence. We further demonstrated that the neural microenvironment upregulates STMN2, a neuron-specific microtubule-destabilizing protein, in SCLC cells. Mechanistically, STMN2 activates the β-alanine metabolic pathway in a concentration-dependent manner, leading to increased intracellular β-alanine levels and thereby enhancing tumor cell migration and invasion. In vivo experiments confirmed that STMN2 knockdown significantly suppresses neural invasion in SCLC, an effect reversible upon exogenous β-alanine supplementation. Together, these findings identify a neural-STMN2-β-alanine-invasion axis in SCLC. This work not only provides novel mechanistic insights into neural invasion but also highlights a promising therapeutic target for countering PNI, underscoring its substantial translational relevance.

overall_design: DMS114 cells were treated with β-alanine at a final concentration of 2.5 mM for 48 hours. Cells treated with an equal volume of vehicle served as controls. After that cells were subjected to next-generation sequencing followed by differential gene expression analysis

first: Yan

last: Zhou

first: Runbo

last: Zhong

first: Yanwei

last: Zhang

first: Michael

middle: R

last: Shurin

first: Galina

middle: V

first: Liwen

last: Xiong

first: Tianqing

last: Chu

first: Baohui

last: Han

first: Haohua

last: Teng

first: Yu

last: Yang

first: Erpeng

last: Wu

first: Chi

first: Lincheng

first: Wanting

first: Yuqing

last: Lou

first: Jun

last: Lu

first: Hua


title: PI3K-AKT activation determines oncogenic RAS-induced hypertranscription and replication stress [ChrRNAseq]

city: Liverpool

first: Claire

last: Wilson

address: Ashton Street

country: United Kingdom

institute: University of Liverpool

summary: Oncogenic RAS activation, hypertranscription and transcription-replication conflicts (TRCs) are frequent features of cancer cells that are under investigation for therapeutic targeting. RAS oncogenes promote RNA polymerase activity and hypertranscription for cell growth and proliferation, which can the lead to TRCs. Here, we investigated hypertranscription and replication stress induced by the HRAS and KRAS oncogenes, and their downstream signalling through the MAPK and PI3K pathways. We show that HRAS causes more TRCs than KRAS because HRAS specifically hyperactivates the PI3K-AKT signalling pathway. MAPK signalling is insufficient to cause hypertranscription and TRCs, which require PI3K activity. PI3K hyperactivation was associated with increased E2F and MYC transcription programmes, increased S phase entry, increased nascent transcription of genes encoding ribosome biogenesis factors and expression of small nucleolar RNAs (snoRNAs). Inhibiting S phase entry alone did not rescue hypertranscription and replication stress, which required both MAPK and PI3K signalling. We report that a main downstream effector of PI3K-induced hypertranscription is AKT-mediated phosphorylation of GSK3b, which stabilises MYC. Cancer datasets further support that PI3K mutations may be associated with hypertranscription and replication stress signatures. Our findings suggest a mechanistic explanation for replication stress variability across RAS models and identify potential new predictors of TRCs in cancer.

overall_design: Chromatin-bound RNA (ChrRNAseq) was performed to quantify nascent transcripts after HRASG12V or KRASG12V induction for 48h. Induction was triggered by supplementing the culture media with 500 nM 4-hydroxytamoxifen (4-OHT). Non-induced cells (0h) were used as controls.

first: Richard

middle: D

last: Kelly

first: Aditi

last: Kanhere

first: Eva

last: Petermann


title: Regulation of neuronal invasion of small cell lung cancer by STMN2/β-alanine-controlled metabolic reprogramming [Proximal_and_Distal-RNA-seq]

first: Yongjie

last: Wang

overall_design: Tumors from proximal and distal invasive regions of a sciatic nerve invasion model using DMS114 cells were subjected to next-generation sequencing followed by differential gene expression analysis

first: Yan

first: Runbo

first: Yanwei

first: Michael

first: Galina

last: Liu


title: PI3K-AKT activation determines oncogenic RAS-induced hypertranscription and replication stress [RNASeq]

overall_design: To investigate the role of HRAS- and KRAS-induced replication stress,  RNAseq was performed. Sequencing was performed on human BJ-hTert cells after 24h and 48h HRASG12V or KRASG12V induction. Induction was triggered by supplementing the culture media with 500 nM 4-hydroxytamoxifen (4-OHT). Non-induced cells (0h) were used as controls.


title: CD47 promotes MAPK and epithelial-to-mesenchymal transition molecular programs to drive pro-metastatic phenotypes in non-small cell lung cancer

city: Toronto

first: Kelsie

last: Thu

state: ON

address: 209 Victoria Street

country: Canada

institute: Keenan Research Centre for Biomedical Science

summary: CD47 is best known for its role in tumor immune evasion; however, studies in diverse cell models indicate that it also has cell-autonomous, tumor-promoting functions which are cell type- and context-specific. Motivated by the prognostic and therapeutic significance of CD47 and the limited knowledge regarding its roles beyond immune evasion in non-small cell lung cancer (NSCLC), we sought to define the cellular and molecular processes driven by intrinsic CD47 signaling in NSCLC. Transcriptome profiling of CD47 wildtype and knockout NSCLC cells implicated its regulation of genes enriched for signatures of MAPK signaling and epithelial-to-mesenchymal transition (EMT). A significant positive association between CD47 and MAPK/EMT expression signatures was also evident in large cohorts of NSCLC cell lines and tumor tissues. Functional studies indicated that CD47 does not regulate cell proliferation in NSCLC cells like it does in other cancer types. Instead, CD47 regulates cell adhesion and migration through an ERK and EMT axis, validating our transcriptomic findings. Moreover, CD47 loss-of-function significantly diminished the ability of NSCLC cells to metastasize in vivo, demonstrating the physiological relevance of cell-intrinsic CD47 signaling in lung cancer cells. Our data reveal a novel role for CD47 in relaying signals through ERK to promote EMT expression programs and pro-metastatic phenotypes in NSCLC. Although additional mechanistic studies are needed to further decipher the CD47-ERK-EMT signaling pathway, our findings reinforce the therapeutic potential of CD47, rationalizing further research to develop CD47 blockade as a multimodal therapy for NSCLC.

overall_design: RNA-seq profiling of clonal wild-type and CRISPR-mediated CD47 knockout Lewis Lung Carcinoma cells (LLC) collected in vitro.

first: Asa

middle: P

last: Lau

first: Lilian

last: G Zhai

first: Ryunosuke

last: Hoshi

first: Zackary

last: Rousseau

first: Abraam

last: Zakhary

first: Yin Fang

first: Rola

last: Saleeb

first: Heyu

last: Ni

middle: L


title: Single cell and spatial sequencing analysis of cancer associated fibroblasts in the brain metastasis tumor microenvironment [Multiome]

city: Los Angeles

first: Bodour

last: Salhia

state: CA

address: 1450 Biggy Street

department: Department of Cancer Biology

institute: University of Southern California

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Brain metastasis (BM) remains largely incurable. Cancer-associated fibroblasts (CAFs) can either support or inhibit tumor growth in the tumor microenvironment (TME), yet their roles in BM remain under-described. Here we report a single-cell and spatial sequencing analysis of human BM tissues and define four transcriptionally distinct CAF subpopulations. BM CAF subpopulations are characterized by either extracellular matrix (ECM), immune, contractile, or neural features, and show distinct spatial distributions within the BM TME. Further analyses reveal that BM CAFs engage extensively in cell-cell communication and adopt distinct cell states, including an ECM CAF cell state marked by high levels of immunoglobulin superfamily containing leucine rich repeat expression (ISLR-CAFs). Functionally, ISLR-CAFs reduce BM tumor cell viability in vitro, consistent with a tumor-inhibitory role. These findings highlight the heterogeneity and plasticity of CAFs in BM, emphasizing the importance of precision in defining stromal contributions to disease progression and therapeutic response.

overall_design: In this study, we performed single-cell Multiome sequencing (scMultiome-seq; RNA and ATAC) on nine human BM tissue samples to evaluate cellular heterogeneity within the TME. Seven samples were from lung-to-brain metastases (LBM) and two from breast-to-brain metastases (BBM)

first: Thomas

last: Simon

first: David

middle: N

last: Buckley

first: Zeyi

last: Yang

first: Chikako

last: Matsuba

first: Ben

middle: Y

last: Tew

first: Gerald

middle: C

last: Gooden

first: Kyle

last: Hurth

first: Steven

middle: A

last: Toms

last: Tran

first: Gabriel

last: Zada

first: Matthew

last: Salomon


title: Expression data from WT and Hela S3 cells

city: Suzhou

first: Gongjie

last: Wu

address: Renai Road

institute: Xi'an Jiaotong-Liverpool University

summary: The goal of this study was to define how loss of full-length DEK reshapes global transcriptional programs in HeLa S3 cells by comparing gene-expression profiles of wild-type (WT) HeLa S3 and the 1E7 DEK-knockout cells generated by TALEN-mediated genomic disruption. The 1E7 clone lacks detectable full-length DEK, while retaining a low-abundance truncated DEK species, consistent with an alternative transcriptional start site/allelic expression.

overall_design: Transcriptome profiling was performed using the Affymetrix GeneChip Human Transcriptome Array 2.0 (HTA 2.0) with total RNA isolated by QIAGEN RNeasy Mini Kit, and raw CEL files were processed using RMA background correction and normalization in Transcriptome Analysis Console to identify differentially expressed genes. The objective was to generate a robust, genome-wide expression dataset enabling statistical identification of DEK-dependent expression changes (e.g., clear WT vs 1E7 separation by PCA and DEGs at FDR < 0.05), thereby providing a resource to link DEK loss to specific transcriptional pathways and regulatory programs.

first: Ferdinand

last: Kappes


title: DNA methylation stochasticity is linked to transcriptional variability and identifies convergent epigenetic disruption across genetically-defined subtypes of AML [PrimaryAML_scATACseq]

city: Baltimore

first: Andrew

last: Feinberg

state: MD

address: 855 North Wolfe Street

department: Center for Epigenetics

institute: Johns Hopkins University

summary: Epigenetic regulators are frequently mutated in acute myeloid leukemia (AML). We investigated DNA methylation stochasticity in genetic subtypes of AML using WGBS data and publicly-available ERRBS data, and identified transcriptional dysregulation and altered chromatin accessibility related to methylation entropy using single-cell RNA-seq and single-cell ATAC-seq data.

overall_design: Single-cell ATAC-seq of primary patient acute myeloid leukemia samples and normal CD34+ and GMP progenitor samples.

