title: HnRNPA2B1 tunes antimycobacterial immune responses in macrophages through alternative splicing of Irgm1

city: Nashville

first: Kristin

last: Patrick

state: Tennessee

department: Pathology, Microbiology, and Immunology (PMI)

institute: Vanderbilt University Medical Center

summary: Alternative splicing (AS) has been implicated in host responses to diverse pathogens, but our understanding of its role in Mycobacterium tuberculosis infection remains incomplete. Using RNA-sequencing and splicing-aware computational pipelines, we quantified AS in Mtb-infected bone marrow-derived macrophages (BMDMs). We found that ~5% of expressed macrophage genes exhibit one or more splicing changes at 8h post-Mtb infection, highlighting AS as a key layer of regulation in the macrophage response to intracellular pathogens. We next sought to identify RNA binding proteins that play an outsized role in shaping the macrophage transcriptome during Mtb infection. We discovered that the splicing factor hnRNPA2B1 promotes the early induction of inflammatory genes while dampening several type I interferon-stimulated genes. HnRNPA2B1 also controls alternative splicing of transcripts, notably Irgm1, a critical immunity-related GTPase. We report that the abundance of Irgm1-long, the canonical isoform, and Irgm1-short, an isoform lacking a lysosomal targeting domain is differentially regulated in response to diverse inflammatory cues. Macrophages overexpressing Irgm1-short are defective in restricting Mtb replication and accumulate LC3+ bacilli. These data highlight a key role for AS in shaping the macrophage transcriptome and pinpoint hnRNPA2B1 as a novel restriction factor in the cell-intrinsic response to Mtb.

overall_design: RNA-seq profiling of unifected and 8 hours post mycobacterium tuberculosis infected primary bone marrow derived macrophages and myeloid specifc A2B1 KO primary bone marrow derived macrophages.

first: Morgan

last: Chapman

first: Jessie

last: Huskey

first: Kaitlyn

middle: S

last: Armijo

first: Jaylen

middle: T

last: Spellman-Reliford

first: Aja

last: Coleman

first: Cory

last: Mabry

first: Stacy

last: Hahn

first: Robert

middle: O

last: Watson


title: Discovery of Competent Chromatin Regions in Human Embryonic Stem Cells [ChIP-Seq 2]

status: Public on Dec 12 2025

city: New York

first: Julian

last: Pulecio

state: NY

department: Developmental Biology

country: USA

institute: Memorial Sloan Kettering Cancer Center

type: Genome binding/occupancy profiling by high throughput sequencing

summary: It remains unclear how ESCs rapidly establish enhancer regulatory elements to activate lineage-specific genes at the earliest differentiation stages. Previous studies suggest that some tissue-specific enhancers are pre-marked in ESCs with permissive features that support their competence to induce gene expression in response to the binding of lineage transcription factors (TFs). However, the absence of functional assays to identify chromatin regions with the competence to activate gene expression upon TF binding has impeded to better define any pre-marking features and their role on the transcriptional competence of the chromatin. To address this, we leveraged a CRISPR-activation(a) system to systematically interrogate the presence of chromatin regions able to induce the expression of 5 lineage-specific genes that are inactive in human ESCs (hESCs). Multiple CRISPRa screens discovered 42 regions, which we termed Competent Chromatin Regions (CCRs), that can induce the expression of the 5 lineage genes upon CRISPRa targeting. ChIP seq analyses showed that CCRs do not represent primed or active enhancers, since they are not enriched in H3K4me1 or H3K27ac histone modifications. Instead, CCRs are characterized by the enriched occupancy of OCT4, NANOG and demethylation factors TET1 and QSER1, which prevent DNA hypermethylation. Although CCRs are exclusively found at the topologically associated domains shared with their related genes, we found they do not require an enrichment in pre-established chromatin contacts to exert their function. To assess the extent of our findings, we simulated the earliest stages of lineage differentiation by inducing the expression of the lineage TF FOXA2 in undifferentiated hESCs and we found it preferentially binds to the regions predicted as CCRs. Our results suggest that the factors enriched at CCRs facilitate the binding of lineage TFs and enable ESCs to promptly activate the expression of lineage genes. This study advances our understanding of pluripotency regulation in ESCs by identifying a novel type of regulatory regions that supports their developmental plasticity.