first: Eleanor

last: Hilgart

first: Michael

last: Koldobskiy


title: Mild cold exposure mitigates ischemic heart injury through indole-3-acetamide–AHR-mediated SMAD2 proteolysis

city: Nanjing

first: Ying

last: Wang

address: No. 2 Southeast University Road, Jiangning District, Nanjing

institute: Southeast University

summary: Ambient temperature profoundly influences organismal metabolism, physiology, and disease. While cold exposure is a well-recognized risk factor for the onset of myocardial infarction (MI), its impact on the injured heart after MI remains unexplored. Here, we demonstrate that a mild cold exposure (MCE) regimen at 16oC improved mice systolic function post-MI compared to the controls kept at room temperature. Integrative multi-omics analyses revealed a critical link between a cold-inducible tryptophan metabolite indole-3-acetamide (I3AM) and cardiac fibrosis in post-MI hearts. MCE elevated I3AM levels in serum and heart tissue and upregulated gut indole-related metabolic pathways; which was necessary to attenuate cardiac fibrotic remodeling by inhibiting SMAD2/3 signaling and fibroblast activation. Mechanistically, I3AM acts as a ligand for the aryl hydrocarbon receptor (AHR). I3AM-bound AHR promoted ubiquitination and proteasomal degradation of SMAD2. Treatment with I3AM recapitulated the cardioprotective effects of MCE, while genetic or pharmacological inhibition of AHR abolished the benefits of both MCE and I3AM in MI mice. In patients with and without heart failure, serum I3AM levels correlated positively with cardiac function, underscoring its clinical relevance. Our findings unveil a previously unrecognized metabolic-proteolytic axis triggered by MCE and identify the temperature responsive I3AM-AHR-SMAD pathway as a potential therapeutic target for ischemic hearts.

overall_design: Heart tissues were harvested and washed in pre-cooled PBSE. Nuclei were isolated using the GEXSCOPE® Nucleus Separation Solution following the manufacturer’s protocol. To reduce technical variability, heart tissues from three mice per group were pooled as one biological sample for nuclei isolation. Isolated nuclei were filtered, quality-controlled (DAPI staining, >80% viability), and used for single-nucleus RNA-seq library preparation with the GEXSCOPE® Single Nucleus RNA-seq Kit. Libraries were sequenced on an Illumina NovaSeq 6000 platform to generate 150 bp paired-end reads.

first: Lei

last: Li


title: Engineering of Amino Acid-Derived Ionizable Lipids Enables Inhaled Base Editing for Therapeutic Gene Correction in the Lung

first: Fanglin

last: Gong

address: 144 College St

institute: University of Toronto

summary: CRISPR-based gene editing holds promise for treating genetic diseases, yet its application to lung disorders has been hindered by the challenges of pulmonary delivery. Inspired by the modularity and biocompatibility of amino acid-derived chemistries, we report the combinatorial synthesis of 960 ionizable lipids incorporating chemically diverse backbones from both proteinogenic and non-proteinogenic α-amino acids. Through high-throughput screening and structure-function analysis, we identify CHCha-10, a cyclohexyl amino acid-derived lipid that forms biodegradable nanoparticles capable of efficiently delivering mRNA-based gene editors to lung epithelial cells. Following intratracheal administration, CHCha-10 nanoparticles exhibit enhanced mucus penetration, and epithelial-specific transfection in both mice and ferrets. As a functional application, we demonstrate the first instance of in vivo base editing in the lung via inhalation. Delivery of adenine base editor mRNA and guide RNA targeting the CFTR G542X mutation restores CFTR expression and chloride channel function in G542X human airway epithelial cells, mouse-derived intestinal organoids, and the lungs of cystic fibrosis mice.

overall_design: Amplicon sequencing results of the Cftr locus from treated B6-Tg(CFTRG542X)Cwr (CFTR G542X) mice lung. Mice were intratracheally administered CHCha-10 LNPs encapsulating ABE/A9. Lung tissues were harvested 3 days post-dosing, and genomic DNA was extracted to assess base-editing efficiency.


title: Identification of a human hematopoietic stem cell subset that retains memory of inflammatory stress [XenoProg_Myeloid]

first: Alexander

middle: James

last: Murison

state: Ontario

address: 101 College Street, TMDT room 11-706

institute: University Health Network

summary: Hematopoietic stem cells (HSCs) survive a lifetime of infection to sustain life-long blood production. Inflammation activates many blood cell types, driving aging and malignancy. To understand HSC adaptation to inflammation, we performed single cell multi-omics on human HSC in xenograft inflammation-recovery models. Two transcriptionally and epigenetically distinct HSC subsets expressing canonical HSC programs were identified. Only one showed sustained transcriptional and epigenetic changes after recovery from inflammatory treatments. This HSC inflammatory memory (HSC-iM) program is enriched in memory T cells and HSC from recovered COVID-19 patients. Importantly, HSC-iM accumulates with age and in clonal hematopoiesis. Overall, heritable molecular alterations in a subset of human HSCs, an adaptation to long-term inflammatory stress, may predispose to heightened age related risk of blood cancer and infection.

overall_design:
All samples were stored as viable cryopreserved cell products. Bone marrow samples were collected from patients undergoing hip replacement surgery for middle and older aged samples, and were purchased from a vendor for younger adult donors. Xenograft samples were pooled from multiple mice treated with the same conditions (as listed) from three separate cord blood engraftment pools. Fresh cord blood was collected from multiple donors. 10x Multiome was performed on cells sorted by FACS after thawing and processing as necessary. For LT-HSC, cells from a large number of donors (>20) were pooled and subjected to scRNAseq (BD Rhapsody).

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Raw files for human/patient samples were not submitted to GEO due to concerns about submitting personally identifiable sequence data for open access.
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title: DNA methylation stochasticity is linked to transcriptional variability and identifies convergent epigenetic disruption across genetically-defined subtypes of AML [PrimaryAML_WGBS]

type: Methylation profiling by high throughput sequencing

overall_design:
WGBS of primary patient acute myeloid leukemia samples and normal CD34+, CMP, GMP, and MEP samples.

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The table below lists GEO accessions reused/reanalyzed for this study.
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title: A Neuronal GPCR Modifies Neuropeptide Signaling to Suppress Proteostasis in distal tissues [AM140]

city: Jerusalem

first: Inbar

last: Plaschkes

address: The Hebrew University of Jerusalem - Ein Kerem

department: The Faculty of Medicine - Ein Kerem

country: Israel

institute: HUJI

summary: The proteostasis network (PN) safeguards the integrity of proteins by the promotion of various cellular activities. However, with aging the competence of the PN declines and aggregated proteins accrue within cells. This process can underlie the development of neurodegenerative disorders, such as Alzheimer’s and Huntington’s diseases. Although the activities of the PN occur within the cell, this network is regulated across the organism by inter-tissue communication, which is partially governed by neurons. We discovered previously, that reducing the expression of gtr-1, which encodes a neuronal GPCR, protects worms from the toxicity of the Alzheimer’s-causing, Aβ peptide. Here we investigated the mechanism that is acted upon the knockdown of gtr-1 and found that gtr-1 RNAi treatment modulates the expression of neuropeptide-coding genes, differentially modifies the aggregation of proteotoxic proteins, and enhances protein degradation. These findings highlight the roles of neuropeptides as coordinators of proteostasis across the organism.

overall_design: three sets of Six thousand synchronized AM140 worms were grown on IPTG induced bacteria expressing either EV or gtr-1 3’UTR RNAi. At day 1 of adulthood, the worms were bleached and eggs were placed on the same RNAi as their parental generation. Worms of the second generation were washed daily to get rid of progeny, harvested at day 6 of adulthood and RNA was extracted using kit (Macherey-Nagel # 740961) according to the manufacturer’s instructions, RNA integrity was tested and the samples were submitted for Nova-seq RNA sequencing.

first: Joud

last: Hirbawi

first: Ehud

last: Cohen

first: Adam

last: Zeretsky

first: Huadong

last: Zho

first: Emmanuelle

last: Merquiol

first: Atif

last: Ahmed Siddiqui

first: Irit

first: Galia

last: Blum

first: Gourab

last: Dey


title: Harnessing the Innate Tropism of Migratory Interneuron Precursors as a Neuron-Based Therapeutic Platform to Treat Glioblastoma

first: Thomas

last: De Raedt

state: PA

address: 3501 Civic Center boulevard CTRB 4028

institute: CHOP

summary: We found that transplanted post-mitotic Migratory Inhibitory Interneuron Precursors (MIPs), which are currently in clinical trial for refractory epilepsy, are chemoattracted to glioblastoma and can be modified to deliver therapeutic proteins that greatly enhance survival of xenografted mice. Engrafted MIPs position themselves at the tumor-brain interface in multiple models of HGG and other brain tumors. We conclude that MIPs can become neuron-based platform for localized, sustained anti-tumor therapy.

overall_design: RNAseq on MIP differentiation cultures at different time points

first: Stephanie

last: Brosius

first: William

last: Manley

first: Isabella

last: Distefano

first: Theodore

last: Linde-Stamos

first: Kyra

last: Harvey

first: Amritanshu

last: Rana

first: Brandi

last: Nelson

first: Jamie

last: Galanaugh

first: Deborah

last: Rohacek

first: Jessica

middle: B

last: Foster

first: Stewart

last: Anderson


title: Medullary cavity expansion is mediated by distinct cell populations during fetal bone development

city: Fukuoka

first: Shinichiro

last: Sawa

address: 3-1-1 Maidashi, Higashiku

country: Japan

institute: Kyushu University

summary: Single cell RNA-sequencing was performed to examine the transcriptomic identity of RANKL-expressing cells during fracture healing in mice. RANKL fate-mapping mice were used to isolate tdTomato-labeled cells from tibiae at post-fracture day 14.

overall_design: Tnfsf11-tTA; LC1; R26-tdTomato mice  were treated with doxycycline in the drinking water from embryonic day 0 to postnatal day 14.  A fracture was surgically induced in the right tibia at postnatal day 21. Tibiae were isolated at post-fracture day 14, and single-cell suspensions were prepared. CD45.2-Ter119-CD31-tdTomato+ cells were sorted from four mice and pooled. Sorted cells were subjected to single-cell RNA sequencing using the 10x Genomics　Chromium Single Cell 3′ Gene Expression v3.1 platform.

first: Eriko

last: Sumiya


title: DNA methylation stochasticity is linked to transcriptional variability and identifies convergent epigenetic disruption across genetically-defined subtypes of AML [CellCulture_WGBS]

first: Andrew

last: Feinberg

summary: Epigenetic regulators are frequently mutated in acute myeloid leukemia (AML). We investigated DNA methylation stochasticity in genetic subtypes of AML using WGBS data and publicly-available ERRBS data, and identified transcriptional dysregulation and altered chromatin accessibility related to methylation entropy using single-cell RNA-seq and single-cell ATAC-seq data. We demonstrated that the hypomethylating drug decitabine (DAC) leads to reduction of DNA methylation entropy specifically in IDH2-mutant AML cells.

overall_design: WGBS of IDH2-mutant isogenic TF-1 cells and parental TF-1 erythroleukemia cells treated with decitabine (DAC) or DMSO control.

first: Eleanor

last: Hilgart

first: Michael

last: Koldobskiy


title: Identification of a human hematopoietic stem cell subset that retains memory of inflammatory stress [TARGET-seq}

first: Alexander

last: Murison

summary: Inflammation activates blood cells, driving aging and malignancy. Hematopoietic stem cells (HSCs) survive a lifetime of infection, sustaining life-long hematopoiesis. Yet, how human HSCs respond and adapt to inflammatory stress is largely unknown. We developed xenograft inflammation-recovery models and performed single-cell multiomics on human HSCs to empirically understand HSC adaptation to inflammatory stress. Two transcriptionally and epigenetically distinct HSC subsets were identified with one, termed HSC inflammatory memory (HSC-iM), retaining molecular memory of prior inflammatory treatments. The HSC-iM molecular program was enriched in HSC from adult and pediatric samples across conditions ranging from COVID-19 recovery, aging, sickle cell disease, and clonal hematopoiesis (CH), establishing both the validity of our xenograft models and the physiological relevance of HSC-iM. The HSC-iM subset exhibited quiescence, hallmark inflammatory signatures, restrained hematopoietic output, and the transmission of the pro-inflammatory HSC-iM transcriptional program to downstream myelo-lymphoid progeny. These heritable molecular alterations represent an adaptation to inflammatory stress and some were found to be relieved by CH mutation acquisition. Finally, HSC-iM programs in circulating blood cells were associated with a risk score for all-cause mortality in population cohort analyses, underscoring the importance of this newly identified HSC subset in characterizing heterogeneous health outcomes across a lifetime.

overall_design:
Bone marrow samples were collected from patients undergoing hip replacement surgery and stored as viable cryopreserved cell products. Xenotransplantation of 5 adult bone marrow samples was performed into irradiated NSG-SGM3 animals with 55,000-90,000 CD34-enriched cells per animal. Mice were treated with PBS or hTNFa at 2w and 10w post-transplantation. Human engraftment was assessed at 12w post-transplantation and cells cryopreserved for downstream analysis. For analysis of primary bone marrow samples, cells were thawed, stained for FACS, index sorted and TARGET-seq+ performed. For xenograft samples, cells were thawed and pooled from multiple mice for each BM donor treated with either PBS or hTNFa. After mouse depletion, samples were stained for FACS and hCD45+hCD34+hCD19-hCD33- HSPCs and hCD45+CD33+CD34-CD19- myeloid cells were index sorted and TARGET-seq+ performed.

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Raw data requires controlled access
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first: Niels Asger

last: Jakobsen


title: Transcriptomic and proteomic analyses show minimal role for mast cell ST2 in primary Heligmosomoides polygyrus bakeri infection

city: Dundee

first: Henry

middle: J

last: McSorley

address: Dow Street

department: Cell Signalling & Immunology

institute: University of Dundee

summary: The IL-33/ST2 pathway is important as part of the type 2 immune response against helminth infections. Mast cells express the highest levels of the IL-33 receptor subunit ST2 of any immune cell, and mast cells can mediate type 2 immune inflammation, however the role of IL-33-driven mast cell responses in helminth infection is poorly understood. We sought to determine the role of mast cell ST2 expression during Heligmosomoides polygyrus bakeri (Hpb) infection by generating mast cell conditional ST2 knockout (MCPT5Cre x ST2f/f, cKO) mice. These mice have normal frequencies of mast cells at steady state, but show specific and strong (albeit incomplete) knockdown of ST2 expression on mast cells. On Hpb infection, faecal egg and adult worm burden were similar between cKO and littermate controls, as were mast cell degranulation markers, serum IgE and goblet cell hyperplasia. Therefore, we conclude that mast cell ST2 does not play a dominant role in Hpb infection. To further investigate the immune response to infection in cKO and littermate controls, transcriptomic and proteomic changes were assessed in duodenal tissues in infected versus naïve mice in cKO and control mice. Minimal transcriptomic and proteomic changes were seen between genotypes, whereas substantial changes were seen between naïve and infected mice, regardless of genotype. Hpb infection induced local increases at the transcript and protein level for mast cell proteases (MCPT1 and MCPT2), resistin-like molecules (RELMα and RELMβ), and markers such as the phospholipase PLA2G4C and the pore-forming protein gasdermin C. Bulk proteomic analysis was also searched against the Hpb genome to identify Hpb proteins present in the duodenal tissues. A list of 60 Hpb proteins of interest were identified in infected duodenal samples, of which 18 contain a signal peptide and are present in the excretory/secretory products of Hpb (HES) (likely secretory products including immunomodulatory proteins); 28 proteins are present in HES but do not contain a signal peptide (likely excretory products); and 14 proteins are not present in HES (likely proteins present in the remnants of Hpb within the duodenum). This work thus provides datasets for changes in the mouse intestine due to Hpb infection, at both the transcript and protein level, as well as a dataset of Hpb proteins detectable in the mouse duodenum at day 14 of infection.

overall_design: Bulk RNA-seq profiling of duodenal tissue from MCPT5Cre x ST2f/f and MCPT5WT x ST2f/f mice, either naïve or at day 14 of Heligmosomoides polygyrus bakeri infection

first: Nicole

last: Ong

first: Suzanne

middle: H

last: Hodge

first: Christa

last: Baker

first: Danielle

last: Smyth

first: Tania

last: Frangova


title: Immune-microbiome coordination defines interferon setpoints in healthy humans [bulk RNA-seq]

city: San Francisco

first: Ravi

last: Patel

address: 513 Parnassus Ave

department: CoLabs

institute: University of California San Francisco

summary: Human immune systems are highly variable, with most variation attributable to non-genetic sources. The gut microbiome crucially shapes the immune system; however, its relationship with the baseline immune states of healthy humans remains incompletely understood. Therefore, we performed multi-omic profiling of 110 healthy participants through the ImmunoMicrobiome study. A factor-based integrative approach identified coordinated variation, revealing that the interferon response was amongst the most variable immune features in healthy participants. Microbiome composition, pathways, and stool metabolites varied concomitantly with interferon response pathways. Longitudinal data spanning more than a year indicated the significant stability of these parameters within individuals over time. Our study provides extensive data to examine the relationship between the immune states and microbiomes of healthy individuals at steady state, which paves the way for delineating inter-individual differences relevant for disease susceptibility and responses to therapy.

overall_design: PBMC samples were collected from healthy individuals and gene expression and variant calls were profiled using bulk RNA-seq.

first: Joel

last: Babdor

middle: K

first: Gabriela

last: Fragiadakis

first: Matthew

last: Spitzer


title: Immune-microbiome coordination defines interferon setpoints in healthy humans

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

overall_design: Refer to individual Series


title: PI3K-AKT activation determines oncogenic RAS-induced hypertranscription and replication stress

first: Claire

last: Wilson

first: Richard

last: Kelly

first: Aditi

last: Kanhere

first: Eva

last: Petermann


title: Wollemia nobilis transcriptome sequencing

city: Cold Spring Harbor

first: W. Richard

last: McCombie

state: NY

address: 1 Bungtown Road

institute: Cold Spring Harbor Laboratory

summary: We performed gene expression profiling analysis using data obtained from RNA-seq of 3 different tissues: leaf, ovule and pollen

overall_design: Gene expression profiling analysis of RNA-seq data for leaf, ovule and pollen tissues in triplicate.

first: Srividya

last: Ramakrishnan

first: Cristiane

last: de Santis Alves Rosa

first: Lais

last: Araujo Coelho

first: Melissa

last: Kramer

first: Sara

last: Goodwin

first: Olivia

last: Mendivil Ramos

first: Gil

last: Eshel

first: Veronica

middle: M

last: Sondervan

first: Samantha

last: Frangos

first: Cecilia

last: Zumajo-Cardona

first: Katharine

last: Jenike

first: Shujun

last: Ou

first: Xiaojin

last: Wang

first: Yin

last: Peng Lee

first: Stella

last: Loke

first: Maurizio

last: Rossetto

first: Hannah

last: McPherson

first: Sebastiano

last: Nigris

first: Silivia

last: Moschin

first: Damon P.

last: Little

first: Manpreet

middle: S

last: Katari

first: Kranthi

last: Varala

first: Sergios-Orestis

last: Kolokotronis

first: Barbara

last: Ambrose

first: Larry

last: Croft

first: Gloria

last: Coruzzi

first: Dennis

middle: W

last: Stevenson

first: Michael

last: Schatz

first: Robert

last: Martienssen


title: Early life B cell memory is archived in the mouse B-1 cell compartment and drives Chronic Lymphocytic Leukemia-like disease [scRNA-seq]

city: Lund

first: Joan

last: Yuan

address: Sölvegatan 19

country: Sweden

institute: Lund University

summary: The adult B cell pool is a developmental mosaic comprising short-lived naïve B cells and long-lived memory. Using genetic time-stamping, we previously showed that early life origin (ELO) B cells contributed substantially to the adult mouse immune system. In this study, we found that ELO B cells share a memory-like signature. Notably, B-1 cells exhibited an enrichment for the PD-L2/CD80 double positive (DP) immunophenotype associated with highly differentiated memory. Indeed, microbial antigen exposure in neonates expanded distinct specificities within the DP B-1 cell compartment, identifying the latter as a reservoir of IgM memory. B cell chronic lymphocytic leukemia (CLL) is a disease marked by the accumulation of memory-like B cells. By applying time-stamping to a mouse model for unmutated CLL (U-CLL), we demonstrated that leukemic expansion is driven by B-1 cell clones arising prior to postnatal day 10. Importantly, B-1 cells in mice and man closely mirrored the molecular profile of U-CLL. These results suggest that an age-associated leukemia can originate from ELO B cells.

overall_design: Single-cell RNA-Seq from Tom+ (early life origin) and Tom– (adult life origin) FACS sorted B cells from postnatal day 19 time-stamped mice.

first: Niklas

last: Segrén

first: Stefano

last: Vergani

first: Giorgia

last: Montano

first: Shamit

last: Soneji


title: Early life B cell memory is archived in the mouse B-1 cell compartment and drives Chronic Lymphocytic Leukemia-like disease


title: Immune-microbiome coordination defines interferon setpoints in healthy humans [CITE-seq and WMS]

first: Ravi

last: Patel

overall_design: PBMC samples were collected from healthy individuals and surface protein and gene expression were analyzed using CITE-seq. The dataset contains PBMC samples collected at two timepoints from the ImmunoMicrobiome cohort (110 healthy donors): baseline dataset (pool1 - pool7) and follow-up dataset (pool8 - pool14). The follow-up dataset was generated for a subset of the cohort, with their corresponding baseline samples repeated to reduce technical biases. The 10X 5' NEXT GEM, v1.1 kits were used for baseline dataset and 5' NEXT GEM, v2 for follow-up dataset. The WMS dataset contains stool samples collected from participants in the ImmunoMicrobiome cohort at two timepoints. Baseline samples are denoted by the suffix "BLE", while follow-up samples are denoted by "AAE6" or "ABE4." The follow-up dataset was generated for a subset of participants, with their corresponding samples repeated to reduce technical bias (denoted by suffix "BLE2"). WMS libraries were prepared from DNA extracted from human stool samples using standard chemical and mechanical lysis methods. Libraries were generated using the Nextera XT kit (Illumina) and sequenced as paired-end reads on an Illumina NovaSeq S4 flow cell.