overall_design: 2 biological replicates for each condition (ESC and DE timepoints) and WT and TET1/QSER1 DKO cells.

first: Danwei

last: Huangfu


title: Identification of Novel Regulators of LINE-1 Expression via CRISPR/Cas9 Screening

city: Lexington

first: Liyang

last: Diao

state: MA

address: 36 Ledgelawn Ave

institute: Sprout Informatics

type: Expression profiling by high throughput sequencing

summary: Background Long Interspersed Nuclear Elements-1 (LINE-1, L1) are transposable elements that make up roughly 17% of the human genome. These elements can copy and insert themselves into new genomic locations [1]. Typically, LINE-1 is repressed in healthy tissues but may become activated in various human diseases. LINE-1 expression has been associated with aging [2–4], neurodegenerative disorders [5–7], cancer [8–10], and autoimmune diseases [11,12]. Despite the strong association between LINE-1 expression and disease, the regulatory mechanisms controlling the expression of LINE-1-encoded ORF1p and ORF2p and the link between LINE-1 activity and cancer cell survival remain poorly understood. Gaining insights into these regulatory pathways may help elucidate how LINE-1 contributes to disease pathogenesis.  Results To identify upstream regulators of LINE-1 and genes associated with LINE-1 activity-dependent lethality, we developed a dual-reporter system that simultaneously monitors the protein levels of LINE-1-encoded ORF1p and ORF2p (wild-type or catalytically inactive EN/RT mutant). Using genome-wide CRISPR/Cas9-based screens with this system, we identified candidate genes that may influence LINE-1 regulation at multiple levels, including RNA and protein expression. Alongside known factors such as the HUSH complex, the screens revealed additional genes not previously linked to LINE-1 regulation, suggesting possible new regulatory mechanisms for ORF1p and ORF2p expression. We also identified genes whose loss correlated with reduced viability in a manner dependent on LINE-1 activity. These findings collectively provide a broad resource for exploring cellular factors that may modulate LINE-1 expression and activity.  Conclusion This study provides a resource for investigating the cellular regulation of LINE-1, highlighting distinct candidate factors that may modulate ORF1p and ORF2p expression and influence LINE-1 activity-associated cytotoxicity. While functional validation of these candidate regulators remains necessary, the findings offer a foundation for future studies aimed at experimentally confirming their roles and elucidating the molecular mechanisms underlying LINE-1 regulation and its potential contributions to disease contexts.

overall_design: HCT116-Cas9 cells lacking a reporter, or stably expressing the L1 WT or L1 EN/RT mutant reporters, were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin–streptomycin, and 1 mM L-glutamine at 37 °C in a humidified incubator with 5% CO₂. Cells were harvested at 3 × 10⁶ cells per tube for RNA-seq and submitted to Azenta Life Sciences for downstream RNA isolation, library preparation, and sequencing.

first: Ozgur

last: Oksuz

first: Chong

last: Chu

first: Cedric

last: Arisdakessian

first: Dennis

last: Zaller

first: Kimberly

middle: K

last: Long

first: Heike

last: Keilhack

first: Sarah

last: Knutson


title: Discovery and Functional Characterization of Novel Non-Coding Variants in a Case of Late-Onset Familial Cerebral Cavernous Malformation [RNA-seq]

city: Cambridge

first: Gavin

middle: R

last: Schnitzler

state: Massachusetts

address: 415 Main St., Broad Institute

department: CVDi

institute: Broad Institute

summary:
Background: Cerebral Cavernous Malformation (CCM) is a severe neurovascular disease that shows autosomal dominant inheritance in families. The disease shows wide phenotypic variability, with some patients developing vascular lesions in infancy and others never experiencing symptoms. The majority of familial CCM cases are caused by mutations in KRIT1, CCM2, or PDCD10. However, 5–15% of familial cases and ~40% of sporadic cases with multiple lesions do not exhibit variants in these genes. It remains unknown whether non-coding variants or variants in novel genes contribute to genotype-negative disease or phenotypic variability.
Methods: We performed whole genome sequencing (WGS) on a family with two siblings with late-onset CCM and an unaffected third sibling. Next we identified potential causal variants that segregated with disease, were rare (allele frequency <0.001), and were either predicted loss-of-function or in CCM pathway genes. Candidate variants were assessed using in silico splice prediction tools and functional assays to evaluate their effects on protein structure and gene expression.
Results: The prioritized causal variants identified with WGS in this family were in the KRIT1 and PIK3CA genes. First we prioritized a single base pair substitution in intron 5 of KRIT1 (c.262+115A>G) predicted to affect splicing. We found the variant leads to inclusion of a novel 129-bp exon containing multiple stop codons in KRIT1 mRNA, likely causing loss of protein function via nonsense-mediated decay or generation of nonfunctional protein. This variant was absent from population databases (ClinVar) and segregated with disease in the family. A second potential disease-relevant variant was in intron 1 of PIK3CA (c.-77+1G>A). Prime editing of this variant in a human aortic endothelial cell line resulted in a 1.5-fold increase in PIK3CA expression.
Discussion: This is the first report of WGS identifying causal non-coding single base pair variants in a case of genotype-negative familial CCM. Our findings highlight the diagnostic limitations of exon-focused sequencing and support the application of WGS for variant-negative cases. In this case there is a possible contribution of two non-coding variants that have functional effects on known CCM risk genes. Expanding genetic screening to noncoding regions may improve diagnostic yield, genetic counseling, and CCM risk stratification.

first: William

middle: E

last: Schwartzman

first: Jathin

last: Rao

first: Lily

last: Widdup

first: Jiahao

last: Jiang

first: Rajat

middle: M

last: Gupta


title: Bulk RNA-seq of colonic epithelial stem cell-derived organoids from healthy epithelial HO-1 knockout and control mice exposed to hemin vs vehicle

city: Aurora

first: Joseph

last: Onyiah

state: CO

institute: University of Colorado

summary: This dataset contains RNA sequencing profiles of colonic epithelial organoids isolated from mice with intestinal epithelial-specific deletion of Hmox1 and littermate controls. Organoids were exposed to hemin for 24 hours to assess the influence of epithelial HO-1 on transcriptional responses to heme-induced stress.

overall_design: RNA-seq profiling of epithelial organoids from Hmox1fl/fl (control) and Hmox1ΔIEC (epithelial HO-1 knockout) mice that were exposed to hemin (200 µM) for 24 hours vs vehicle.


title: Metabolic adaptation of stromal cells to fatty acid drives metastasis of oral squamous cell carcinoma