first: Joel

last: Babdor

first: Gabriela

last: Fragiadakis

first: Matthew

last: Spitzer


title: Early life B cell memory is archived in the mouse B-1 cell compartment and drives Chronic Lymphocytic Leukemia-like disease [bulkVDJ-seq]

overall_design: Bulk VDJ-Seq (IgHM repertoire) from Tom+ (early life origin) and Tom– (adult life origin) FACS sorted B cells from postnatal day 10 or day 19 time-stamped Eµ-TCL-1tg and WT control mice.


title: A genome wide code to define cell-type specific CTCF binding and chromatin organization

city: New York

first: Lara

last: Ladney

address: 550 1st Ave

department: Pathology

institute: NYU Langone Health

summary: CTCF-mediated chromatin folding plays a key role in gene regulation, however the mechanisms underlying cell type-specific control are not fully elucidated. Comprehensive analyses reveal that CTCF binding stability and cohesin overlap in mice and humans is regulated by species specific differences in accessibility, the presence of CTCF extended Up and Downstream binding site sequences and motifs corresponding to expressed TFs enriched at most bound sites. CTCF repositions nucleosomes asymmetrically, with linker spacing altered by accessibility, while cohesin-mediated nucleosome phasing is affected by surrounding motifs which also impact chromatin insulation. Importantly, cell type-specific transcriptional programs determine motif enrichment at CTCF bound sites, reflecting the stabilizing/destabilizing effect of individual TFs. These studies identify mechanisms underlying cell type-specific CTCF profiles, linked to local and long-range chromatin organization.

overall_design:
WGBS-NOMe-seq was performed on mESCs with a auxin-inducible degron and dox inducible expression transgene WT or R342C mutant CTCF system. The nuclei were treated with a GpC methyltransferase prior to DNA  bisulfite conversion to assess both endogenous (WCG context) and exogenous (HGC context) methylation to assess DNA methylation and nucleosome occupancy at a single molecule level.
Nano-NOMe-seq was performed to assess nucleosome occupancy at a single molecule level (exogenous methylation,5mC in HGC context) in mESCs with a CTCF auxin-inducible degron and dox inducible expression transgene WT CTCF and in Hela with a SCC1 auxin-inducible degron.

first: Catherine

last: Do

first: Guimei

last: Jiang

first: Paul

last: Zappilie

first: Jane

last: Skok


title: m6A Methylation Suppresses anti-tumour immune responses in Colorectal Cancers by Inhibiting Double-stranded RNA accumulation from Alu Repeats [RNAseq2]

city: Oxford

first: Yucheng

last: Wang

state: Oxfordshire

address: Roosevelt Drive, Headington

institute: University of Oxford

summary: How cancer cells evade immune detection despite expressing immunostimulatory retroelement (RE) transcripts remains an open question in tumour immunology. In cancer cells, endogenous REs that escape histone and DNA silencing are transcribed into RNA and can form double-stranded RNA (dsRNA), triggering innate immune responses through a process known as viral mimicry. However, RNA-level checkpoints can limit this effect. Here, we identify the m6A RNA methyltransferase METTL3 as a key regulator of this suppression in colorectal cancer (CRC). Targeting METTL3 enhances the accumulation of dsRNA derived from both pre-existing and newly transcribed REs, thereby amplifying immunostimulatory signalling and activating cell-intrinsic anti-tumour immunity. Notably, CRCs exhibit differential sensitivity to METTL3 inhibition: tumours with high basal dsRNA levels and RNA methylation respond robustly to METTL3 blockade alone, whereas those with low endogenous dsRNA require combination therapy to achieve comparable immune activation. Co-treatment with DNA methyltransferase inhibitors (DNMTis) restores immune activation in these otherwise resistant tumours. Together, these findings uncover a novel mechanism by which METTL3 functions as an RNA-level immune checkpoint and suggest that combining METTL3 and DNMT inhibition may overcome resistance and serve as an effective therapeutic strategy in CRC.

overall_design: RNA-Seq

first: Parinaz

last: Mehdipour


title: Nuclear Speckles enable processing of RNA from GC-rich Isochores [RNA-Seq]

city: Berlin

first: IBRAHIM

middle: AVSAR

last: ILIK

state: Berlin

address: Ihnestraße 63-73

country: Germany

institute: Max-Planck-Institut für molekulare Genetik

summary: Nuclear Speckles are large, multi-component condensates in the mammalian nuclei that are thought to be involved in various stages of gene expression, including transcription, splicing, and export. Investigating the direct role of Speckles in these processes have been intractable due to their heterogeneous, multi-component nature. Here we show that Speckles are necessary for the splicing of GC-rich, levelled intron/exon architecture. Acute removal of Speckles leads to a drastic downregulation of GC-rich genes which are clustered within GC-rich isochores. Genes outside these domains are physically away from Speckles and are not affected from loss of Speckles. Animal genomes that do not have GC-rich isochores, do not have GC-rich levelled intron/exon architecture and do not have Speckles. Our results suggest that the expansion of Speckles core proteins’ intrinsically disordered domains during vertebrate evolution facilitated GC-content elevation in Amniotes by creating a condensate necessary to splice the by-products of this process: GC-rich, levelled intron/exon architecture.

overall_design: Gene expression profiling of HEK293 cells stably expression various SON variants in endogenous-SON-depleted cells.. Libraries were prepeared from pA(+)-selected RNA, 27 libraries in total.

first: Michal

last: Malszycki

first: Lisa

last: Martina

first: Ibrahim

last: Ilik

first: Tugce

last: Aktas


title: Nuclear Speckles enable processing of RNA from GC-rich Isochores [4sU]

overall_design: Nascent transcription profiling of HCT116 cells subject to SRRM2-depletion, SON-depletion, SRRM2+SON-depletion or control treatment. Libraries were prepeared from 4sU-labelled RNA, 8 libraries in total.


title: Early life B cell memory is archived in the mouse B-1 cell compartment and drives Chronic Lymphocytic Leukemia-like disease [bulkRNA-seq]

first: Joan

last: Yuan

overall_design: Bulk RNA-Seq from Tom+ (early life origin) and Tom– (adult life origin) FACS sorted B cells from postnatal day 10 time-stamped Eµ-TCL-1tg and WT control mice.

first: Niklas

last: Segrén

first: Giorgia

last: Montano

first: Shamit

last: Soneji


title: Nuclear Speckles enable processing of RNA from GC-rich Isochores [Fish_RNAseq]

overall_design: Gene expression profiling of Fathead minnow (fish) EPC cells cells subject to human SON-overexpression  or control treatment.. Libraries were prepeared from pA(+)-selected RNA, 6 libraries in total.


title: Human neuromodulatory assembloids to study serotonin signaling and disease

city: Stanford

first: Sabina

last: Kanton

address: 318 Campus Drive

department: Psychiatry and Behavioral Sciences

institute: Stanford University

summary: Neuromodulators influence critical functions of the developing brain and regulate behavioral states. Dysfunction of these systems is often involved in neuropsychiatric disease and many drugs for these conditions act on these signaling pathways. Recent advances in stem cell biology have made it possible to derive a wide range of cells across the developing human nervous system in organoids and to functionally integrate them into assembloids, however they currently do not systematically incorporate neuromodulation. Here, we generate a midbrain-hindbrain organoid (hMHO) from human induced pluripotent (hiPS) cells and fuse them with cortical organoids (hCO) to form neuromodulatory assembloids (hNMA). We focus on serotonin (5-hydroxytryptamine, 5-HT) as one key neuromodulator and find characteristic gene expression patterns and properties of the 5-HT neurons in the hMHO. In hNMA, 5-HT neurons project into hCO, release 5-HT and change cortical network activity. To explore the utility of this system, we study 22q11.2 deletion syndrome (22q11.2DS), a common microdeletion associated with high risk for neuropsychiatric disease and defects in 5-HT signaling. We found differences in 5-HT dynamics in hNMA from patients that can be rescued by a selective serotonin reuptake inhibitor (SSRI). Taken together, hNMA can be used to study human 5-HT dynamics and uncover disease phenotypes which would facilitate therapeutic development.

overall_design: scRNA-Seq of human midbrain-hindbrain organoids (hMHO)

first: Fikri

last: Birey

first: Xiangling

last: Meng

first: Chunyang

last: Dong

first: Sergiu

last: Pasca


title: Herpes simplex virus type 1 (HSV-1) R-loops are targets for APOBEC-mediated mutagenesis

city: Debrecen

first: Márton

last: Miskei

address: Egyetem tér 1.

department: Department of Molecular Biotechnology and Microbiology

country: Hungary

institute: University of Debrecen

summary: APOBEC3 enzymes are key components of antiviral immunity that introduce mutations into viral genomes. We show that R-loops serve as preferred substrates for APOBEC3 mutagenesis during herpes simplex virus type 1 infection. APOBEC3 enzymes are recruited to viral R-loops, where they generate clustered C-to-T mutations in genes critical for viral assembly. Importantly, R-loops alone are not mutagenic, and APOBECA3 enzymes do not edit HSV-1 without an R-loop; mutagenesis occurs when R-loops and APOBEC3 enzymes interact. These findings identify endogenous R-loops formed during the HSV-1 life cycle as focal vulnerabilities and highlight R-loop–APOBEC3 coupling as a mechanism for antiviral mutagenesis.

overall_design: ChIP-seq and DRIP-seq experiments to determine the APOBEC binding site and R-loops on the HSV-1 genome.

first: Marton

first: Lorant

last: Szekvolgyi


title: Wnt-materials sustain H3K14-acetylation in human skeletal stem cells for tissue engineering and bone repair [RNA-Seq]

city: Hangzhou

first: Dong

last: Fang

state: Zhejiang

address: 866 Yuhangtang Road

department: Life Sciences Institute

institute: Zhejiang University

summary: Engineering functional tissues for transplantation requires insight into epigenetic mechanisms that regulate stem cell fate. We have developed the Wnt-induced osteogenic tissue model (WIOTM), a platform that recapitulates human osteogenesis, and identified acetylation of histone H3 at lysine 14 (H3K14ac) as a critical epigenetic regulator in human skeletal stem cells (hSSCs). In WIOTM, localized Wnt signals drive asymmetric cell division (ACD), yielding a proximal hSSC with high H3K14ac and a distal daughter with reduced H3K14ac that migrates into the 3D collagen matrix and initiate osteogenic differentiation. Disrupting H3K14ac in hSSCs abrogates ACD and WIOTM formation. To test whether hSSCs maintain H3K14ac in vivo, we formed the WIOTM on Wnt-functionalized polymer bandages and transplanted them into calvarial defects. The WIOTM contributed to bone repair, and human cells adjacent to the bandages retained high H3K14ac despite the injury environment. These findings establish WIOTM as both a mechanistic and translational platform for regenerative medicine.