city: Wuhan

first: Hui

last: Zhao

address: Luoyu Road

country: China

institute: Wuhan university

summary: Metabolic adaptation enables cancer cell survival under nutrient stress, but how stromal cells in tumor microenvironment (TME) withstand similar metabolic change remains elusive. Here, we report that fatty acids (FAs), a nutrient with dual roles in energy provision and lipotoxicity, orchestrate a pro-metastatic adaptation program in cancer-associated fibroblasts (CAFs). In patients with oral squamous cell carcinoma (OSCC), the FA transporter CD36 was upregulated not only in cancer cells but also in myofibroblastic CAFs (myoCAFs), where it correlated with more advanced metastasis stage. Among 5 kinds of FAs enriched in OSCC tissues, palmitic acid (PA) potently activated myoCAFs phenotypes across 2D, 3D, and organoid co-culture models. In vivo, PA promoted lymph node metastasis in orthotopic tumors comprising OSCC cells and CAFs, an effect abolished by CD36 knockdown in CAFs. Mechanistically, PA engaged chromatin occupancy of H3K27ac at multiple genes including IRE1 and TMBIM6, two stress-adaptive regulators. Transcription of IRE1 and TMBIM6 in CAFs was regulated by CCAAT/enhancer-binding protein γ (CEBPG) through an enhancer-dependent manner. Disruption of the CEBPG–IRE1/TMBIM6 axis attenuated myoCAFs properties and abrogated PA-driven metastasis. Our results unveil a stromal metabolic checkpoint and establish CEBPG-enhanced stress resilience as a therapeutic target to curtail metastasis.

overall_design: Live CAFs treated with PBS or 50 μM PA for 1 day

first: Yiling

last: Duan

first: Yitong

last: Li

first: Yufei

last: Wu

first: Rui

first: Xiao

last: Yang

first: Zhengjun

last: Shang


title: Pa01c cells treated with ERK and MK2 inhibitors

status: Public on Dec 13 2025

city: Saint Louis

first: Iftikhar Ali

last: Khawar

state: Missouri

address: McDonnell Medical Sciences Building, Room 502 4566 Scott Ave

department: Internal Medicine

institute: Washington University in Saint Louis

summary: We trested Pa01c cells with DMSO and, BVD-523 and ATI-450 alone or in combination for 48 hours

overall_design: Bulk RNAseq analysis of Pa01c cells after DMSO and drugs treatment

first: Kian

middle: H

last: Lim

first: Iftikhar

middle: A


title: Mycobacterial Metallophosphatase MmpE Functions as a Nucleomodulin to Regulate Host Gene Expression and Promote Intracellular Survival

first: Liu

last: Chen

address: No.1 Shizishan Street, Hongshan District

department: College of Animal Science & Technology

institute: Huazhong Agricultural University

summary: Nucleomodulins are bacterial effectors that target host cell nuclei and play crucial roles as virulence factors in mycobacterial pathogenesis. Mycobacterium tuberculosis, the causative agent of tuberculosis, presents a significant global health challenge; however, the mechanisms of nucleomodulins in Mtb remain unclear. In this study, we characterize the hypothetical protein Rv2577 (designated MmpE) as a critical nucleomodulin involved in immune evasion and intracellular persistence. We performed ChIP-seq in HEK293T cells transfected with EGFP-MmpE, identifying 2,903 potential MmpE-binding sites: 537 in intergenic regions and 1,144 in intragenic regions. Remarkably, 99.7% (2,894) of these sites were associated with protein-coding regions. Among them, 1,013 sites were located within 3 kb of transcription start sites (TSS), primarily targeting promoter regions (<1 kb: 63.18%; 1–2 kb: 22.9%; 2–3 kb: 13.92%), indicating MmpE’s role in transcription regulation. Further analysis of the 1,013 potential target genes revealed significant enrichment in protein kinase-related genes, such as PRKCB, PLCG2, and PIK3CB, involved in immune activation and inflammatory responses via the PI3K–Akt and mTOR signaling pathways. Additionally, we identified four transcription factors (CREBBP, VDR, GREB1, and FOXP4) that MmpE directly targets in their promoters. This finding points to the critical role of MmpE in shaping host immune responses and highlights its potential as a therapeutic target in combating Mycobacterium tuberculosis infections.

overall_design: This study used CUT&Tag to identify genome-wide binding sites of the MmpE protein in HEK293T cells. Cells were transfected with either MmpE-pEGFP or empty pEGFP-N1 vector as a control. After 36 hours, cells were harvested and subjected to CUT&Tag using an anti-GFP antibody to target MmpE-bound chromatin regions. Libraries were sequenced on the Illumina NovaSeq 6000 platform. Peaks were identified using MACS2, and downstream analyses including genomic annotation, GO enrichment, and motif discovery were performed using HOMER and TOMTOM.