overall_design: RNA-Seq in hSSCs cultured on Inactive and Active Wnt.


title: Wnt-materials sustain H3K14-acetylation in human skeletal stem cells for tissue engineering and bone repair [CUT&Tag]

overall_design: CUT&Tag for histone modifications H3K14ac and H3K27me3 in hSSCs cultured on Inactive and Active Wnt.


title: Single-nucleus RNA sequencing of colon tissue from Pink1 knockout and wild-type mice

city: Suwon

first: Eun

middle: Jeong

last: Lee

state: Gyeonggi-do

address: 206 World cup-ro, Yeongtong-gu

department: Department of Brain Science

country: South Korea

institute: Ajou University

summary: Mutations in PTEN-induced kinase 1 (Pink1), have been associated to mitochondrial dysfunction and gastrointestinal abnormalities. We generated single-nucleus RNA sequencing (snRNA-seq) profiles from colon tissues of wild-type (WT) and Pink1 knockout (KO) mice to characterize cell-type specific transcriptional changes associated with loss of Pink1.

overall_design: Colon tissue was collected from 15-week-old male Pink1 knockout (KO) mice (C57BL/6) and age and sex-matched wild-type (WT) littermates as controls. The colon was detached from the mesenteric tissues, and nuclei were isolated by detergent-based lysis. Sorted nuclei were pooled and loaded onto 10x Genomics Chromium single-cell 3′ library preparation followed by Illumina sequencing. Obtained raw reads were processed with standard snRNA-seq pipline to generate the gene and barcode UMI count metrics.

first: Muhammad

last: Junaid

first: Soo Jung

last: Park

first: Yiseul

last: Bae

first: Eun Jeong

first: Su Bin

last: Lim


title: Nuclear Speckles enable processing of RNA from GC-rich Isochores [Df1 RNA-Seq]

first: IBRAHIM

last: ILIK

overall_design: Gene expression profiling of Df1 (Chicken) cells subject to SON-depletion  or control treatment.. Libraries were prepeared from pA(+)-selected RNA, 6 libraries in total.

first: Michal

last: Malszycki

first: Ibrahim

last: Ilik

first: Lisa

last: Martina

first: Tugce

last: Aktas


title: Exposed phosphatidylserine is a non-classical inhibitory molecule during CD8 T cell exhaustion [scRAN-seq]

city: Tampa

first: Christopher

middle: Bernardo

last: Medina

state: FL

address: 3011 W Holly Dirve

department: Immunology

institute: Moffitt Cancer Center

summary: In cancer and chronic infection CD8 T cell exhaustion is hallmarked by expression of inhibitory surface receptors such as PD-1, TIM-3, CTLA-4, LAG-3, and TIGIT. Thus, inhibitory receptor focus has been limited to surface expressed proteins and an expansion into different classes of inhibitory molecules is needed. In this study we identify the lipid metabolite phosphatidylserine (PS) as a regulator of T cell exhaustion. PS localizes to the inner plasma membrane of live cells but is externalized to the outer membrane of apoptotic cells where it can function as a potent inhibitory ligand. However, the role of PS on live immune cells is less clear4. We show that live, exhausted, antigen specific CD8 T cells externalize PS during chronic lymphocytic choriomeningitis virus (LCMV) infection. PS exposure is initiated upon T cell activation and chronic antigen stimulation led to continued externalization. PD-1+ Tcf1+ stem-like, Tim-3+ transitory, and Tim3+ terminally differentiated/exhausted cells all expose PS. Furthermore, transcriptomic and lipidomic analyses identified increased PS metabolism during exhaustion. To evaluate the inhibitory function of exposed PS in exhaustion we treated chronically infected mice with the PS-targeting antibody (mch1N11)5 and found that monotherapy alone expanded the PD-1+ LCMV-specific CD8 T cell immune response. Most significantly, PD-1+ Tcf1+ stem-like CD8 T cells downregulated quiescence-associated gene modules and increased proliferation and self-renewal after treatment. Mechanistically, we found that exposed PS from exhausted T cells could suppress co-stimulatory molecule expression on neighboring dendritic cells to limit PD-1+ CD8 T cell proliferation. Combination of PS-targeting with aPD-L1 synergized to further increase the CD8 T cell effector response and improve viral control. Lastly, we identify that CD8 tumor infiltrating lymphocytes from human clear cell renal cell carcinoma (ccRCC) and human non-small cell lung cancer (NSCLC) patient tumors similarly expose PS indicating its translational potential. In fact, clinical trials combining PS-targeting antibodies and PD-L1 antibodies have recently been used in combination therapies. Therefore, our work details live PS CD8 T cell biology and provides a potential mechanism of action by which exposed PS facilitates suppressive T cell – DC interactions to function as a ‘non-classical’ inhibitory molecule during exhaustion.

overall_design: LCMV Cl. 13 chronically infected mice were treated with control IgG or mch1N11 PS-targeting antibodies every 3 days at 200ug per injection IP for one week. Spleen were isolated post-treatment and CD11b+ (Tcell/NKcell/Bcell lineage neg) cells were isolated from respective groups and used for scRNAseq

first: Rafi

last: Ahmed


title: Nuclear Speckles enable processing of RNA from GC-rich Isochores [Pore-C]

summary: Nuclear Speckles are large, multi-component condensates in the mammalian nuclei that are thought to be involved in various stages of gene expression, including transcription, splicing, and export. Investigating the direct role of Speckles in these processes have been intractable due to their heterogeneous, multi-component nature. Here we show that Speckles are necessary for the splicing of GC-rich genes with short introns clustered within GC-rich isochores (levelled intron/exon architecture). Acute removal of Speckles leads to a drastic downregulation of this class of  genes, whereas genes outside these domains are physically away from Speckles and are not affected from loss of Speckles. Animal genomes that do not have GC-rich isochores, do not have GC-rich levelled intron/exon architecture and do not have Speckles. Our results suggest that the expansion of Speckles core proteins’ intrinsically disordered domains during vertebrate evolution facilitated GC-content elevation in Amniotes by creating a condensate necessary to splice the by-products of this process: GC-rich, levelled intron/exon architecture.

overall_design: HCT116 cells, Pore-C

first: Michał

last: Małszyck

first: İbrahim

last: Avşar Ilık

first: Daniela

last: Salgado Figueroa

first: Nirmalya

last: Dasgupta

first: Menşura

last: Feray Çoşar

first: Keun-Tae

last: Kim

first: Gil Daniel

last: Bregieiro Carraco

first: Beatrix

last: Fauler

first: David

last: Meierhofer

first: Thorsten

last: Mielke

first: Hiroo

last: Imai

first: Cantaş

last: Alev

first: Ferhat

last: Ay

first: Tuğçe

last: Aktaş


title: Wnt-materials sustain H3K14-acetylation in human skeletal stem cells for tissue engineering and bone repair [ATAC-Seq]

first: Dong

last: Fang

overall_design: ATAC-Seq in hSSCs cultured on Inactive and Active Wnt.


title: Enhanced interferon-stimulated gene signatures in B cells following influenza B virus infection compared to influenza vaccination

city: Melbourne

first: Lachlan

last: Dryburgh

state: Victoria

address: 792 Elizabeth Street

department: The Department of Microbiology and Immunology,  The University of Melbourne

country: Australia

institute: The Peter Doherty Institute for Infection and Immunity

summary: Despite influenza vaccination, some individuals succumb to life-threatening influenza or even death. Yet our understanding of differential immune features generated by vaccination versus infection by influenza A and influenza B viruses (IAVs, IBVs) is limited. We sought to define molecular signatures of influenza-specific B cells elicited following influenza virus infection or vaccination towards both IAV and IBV subtypes. Using barcoded fluorescently-labelled haemagglutinin (HA)-specific probes, we performed single-cell RNA sequencing of influenza-specific B cells on day 0, 7 and 28 following influenza inactivated vaccination, in comparison to hospitalized patients during acute IAVor IBV as well as at 1-month convalescence. Comparing B cell responses elicited by infection and vaccination, the most striking findings stemmed from increased interferon-stimulated gene signatures, especially IF44L, IFITM1 and XAF1, in total B cells during acute IBV infection, which decreased at 1 month following patients’ recovery. These interferon-stimulated genes were not observed in B cells following influenza vaccination encompassing both IAV and IBV components or IAV infection in our patients. For HA-specific B cells, transcriptomic analysis revealed phenotypic differentiation and isotype class-switching following vaccination, with evidence of clonal sharing between memory and atypical B cell phenotypes. In in vitro influenza virus infection experiments, IBVs showed higher infectivity of human peripheral blood mononuclear cells, including B cells, in comparison to IAVs. Additional co-culture of infected PBMCs with plasma obtained from patients hospitalized with IAV, IBV, COVID-19 and RSV increased the infectivity of immune cells by both IBV and IAV, suggesting that B cell infectivity we observed might also be driven by plasma mediators. IBV infection also resulted in reduced B cell proliferation compared to IAV, potentially associated with the antiproliferative effect of IFITM1. Our findings comparing and contrasting influenza-specific B cell responses elicited following acute influenza virus infection versus vaccination provide key insights into understanding B cell immunity towards IBV versus IAV infections, in contrast to vaccination responses, to inform rational vaccine design and therapeutic targets.

overall_design: Using barcoded fluorescently-labelled haemagglutinin (HA)-specific probes, we performed single-cell RNA sequencing of influenza-specific B cells, as well as Tfh cells on day 0, 7 and 28 following influenza inactivated vaccination, in comparison to hospitalized patients during acute IAV or IBV as well as at 1-month convalescence.