first: Chen

last: Liu

first: Baojie

first: Qiang

first: Yifan

last: Wang

first: Yingyu

first: Lei

last: Zhang

first: Aizhen

last: Guo


title: Spatial analyses of early, untreated SSc skin identify a proinflammatory vascular niche of macrophage-fibroblast signaling

city: Lebanon

first: Helen

middle: C

last: Jarnagin

state: NH

address: WTRB 6-84 1 Medical Center Drive

institute: Geisel School of Medicine

summary: Uncovering the early interactions and spatial distribution of dermal fibroblasts and immune cells in treatment-naïve diffuse cutaneous systemic sclerosis (dcSSc) patients is critical to understanding the initiating events skin fibrosis. We generated an integrated multiomic dataset of early, treatment naïve dcSSc skin. Skin biopsies were analyzed by single-nuclei multiome sequencing (snRNA-seq and snATAC-seq) and two different paired spatial transcriptomic platforms to comprehensively describe the molecular changes in these individuals. We identified a proinflammatory niche within papillary, hypodermis, and vascular niches, that are enriched for activated myeloid cells and proinflammatory fibroblasts characterized by expression of proinflammatory cytokines. Pathway analyses showed significant enrichment of PI3K-AKT-mTOR signaling pathway expression in these cellular niches, driven by profibrotic growth factor signaling networks. Macrophage subclustering showed SSc-specific macrophage activation of IL6-JAK-STAT signaling and the enrichment of oxidative phosphorylation pathways. Ligand-receptor analysis revealed that SSc macrophages secrete PDGF and TGFB to activate the inflammatory SSc-dominant fibroblast subclusters. Spatial transcriptomic analyses showed infiltrating macrophages secreted PDGF and TGFB, modulating fibroblast activation in these niches. Multiomic data integration and spatial transcriptomic neighborhood analysis showed co-localization of fibroblasts, macrophages, and T cells around the vasculature. These data suggest that interactions between activated immune cells and proinflammatory fibroblasts around vascular niches are an early event in scleroderma pathogenesis.

overall_design: 10 samples

first: Jarnagin

last: Helen

first: Whitfield

middle: L

last: Michael


title: Integrative proteomics and transcriptomics identify novel fibrosis-related biomarkers of patients with post-HBV hepatitis cirrhosis

city: Changchun

first: Yutong

last: Dong

state: Jilin

address: NO.1 Xinmin Street

department: Hepatology Department, Center of Infectious Diseases and Pathogen Biology

institute: First Hospital of Jilin University

summary: Background: Hepatitis B virus (HBV) infection is an important public health burden. Chronic HBV infection always leads to chronic hepatitis, liver fibrosis and increases the incidence of liver cancer. However, the mechanism of liver fibrosis caused by persistent HBV infection remains unclear.  Methods: In present study, we isolated and enriched primary human hepatic stellate cells (HSCs) from chronically HBV infected patients with liver cirrhosis. The differences expression of mRNA and protein were analyzed by combining proteomics, transcriptomics, and biological network analysis.   Results: After bioinformatics analysis with transcriptomics and proteomics data, a total of 2,156 genes and 711 proteins were differently expressed between the liver cirrhosis group and control group. GO enrichment analysis and KEGG analysis indicated those differently expressed molecules were mainly involved in oxidative stress, glycometabolism, and fibrous repair. Moreover, a total of 110 overlapping biomarker were identified and two of them (PKM2 and EHD2) were validated as candidates correlated with liver fibrosis by qRT-PCR and immunofluorescence from primary human HSCs and in vitro cellular hepatic fibrosis model.  Conclusion: Our results indicated that PKM2 and EHD2 were overexpressed in chronically HBV infected patients with liver cirrhosis, suggesting them as potential biomarkers and therapeutic targets for liver fibrosis.

overall_design: Liver biopsy samples were obtained from patients who admitted to the first hospital of Jilin university between 2020 to 2021. Chronically HBV infected patients with liver cirrhosis underwent liver transplantation were defined as cirrhosis group and hepatic hemangioma partial hepatectomy were defined as control group. All HBV-infected patients were diagnosed with the criteria recommended by the Asian Pacific Association for the Study of the Liver (APASL). Primary human HSCs were isolated from resectable liver sample using an established method.Total RNA was extracted from primary human HSCs with the RNeasy Mini kit (Qiagen, Germantown, Maryland, USA) according to manufacturer’s protocol.RNAsamples were on sequenced Illumina Novaseq 6000 (Illumina, , California, USA).Protein preparation for each sample was performed as previous described.Proteomic analysis was performed by DDA mass spectrometry assay.   The microarray datasets were processed with rigorous quality control procedures to ensure the highest possible level of data quality using the R 4.2.1 and the R package of “Bioconductor”.Through the “ClusterProfiler” package in R, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to discover the biological function and significant signaling pathways correlated with liver fibrosis.To identify more key genes related to liver fibrosis, a PPI network of differently expressed molecules was established by the online tool STRING (STRING, http://string-db.org) for exploring interactions between genes or proteins.

first: Yuwei

first: Xiaomei

first: Junqi

last: Niu


title: Transcriptomic profiling of brown adipose tissue in high-fat diet-induced obese mice following Pu-erh tea extract intervention

city: Chongqing

first: Xiujuan

last: Jiang

address: Tiansheng road

institute: Southwest University

summary: Background: Obesity driven by high-fat diet (HFD) is a global public health concern, and Pu-erh tea extract (PTE) exhibits anti-obesity potential, but its regulatory mechanism on brown adipose tissue (BAT) metabolism remains unclear. Transcriptomic analysis of BAT is critical to reveal PTE-mediated metabolic remodeling. Methods: C57BL/6J mice were divided into HFD (high-fat diet) and HFD+PTE groups. BAT samples were collected after 16-week intervention for RNA sequencing. Results: Transcriptomic data showed 623 differentially expressed genes (DEGs) in HFD+PTE vs HFD groups, mainly enriched in cAMP/Calcium signaling, Biosynthesis of unsaturated fatty acids, and Glycerolipid metabolism pathways. Value: This dataset provides transcriptomic evidence for PTE regulating BAT function, complementing our previously deposited gut microbiota 16S rRNA data, and facilitating research on natural product-mediated metabolic regulation.