first: Wuji

last: Zhang

first: Kaitlynn

last: Allen

first: Shihan

last: Li

first: Ilariya

last: Tarasova

first: Jan

last: Schroeder

first: Jeremy

last: Crawford

first: Paul

middle: G

last: Thomas

first: Katherine

last: Kedzierska

first: Thi

last: Nguyen


title: Wnt inhibition activates Interferon Stimulated Gene expression by alleviating epigenetic repression of endogenous retroviruses

city: Tarrytown

first: Varenka

last: Rodriguez DiBlasi

address: 777 Old Saw Mill River Road

institute: Regeneron Pharmaceuticals Inc

summary: As the use of immune checkpoint inhibitors for the treatment of cancer has expanded, convincing data have emerged correlating active WNT signaling with resistance to immunotherapies. To identify mechanisms through which WNT signaling limits anti-tumor immunity, we examined the response to WNT inhibition in a variety of human cancer cell lines that harbor distinct WNT pathway mutations. Our data show that inhibition of WNT signaling leads to activation of the TBK1/IRF3 dsRNA-sensing pathway and expression of interferon-stimulated genes (ISGs), independently of IFNa/JAK/STAT signaling. Mechanistically, we show that WNT inhibition leads to increased chromatin accessibility at genomic loci harboring endogenous retroviruses (ERVs), resulting in ERV re-expression and activation of the dsRNA response. Increased ISG expression following WNT inhibition does not involve decreased MAP kinase signaling and therefore differs from reports documenting ISG induction in response to inhibition of other oncogenic pathways. Given the variety of tumor cell lines and WNT pathway mutations examined, these data suggest a mechanism by which WNT may drive immune evasion and several therapeutic avenues to reverse it, including tumor- targeted type 1 interferon stimulation and/or epigenetic therapies.

overall_design: We examined the response to WNT inhibition in a variety of human cancer cell lines that harbor distinct WNT pathway mutations. We performed Assay for Transposase-Accessible Chromatin with Sequencing (ATAC-Seq) on HPAF-II cells treated with Lgk974 for 24 and 48 hours to identify alterations in chromatin structure.

first: Courtney

last: Williams

first: Jessica

last: Harper Calderon

first: Samer

last: Nuwayhid

first: Hannah

last: Cevasco

first: Jinrui

last: Liu

first: Wei

last: Wang

first: Rekha

last: Soni

first: Christina

last: Adler

first: David

last: D’Ambrosio

first: Namita

middle: T

last: Gupta

first: Christopher

last: Daly

first: John

last: Lin


title: Two LEAFY homologues regulate floral patterning and development without affecting flowering time in kiwifruit

city: Auckland

first: Yongyan

last: Peng

state: Auckland

address: 120 Mt Albert Road

department: Kiwifruit New Cultivars

country: New Zealand

institute: Plant and Food Research

summary: Actinidia chinensis (kiwifruit) are economically important fruit-bearing woody perennial vines, but with a long juvenile phase which slows plant breeding efforts. LEAFY (LFY) encodes a plant specific transcription factor central to the reproductive pathway in many plants, and its overexpression can accelerate flowering including in some trees. We identified two kiwifruit LFY (AcLFY) genes, expressed in actively-growing apical buds. Overexpression of AcLFY1 or AcLFY2 promoted flowering in Arabidopsis, but did not accelerate flowering in kiwifruit. Instead, branching at the lower nodes and changes in leaf morphology were observed. CRISPR-Cas9 targeted mutagenesis of AcLFY1 and/or AcLFY2 in a fast-flowering hermaphrodite kiwifruit background generated single and double mutants. The mutants flowered at the same time as control lines. Single mutants developed normal flowers, but double knockout mutations had severe effects on floral patterning and floral organ development. Petals and stamens were strongly affected, impacting on self-pollination, fruit size and shape. RNA-seq showed the two AcLFY genes were differentially regulated by the expression of a floral repressor CENTRORADIALIS gene AcCEN4. These results suggest that the AcLFY genes control plant architecture and contribute together to floral organ development and floral patterning which has implications for successful pollination and fruit set in kiwifruit.

overall_design: RNA-seq profiling of kiwifruit shoot apices over-exprssing the floral repressor AcCEN4 (pHEX2:AcCEN4-6xHA) comapred to the empty vector kiwifruit control. RNA-seq profilling of fast flowering kiwifruit shoot apices (floral repressor AcCEN and/or AcCEN4 CRISPR-Cas9 gene edited) compared to the wild type kiwifruit shoot apices.

first: Charlotte

last: Voogd

first: Cecilia

last: Deng

first: Mikaela

last: Douglas

first: Bo

last: Yang

first: Tianchi

first: Andrew

last: Allan

first: Erika

last: Varkonyi-Gasic

first: Joanna

last: Putterill


title: Single-nucleus RNA-seq analysis of extravillous trophoblast differentiation from human trophoblast organoids cultured under simulated microgravity

city: Sendai

first: Shun

last: Shibata

address: 2-1 Seiryo-chou

department: Graduate School of Medicine

institute: Tohoku University

summary: To investigate extravillous trophoblast (EVT) differentiation mechanisms, we generated human trophoblast organoids (hTOr) from human trophoblast stem cells (hTSCs) and analyzed them by single-nucleus RNA sequencing. Organoids were initially cultured under simulated microgravity using a ClinoStar bioreactor for 11 days to establish proper trophoblast architecture, then embedded in Matrigel and cultured in EVT differentiation medium for 14 days. This study examines the cellular composition and differentiation trajectories during EVT specification, providing insights into human placental development and trophoblast lineage decisions. Two independent hTSC lines (CT27 and CT29) were used as biological replicates to ensure reproducibility and capture potential line-specific variations in EVT differentiation capacity.asts.

overall_design: Single-nucleus RNA sequencing comparison of EVT-differentiated human trophoblast organoids derived from two independent hTSC lines (CT27 and CT29). Organoids were first cultured under simulated microgravity for 11 days, then embedded in Matrigel and cultured in EVT differentiation medium for 14 days. Single-nucleus libraries were prepared using 10x Genomics Chromium Single Cell 3′ v3.1 platform and sequenced on Illumina NovaSeq X Plus

first: Yuki

last: Ishikawa

first: Takahiro

last: Arima


title: Single-nucleus multiome ATAC-seq and RNA-seq analysis of GCM1 knockout in rotational human tropholast organoids

summary: Single nuclei were isolated from organoids to investigate the effects of GCM1 knockout and AAVS1 knockout (as control) on chromatin accessibility and gene expression profiles. Approximately 10,000 nuclei per sample were processed using 10x Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit, enabling simultaneous profiling of chromatin accessibility and gene expression from individual nuclei. Raw sequencing data were processed using Cell Ranger ARC to generate filtered feature-barcode matrices. Data analysis was performed in R (v4.3+) using Seurat (v4) and Signac packages.

overall_design: Comparison of GCM1 knockout and AAVS1 knockout control organoids using single-nucleus multiome sequencing. Single nuclei isolation followed by simultaneous scRNA-seq and scATAC-seq using 10x Genomics platform.

first: Hiroaki

last: Okae


title: Single-nucleus RNA-seq analysis of YAP5SA-induced human trophoblast organoids cultured under simulated microgravity

summary: To investigate the role of YAP activation in human trophoblast differentiation, we generated doxycycline-inducible YAP5SA (constitutively active YAP) expressing human trophoblast stem cells and analyzed them by single-nucleus RNA sequencing. Control and YAP5SA-induced trophoblast organoids were cultured under rotational conditions using simulated microgravity for 15 days. YAP activation resulted in substantial reduction of syncytiotrophoblast population and enhanced expression of cytotrophoblast stemness-associated genes, demonstrating YAP's role as a biomechanical integrator orchestrating trophoblast lineage allocation.

overall_design: Single-nucleus RNA sequencing comparison of doxycycline-treated versus control human trophoblast organoids at day 15. Organoids derived from YAP5SA-inducible hTSCs (CT27 line) were cultured under simulated microgravity using ClinoStar bioreactor. YAP5SA expression was induced with 100 ng/ml doxycycline treatment during rotational culture. Single-nucleus libraries were prepared using 10x Genomics Chromium platform and sequenced on Illumina NovaSeq X Plus


title: Intrinsically disordered regions restrain genomic targeting of RNA and histone demethylases in mammals and plants [additional data]

city: Chicago

first: Long

last: Zhao

state: Il

address: 929 E 57th Street

department: Department of Chemistry

institute: University of Chicago

summary: Intrinsically disordered regions (IDRs) are well-known facilitators of chromatin regulation, but their role as regulatory restraints remains underexplored. Here, we reveal that the C-terminal IDR of mammalian ALKBH5 anchors its main activity to mRNA, limiting chromatin engagement in mouse embryonic stem cells (mESCs). IDR deletion redirects ALKBH5 to chromatin-associated non-coding RNAs, driving widespread chromatin opening and transcriptional activation. This mechanism extends to histone demethylases, where IDR deletion also results in broad removal of repressive histone marks, leading to increased chromatin accessibility and activation of transposable elements. Leveraging these insights, we engineered ALKBH5 and its plant homologs with IDR deletion. Overexpression of these IDR-deleted ALKBH5 demethylases could promote Arabidopsis root growth and increase rice yield. Our findings uncover a conserved role for IDRs in restraining chromatin access by RNA and histone demethylases and establish a new strategy of chromatin programming for crop engineering using plant proteins.

sample_organism:
Arabidopsis thaliana
Mus musculus

platform_organism:
Arabidopsis thaliana
Mus musculus

overall_design: Experiment Description: This dataset contains RNA-binding profiles obtained through eCLIP-seq for endogenous full-length mALKBH5 in WT mESCs and mALKBH5-ΔcIDR in Alkbh5-ΔcIDR mESCs; caRNA MeRIP-seq and ATAC-seq data in WT, Alkbh5-/-, and Alkbh5-ΔcIDR mESCs; H3K4me3 and H3K27ac CUT&Tag, as well as H3K9me3 CUT&RUN in WT and Alkbh5-ΔcIDR mESCs; caRNA-seq, HA-tag CUT&RUN, H3K27me3 CUT&Tag and H3K9me3 CUT&RUN data for mESCs ectopically expressing full-length (FL) and ΔIDR variants of KDM4A, KDM4B, KDM6A, and KDM6B; caRNA MeRIP-seq in Col-0, full-length hALKBH5, and hALKBH5-ΔcIDR transgenic Arabidopsis thaliana lines, along with their respective inactive mutant lines; ATAC-seq, H3K4me3 and H3K27ac CUT&Tag, Flag-tag CUT&RUN in Col-0, full-length hALKBH5, and hALKBH5-ΔcIDR transgenic Arabidopsis thaliana lines.

first: Liudan

last: Jiang

first: Chuan

last: He


title: Cohesin cofactor dosage sets the rate of loop extrusion, rendering genome folding tunable yet vulnerable to genetic disruption

first: Elphege

last: Nora

address: 555 Mission Bay Boulevard South

department: Cardiovascular Research Institute

summary: Genome folding is not static, but emerges from dynamic processes that control transcription, replication, recombination, and repair. DNA loop extrusion by cohesin is central to genome organization, yet it remains unclear how cells can tune extrusion kinetics to achieve precise and functional chromosome folding patterns. Here we show that extrusion rate acts as a tunable biophysical parameter in cells, quantitatively dialed by the respective dosage of the cohesin cofactors NIPBL and PDS5. Modulation of extrusion rate can offset changes in cohesin lifetime to buffer steady-state chromosome structure and transcriptional states, even in the face of abnormal extrusion dynamics. These findings provide a long-sought mechanistic basis for the genetic interactions between cohesin cofactors and for the molecular origin of haploinsufficiency in cohesinopathies, such as Cornelia de Lange syndrome.