overall_design: 1. Study Overview This study aimed to elucidate the transcriptomic alterations in brown adipose tissue (BAT) of high-fat diet (HFD)-induced obese mice following intervention with Pu-erh tea extract (PTE), and to explore the molecular mechanisms underlying PTE-regulated metabolic remodeling of BAT. The transcriptomic data submitted here correspond to BAT samples isolated from 8 mice (4 mice per group), capturing the differential gene expression profiles associated with the anti-obesity effects of PTE.  2. Sample Preparation  Animal Model: Specific pathogen-free (SPF) male C57BL/6J mice (6 weeks old, 17±2 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. All mice were acclimatized for one week under standard conditions (22±2°C, 12-h light/dark cycle) with free access to food and water.  Grouping and Intervention: Mice were randomly assigned to two groups (n=4 per group):  HFD Group: Fed a high-fat diet (60% kcal from fat) for 16 weeks.  HFD+PTE Group: Fed the high-fat diet for 16 weeks with ad libitum access to drinking water supplemented with 0.25% (w/v) PTE. The PTE was prepared from ripened Pu-erh tea (7572, Yunnan TEATEA Group, production year 2006) via sequential hot-water extraction (1:15 and 1:10, w/v), followed by filtration, combination of filtrates, and lyophilization. The resulting extract was stored at -80°C until use.  3. Tissue Collection Following the 16-week intervention and a 12-hour fast, mice were euthanized by CO₂ inhalation. Interscapular brown adipose tissue (BAT) was rapidly dissected, rinsed briefly with ice-cold sterile saline, blotted dry, and immediately snap-frozen in liquid nitrogen. All samples were stored at -80°C until RNA extraction.  4. RNA Extraction and Quality Control Total RNA was extracted from frozen BAT samples using TRIzol® Reagent according to the manufacturer’s instructions. RNA concentration and purity were measured using an ND-2000 spectrophotometer (NanoDrop Technologies). RNA integrity was assessed with a 5300 Bioanalyzer (Agilent Technologies). Only RNA samples meeting stringent quality criteria (OD260/280 ratio of 1.8-2.2, OD260/230 ratio ≥ 2.0, RNA Quality Number (RQN) ≥ 6.5, 28S:18S ribosomal RNA ratio ≥ 1.0, and total mass > 1 μg) were proceeded to library construction.  5. Library Construction, Sequencing, and Data Processing Sequencing libraries were prepared from 1 μg of qualified total RNA using the Illumina® Stranded mRNA Prep, Ligation kit, following the manufacturer’s protocol. This included polyA selection, RNA fragmentation, double-stranded cDNA synthesis, adapter ligation, size selection (~300 bp), and PCR amplification. Library quantification was performed using Qubit 4.0. Pooled libraries were sequenced on an Illumina NovaSeq X Plus platform using a NovaSeq Reagent Kit to generate 150-bp paired-end reads. Raw reads were processed with fastp for quality control and adapter trimming. Clean reads were aligned to the GRCm39 reference genome using HISAT2. Transcript assembly and quantification (in TPM) were performed using StringTie and RSEM, respectively. Differential expression analysis was conducted with DESeq2, with significance thresholds set at |log2FC| ≥ 1 and FDR < 0.05. Alternative splicing events were analyzed using rMATS. Functional enrichment analysis (GO and KEGG) was performed on differentially expressed genes.

first: Jiang

last: Xiujuan

first: Zeng

last: Liang


title: KMT2C deficiency promotes meningioma progression through H3K27ac-mediated HIPPO Pathway Inactivation [CUT&Tag]

city: Nanjing

first: Liuchao

state: Jiangsu

address: 300 Guangzhou Road, Nanjing

institute: Nanjing medical universuty

summary: KMT2C is a frequently mutated gene in meningiomas, however, its functional role in meningioma pathogenesis remains poorly understood. Here we demonstrate that KMT2C expression is reduced in high-grade meningiomas and that its deficiency promotes tumor progression by inactivating the Hippo pathway. Mechanistically, KMT2C loss diminishes H3K27ac levels at transcription start sites (TSS) and disrupts chromatin accessibility, leading to transcriptional downregulation of NF2 and subsequent dysregulation of Hippo signaling. We further reveal that KMT2C deficiency impairs the acetyltransferase activity of CBP/EP300, thereby reducing H3K27ac deposition, while the chromatin binding of CBP/EP300 remains unaffected following KMT2C knockout. Treatment with the histone deacetylase inhibitor TSA restores H3K27ac levels and reactivates NF2 expression in KMT2C-deficient meningiomas. Notably, KMT2C loss sensitizes meningioma cells to ferroptosis, and the ferroptosis inducer erastin significantly suppresses tumor growth both in vitro and in vivo. These findings establish a foundation for developing targeted therapies for meningioma patients with KMT2C deficiency.

overall_design: By knockout the expression of KMT2C in meningioma cells, the role and mechanism of KMT2C in meningioma were explored.