overall_design: Stranded mRNA-seq in mouse embryonic stem cell lines. Cells harbor an auxin-inducible degron or dTAG-inducible (FKBP12) degron on endogenous PDS5A, PDS5B, WAPL, NIPBL alleles and were treated with  dTAG/IAA (PDS5A or PDS5B or WAPL or NIPBL depletion). WT parental cells are E14Tg2a (129 X,Y background).

first: Shah

last: Rini

first: Nora

last: Elphege


title: Female-specific Cx3cr1-driven regulation of ALS and Alzheimer’s risk genes in tauopathy

first: Ari

last: Sudwarts

address: 4001 E Fletcher Ave

institute: USF

summary: By introducing haploinsufficiency of Cx3cr1 in the P301S (PS19) transgenic model of tau pathology, we report remarkable transcriptional changes, including crucial amyotrophic lateral sclerosis and Alzheimer’s disease risk genes, several of which showed co-expression, suggesting gene–gene interactions among these genetic risk factors.

overall_design: Bulk RNAseq profiling of brain tissue from non-transgenic, PS19 and PS19;Cx3cr1+/- mice. Mice were approximately 8.5-months. Both male and female samples (total = 28x samples).

first: Gopal

last: Thinakaran


title: Exposed phosphatidylserine is a non-classical inhibitory molecule during CD8 T cell exhaustion

first: Christopher

last: Medina

overall_design: LCMV Cl. 13 chronically infected mice were treated with control IgG or mch1N11 PS-targeting antibodies every 3 days at 200ug per injection IP for two weeks. Spleen were isolated post-treatment and PD-1+ Tim3- CD73+ Stem-like, PD-1+ Tim3+ CD101- Transitory, and PD-1+ Tim3+ CD101+ TD cells were isolated from respective groups and RNAsequencing was perfromed.

first: Rafi

last: Ahmed


title: ACSS2 plays a role in mediating lasting transcriptomic dysregulation in the dorsal hippocampus and cerebellar vermis of mice chronically prenatally exposed to alcohol

city: St. Louis

first: Gabor

last: Egervari

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

state: MO

address: 4515 McKinley Ave, Couch Building

department: Genetics, Biochemistry and Molecular Biophysics

institute: Washington University School of Medicine

summary: BACKGROUND: Recent findings showed that alcohol-derived acetate contributes to ACSS2-dependent histone acetylation in the brain, and this metabolic-epigenetic mechanism regulates alcohol-associated learning. Further, alcohol-derived acetate is incorporated into histone acetylation in gestating fetal brains following acute maternal alcohol exposure in late pregnancy (E18.5). GOAL: Determine any role of ACSS2 in mediating chronic prenatal alcohol exposure (cPAE)-driven lasting transcriptomic effects in the brain. APPROACH: We used RNA sequencing to investigate the lasting cPAE-related transcriptomic effects in the dorsal hippocampus (dHPC) and cerebellar vermis of adolescent WT and ACSS2 KO mice. These two brain regions were selected because of their implications in hallmark FASD phenotypes (long-term memory impairments and motor deficits, respectively). The chronic prenatal exposure model was a daily i.p. injection (3 g/kg ethanol or equivalent volume saline) to pregnant dams from E0.5 until pups were born. FINDINGS: cPAE resulted in lasting gene and transcript expression changes in both of these brain regions of both genotypes compared to respective saline (cPSE)-treated controls. There were few enriched biological processes, indicating cPAE has widespread lasting transcriptomic effects across a variety of pathways. cPAE-related differentially expressed features (DEFs: DEGs and DETs) were nearly entirely specific to genotype, evidenced by very few DEFs overlapping between genotypes. All but one of these overlapping DEFs were oppositely affected between genotypes. Further, there were hundreds of DEFs exhibiting significant interaction between genotype and prenatal exposure. Taken together, these findings suggest that lasting cPAE-related transcriptomic dysregulation is in part modulated by ACSS2.

overall_design: RNA sequencing of the dorsal hippocampus [dHPC] and cerebellar vermis [Vms] collected from WT and ACSS2 KO mice that were subjected to chronic prenatal alcohol exposure [cPAE] or chronic prenatal saline exposure [cPSE] (control). For each genotype, samples were extracted from 4 biological replicates (mice; 2 females [F] and 2 males [M]) per prenatal exposure condition.

first: Kala

last: Dodson

first: Erica

last: Periandri

first: Anjali

last: Yadav

first: Mariana

last: Lopes

first: Amelia

last: Barfield

first: Anjola

last: Ola

first: Francisca

middle: N

last: Vitorino

first: Cory

last: Cearlock

first: Benjamin

last: Garcia

first: Cheryl

last: Hill

first: Susan

middle: E

last: Maloney


title: Sequence-specific RNA recognition drives Restrictor-mediated termination of extragenic transcription [TTseq]

city: Milano

first: Gaurav

last: Mandana

address: Via Adamello 16

country: Italy

institute: European Institute of Oncology

summary: Mammalian genomes harbour hundreds of thousands of RNA Polymerase II (Pol II) landing pads, enhancers and promoters, from which transcription can initiate bidirectionally. Nevertheless, processive transcription is largely restricted to the small gene-containing fraction of the genome. A highly conserved and essential metazoan complex, Restrictor, composed of WDR82 and ZC3H4, restrains processive Pol II activity at thousands of extragenic transcription units, thus representing a critical enforcer of genome utilization. However, because of the widespread recruitment of Restrictor to both genic and non-genic transcription sites, the identification of the mechanistic basis for its exquisite selectivity for extragenic transcription has remained elusive thus far. Here, we show that while WDR82 tethers Restrictor to sites of transcription initiation, the three C3H1-type zinc fingers of ZC3H4 make sequence-specific interactions with short motifs selectively enriched at the 5’ end of extragenic transcripts, with such interactions being required for transcription termination. Hence, while Restrictor recruitment relies on WDR82-dependent tethering to the initiating Pol II, its selectivity mainly arises from sequence-specific RNA recognition.

overall_design: Nascent transcriptomes were analyzed in multiple experimental conditions to characterize Restrictor-mediated transcriptional regulation of extragenic transcription

first: Danilo

last: Polizzese

last: Madappa Mandana

first: Viviana

last: Piccolo

first: Marta

last: Russo

first: Sara

last: Polletti

first: Elena

last: Prosperini

first: Gioacchino

last: Natoli


title: Sequence-specific RNA recognition drives Restrictor-mediated termination of extragenic transcription [eCLIP]

overall_design: eCLIP was carried out in the indicated experimental conditions in a HCT-116 cell line engineered to endogenously modify ZC3H4 with the addition of a Flag-Tag. Endogenous ZC3H4 was precipitated using an anti-FLAG antibody while HA-ZC3H4 expressed from lentiviral vectors was immunoprecipitated by anti-HA.


title: SadB is a modulator of Pseudomonas aeruginosa PAO1 virulence

city: Nottingham

first: Maria

last: Papangeli

address: University Park

institute: University of Nottingham

summary: Pseudomonas aeruginosa sadB mutants exhibit a hyper-swarming but biofilm defective phenotype. To acquire insights into the contribution of sadB to virulence, we employed bioluminescence imaging and histopathology of mouse soft tissue infection. Compared with the parent PAO1 strain, the ΔsadB mutant was highly attenuated and rapidly cleared from the infection site whereas genetic complementation conferring constitutive expression of sadB resulted in a more invasive phenotype than the parent strain. Transcriptome analysis via RNA-seq of exponential and stationary phase planktonic cells revealed that SadB has a more global role than previously appreciated and acts as a pleiotropic regulator of diverse genes involved in biofilm development, quorum sensing (QS), secondary metabolite production, iron acquisition, virulence, protein secretion and anaerobiosis. In ΔsadB, we observed early induction of the rhl and pqs QS systems, increased production of siderophores and pyocyanin, changes in the expression of genes involved in c-diGMP signalling and a growth defect in under static but not shaking conditions. Since ΔsadB is a hyperswarmer and as swarming requires rhamnolipids, this suggested that deletion of sadB may impact on the timing and level of rhamnolipids produced. In ΔsadB, the rhlA and rhlB genes were induced in log rather than stationary phase resulting in overproduction of rhamnolipids likely as a consequence of the up-regulation of  the rhl and pqs QS systems. Since rhamnolipids also contribute to biofilm maturation and dispersal and can act as anti-adhesives, we deleted the rhlA in ΔsadB and observed that biofilm formation was restored offering mechanistic insight into the biofilm defective phenotype of ΔsadB.

overall_design: RNA-seq profiling of the wild-type PAO1W, the sadB knockout derivative and the overexpressing sadB strain

first: Jeni

last: Luckett

first: Jean-Frederic

last: Dubern

first: Stephan

last: Heeb

first: Morgan R.

last: Alexander

first: Paul

last: Williams


title: Repression of a transcription factor motif cluster within a Tandem Repeat is Required for Immune Homeostasis

city: Vienna

first: Moritz

last: Gaidt

address: Campus-Vienna-Biocenter 1

country: Austria

institute: IMP

summary: The innate immune antiviral protein MORC3 restricts transcription of DNA viruses and is frequently inhibited by viruses. To counteract viral antagonism, MORC3 represses type I interferon (IFN), causing viral inhibition of MORC3 to trigger (>10,000-fold) IFN induction. How MORC3 specifically represses IFN and why IFN induction upon MORC3 loss is so potent without canonical IRF/STAT factors is unknown. Here, we show that MORC3 represses a novel class of bifunctional enhancer DNA elements composed of tandem repeats densely populated with up to 80 transcription factor (TF) motifs. At steady state, the high motif density allows TFs to recruit SUMO and MORC3 inducing repression. Disruption of SUMOylation or MORC3 converts these elements into potent enhancers. Specifically, the human IFN-inducing tandem repeat contains approximately 35 PU.1 binding sites, and PU.1 drives IRF/STAT-independent IFN induction upon MORC3 loss. Our findings uncover a TF motif cluster-driven repression mechanism by MORC3 and SUMO at tandem repeats, which is utilized by the innate immune system to specifically repress IFN and potently activate immune defenses upon viral antagonism of MORC3.

overall_design: The goal of this study was to identify the regulatory mechanism behind the repression and activation of the MORC3-regulated tandem repeat element

first: Luisa

last: Krumwiede

first: Erika

last: Valeri

first: Karina

last: Schindler-Schumitsch

first: Monika

last: Bazyl

first: Johanna

last: Schiedlbauer

first: David

last: Hollaus

first: Nanette

last: Becht

first: Markus

last: Jaritz

first: Jacob

last: Schreiber

first: Bernardo

middle: P

last: de Almeida

first: Siegfried

last: Schloissnig

first: Alexander

last: Stark

first: Dara

middle: L

last: Burdette


title: Sequence-specific RNA recognition drives Restrictor-mediated termination of extragenic transcription [HCT116_4sU-RNAseq]

first: Gaurav

last: Mandana

overall_design: 4sU-RNA seq in a HCT-116 human cell line genetically modified to introduce the AID degron at the C-terminus of ZC3H4. Data were generated with or without Auxin-induced depletion of ZC3H4.

first: Danilo

last: Polizzese

last: Madappa Mandana

first: Viviana

last: Piccolo

first: Marta

last: Russo

first: Sara

last: Polletti

first: Elena

last: Prosperini

first: Gioacchino

last: Natoli


title: Single-nucleus RNA sequencing of human trophoblast-mesoderm assembloids

first: Shun

last: Shibata

summary: Human placental and extraembryonic development involves complex interactions between trophoblast and mesoderm lineages. Here, we performed single-nucleus RNA sequencing of day 11 and day 23 trophoblast-mesoderm assembloids generated by co-culturing human trophoblast stem cells (hTSCs) with extraembryonic mesoderm-primed (ExM) cells under rotational culture conditions. Our analysis reveals distinct cellular populations including cytotrophoblasts, syncytiotrophoblasts, extravillous trophoblasts, and mesodermal derivatives, providing insights into lineage specification and cell-cell interactions during early human development.

overall_design: Single-nucleus RNA sequencing of day 11 and day 23 trophoblast-mesoderm assembloids generated by co-culturing hTSCs with ExM-primed hESCs in ClinoStar bioreactor under rotational culture conditions. Libraries were prepared using 10x Genomics Chromium Single Cell 3' v3.1 with dual indexing and sequenced on NovaSeq X Plus. Raw data processed using Cell Ranger v8.0.1.

first: Yuki

last: Ishikawa


title: Re-analysis of PRO-seq data from HCT116 cells (GSE129501)

summary: This submission provides processed PRO-seq coverage tracks (bigWig files) from the re-analysis of public data (GSE129501, samples GSM3714462 and GSM3714463) from HCT116 human colorectal carcinoma cells. Data were aligned to the hg38 reference genome (GENCODE release 33; https://www.gencodegenes.org/human/release_33.html)  and normalized using bamCoverage (PMID: 27079975). These processed files are intended for visualization and downstream analysis of nascent transcription.

relation:
Reanalysis of: GSM3714462
Reanalysis of: GSM3714463
SubSeries of: GSE300453

overall_design:
Publicly available PRO-seq datasets from wild-type HCT116 cells (GSE129501) were downloaded and re-processed to generate normalized genome-wide coverage tracks (bigWig) on the hg38 reference genome.

***************************************************************
The table below lists GEO accessions reused/reanalyzed for this study.
***************************************************************


title: Sequence-specific RNA recognition drives Restrictor-mediated termination of extragenic transcription [RBNS]

overall_design: To investigate the binding specificity of ZC3H4, we conducted RNA Bind-n-Seq (RBNS) experiments using either a ZC3H4 fragment including the three C3H1 zinc fingers or WDR82-ZC3H4 in complex.


title: Re-analysis of PRO-cap data from HCT116 cells (GSE219977)

summary: This submission provides processed PRO-cap coverage tracks (bigWig files) from the re-analysis of public data (GSE219977, samples GSM6783554 and GSM6783555) from Homo sapiens HCT116 genetically modified (insertion) using CRISPR inserting O. sativa LOC4335696, (insertion) using CRISPR targeting H. sapiens SUPT16H. Data were aligned to the hg38 reference genome (GENCODE release 33; https://www.gencodegenes.org/human/release_33.html)  and normalized using bamCoverage (PMID: 27079975). These processed files are intended for visualization and downstream analysis of nascent transcription.

relation:
Reanalysis of: GSM6783554
Reanalysis of: GSM6783555
SubSeries of: GSE300453

overall_design:
Publicly available PRO-cap datasets from wild-type HCT116 cells (GSE219977) were downloaded and re-processed to generate normalized genome-wide coverage tracks (bigWig) on the hg38 reference genome.

***************************************************************
The table below lists GEO accessions reused/reanalyzed for this study.
***************************************************************


title: Immune Dynamics in Palmoplantar Pustulosis Unveiled by Single-Cell and High-Resolution Spatial Transcriptomics [Spatial Transcriptomics]

city: Seoul

first: Hanjae

last: Lee

address: 103, Daehak-ro, Jongno-gu

institute: Seoul National University

summary: Palmoplantar pustulosis (PPP) is a chronic and relapsing inflammatory skin disorder marked by the appearance of sterile pustules on the palms and/or soles, with an underlying pathogenesis that remains only partially understood. In this study, we mapped the immune landscape underlying PPP progression using single-cell RNA sequencing (scRNA-seq) integrated with high-resolution spatial transcriptomics (ST). Keratinocytes and fibroblasts in PPP showed enhanced activation of the JAK–STAT signaling pathway, alongside a progressive accumulation of myeloid dendritic cells and Th17 cells across disease stages—from healthy control (HC) to non-pustular lesion (NPL), and then to pustular lesion (PL). Cell–cell interaction analysis revealed CCL19–CCR7 signaling between fibroblastic reticular cell (FRC)-like fibroblasts and LAMP3⁺/CCR7⁺ migratory dendritic cells, as well as CCL22/CCL17–CCR4 signaling between these dendritic cells and CD4⁺ T cells, including Th17 subsets. These interactions were spatially validated, with dendritic cells localized both within pustules and in the upper dermis. Within the CCL19⁺ dermal compartment, activated antigen-presenting cells and lymphocytes coalesced into a lymphoid-like immune structure. Moreover, pustular and surrounding keratinocytes emerged as key sources of CXCL1/6/8–ACKR1 signaling, promoting neutrophil infiltration via endothelial engagement. These coordinated cellular interactions contribute to the establishment of a spatially organized and densely populated immune niche during PPP progression. Lastly, transcriptome-based drug prediction analysis highlighted JAK inhibitors and phosphodiesterase-4 (PDE4) inhibitors as promising therapeutic candidates capable of targeting multiple components of PPP pathology. Overall, this work provides an integrative view of immune dynamics in PPP and offers potential avenues for more targeted and effective therapies.

overall_design: Spatial sequencing libraries were generated using the Visium HD Human Transcriptome Kit (10x Genomics, 1000675) according to the manufacturer’s instructions at Macrogen Inc. (Seoul, Korea). FFPE tissue blocks with DV200 values greater than 30% were selected for library preparation, and 5 μm sections were used.


title: An aerosolised dual-action Autotaxin inhibitor-PPARγ agonist for the treatment of pulmonary fibrosis.

city: Athens

first: Vasilis

last: Aidinis

state: Vari

address: Fleming 34

country: Greece

institute: Institute for Fundamental Biomedical Research, BSRC Alexander Fleming

summary: Fibrosis is a significant mortality factor and health concern, promoting organ malfunction as well as immune and chemical resistance. Among the different fibroproliferative diseases, idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic interstitial lung disease (ILD) with limited therapeutic options. Autotaxin, a novel therapeutic target in IPF, is a secreted lysophospholipase D catalysing the extracellular production of lysophosphatidic acid (LPA), a growth-factor-like signalling phospholipid. The pathologic effects of LPA in the lung include the suppression of peroxisome proliferator-activated receptor γ (PPARγ), a therapeutic target in metabolic disorders, frequent comorbidities of IPF associated with unfavourable prognosis. In this report, we introduce EL244, the first-in-class dual ATX inhibitor and PPARγ agonist endowed with drug-like properties. Developed through chemoinformatic repositioning, innovative rational design, synthesis and pharmacological evaluation, EL244 exhibited favourable ADMET and PK/PD profiles. Remarkably, EL244 inhalation, which alleviates systemic toxicity concerns, decreased pulmonary LPA levels and related effects in pulmonary cells, and attenuated bleomycin (BLM)-induced pulmonary fibrosis, restoring respiratory functions. Therefore, EL244 emerges as a promising investigational new drug candidate for the inhaled treatment of IPF and ILDs.

overall_design: RNA-seq profiling of whole mouse lungs from animals treated with the fibrotic factor bleomycin or bleomycin and the atx inhibitor / pparg agonist EL244. Control animals were treated with saline. EL244 was administered via inhalation, control groups received vehicle.

first: Alexios

last: Matralis

first: Elli-Anna

last: Stylianaki

first: Eleni

last: Ladopoulou

first: Paraskevi

last: Kanellopoulou

first: Dimitris

last: Nastos

first: Stefanos

last: Smyrniotis

first: Alexandros

last: Galaras

first: Konstantinos

last: Papavasileiou

first: Sabine

last: Willems

first: Skarlatos

last: Dedos

first: Juan Pablo

last: Rincon Pabon

first: Pantelis

last: Hatzis

first: Eleanna

last: Kaffe

first: Christiana

last: Magkrioti

first: Daniel

last: Merck

first: Argyris

last: Politis

first: Antreas

last: Afantitis

first: Vassilis


title: Bulk RNA-seq of Genetically Diverse Triple-Negative Breast Cancer Mouse Models Treated with Immunotherapy, Chemotherapy, Erlotinib, or Trametinib

city: Chapel Hill

first: Office of Genomics Research

last: UNC Lineberger Comprehensive Cancer Center

state: NC

address: 125 Mason Farm Road, CB #7599 5105-5106 Marsico Hall

institute: University of North Carolina at Chapel Hill

summary: To investigate the molecular determinants of response to therapy, we profiled 26 genetically diverse murine models of mammary cancer using bulk RNA sequencing. Tumors were harvested from mice that were either untreated or treated with immunotherapy (anti-PD-1 and anti-CTLA-4), chemotherapy (Carboplatin + Paclitaxel), EGFR inhibition (Erlotinib), or MEK inhibition (Trametinib) for 7 days. Each model represents a distinct transcriptional subtype and genetic background, enabling the assessment of intertumoral heterogeneity in treatment response. This dataset includes RNA-seq from 2-9 biological replicates per model per condition. Tumor samples were collected prior to or during treatment response assessment, and are accompanied by corresponding survival from similarly-treated tumors. All sequencing was performed on polyA-selected RNA using Illumina platforms.

overall_design: Gene expression of 309 individual mouse mammary tumors from mRNAseq data, either untreated or treated. The tumor models included in this submission are: 2153, 2208, 2224, 2225, 2250, 2336, 4T1, 9263, C3-TAG, E0771, K14-TRT, KPB1-A, KPB1-B, KPB25L, KPB25L-APOBEC, KPB25L-UV, MMTV-Neu, MMTV-PyMT, p53RB-426, p53RB-624, p53RB-626, T11, T11-APOBEC, and T12.

first: Charles

last: Perou