first: Jing

last: Ji


title: Positional Interpretation of Cis-Regulatory Code and Nucleosome Organization with Deep Learning Models

city: Kansas City

first: Melanie

last: Weilert

state: MO

institute: Stowers Institute for Medical Research

summary: Sequence-to-function neural networks learn cis-regulatory sequence rules driving many types of genomic data. Interpreting these models to relate the sequence rules to underlying biological processes remains challenging, especially for complex genomic readouts such as MNase-seq, which maps nucleosome occupancy but is confounded by experimental bias. We introduce pairwise influence by sequence attribution (PISA), an interpretation tool that combinatorially decodes which bases contributed to the readout at a specific genomic coordinate. PISA visualizes the effects of transcription factor motifs, detects undiscovered motifs with complex contribution patterns, and reveals experimental biases. By learning the bias for MNase-seq, PISA enables unprecedented nucleosome prediction models, allowing the \emph{de novo} discovery of nucleosome-positioning motifs and their long-range chromatin effects, as well as the design of sequences with altered nucleosome configurations. These results show that PISA is a versatile tool that expands our ability to train and interpret sequence-to-function neural networks on genomics data and understand the underlying cis-regulatory code.

overall_design: Micrococcal nuclease digestion with deep sequencing (MNase-seq) experiments were conducted in Saccharomyces cerevisiae BY4741 strains across a wildtype set and a CRISPR mutant (3 point-mutations upstream of Pho5) set of replicates.

first: Charles

last: McAnany

first: Grishma

last: Mehta

first: Fahad

last: Kamulegeya

first: Jennifer

last: Gardner

first: Schreiber

last: Jacob

first: Anshul

last: Kundaje

first: Julia

last: Zeitlinger


title: tRNA-seq of DUS1L knockout and wild-type A375 human melanoma cells

city: Beijing

first: Jingkun

last: Yi

address: XueYuan Road No 38

department: School of Basic Medical Sciences

institute: Peking University

type: Non-coding RNA profiling by high throughput sequencing

summary: tRNA-seq was performed on A375 human melanoma cells with CRISPR/Cas9-mediated knockout of DUS1L and wild-type control cells, with three biological replicates per group. Total RNA was extracted using TRIzol, and ~20 μg RNA per sample was fractionated on 12% urea-PAGE to isolate 60–100 nt RNA fragments. These fragments were subjected to demethylation of m1A, m1G and m3C using an RNA Pretreatment Kit, followed by partial alkaline hydrolysis, dephosphorylation and re-phosphorylation. Small RNA libraries were constructed from 19–35 nt fragments using the NEBNext Small RNA Library Prep Set for Illumina and sequenced on an Illumina NextSeq 500 platform. Clean reads were aligned with BWA to human mature and genomic tRNA reference sequences compiled from GtRNAdb, and uniquely mapped reads were summarized to generate tRNA expression profiles for differential expression analysis between DUS1L knockout and wild-type cells.

overall_design: Six tRNA-seq libraries were generated from A375 cells: three biological replicates of wild-type cells and three biological replicates of DUS1L knockout cells. Size-selected small RNAs were used to construct NEBNext-based small RNA libraries, which were sequenced on an Illumina NextSeq 500 platform. Differential expression analysis of tRNA species between DUS1L knockout and wild-type cells was performed using DESeq2.


title: Integrating pharmacokinetics, tissue distribution, RNA sequencing, and network pharmacology identifies STAT1 and SERPINE1 as potential targets of moscatilin in treating liver diseases

city: hefei

first: Yajun

last: Chen

state: anhui

address: No.1688, Lianhua Road, Hefei 230601, Anhui, China.

institute: Hefei Normal University

summary: This study established a quantitative analysis method for moscatilin using ultrahigh-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) to investigate its pharmacokinetics and tissue distribution in Kunming mice. Additionally, RNA sequencing (RNA-seq) and network pharmacology were employed to elucidate the molecular mechanisms underlying moscatilin's therapeutic potential in liver diseases, including non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), viral hepatitis, liver injury, liver fibrosis, liver cirrhosis, and liver cancer. The analytical method was validated for selectivity, carryover, linearity, precision, accuracy, dilution integrity, matrix effect, recovery, and stability following US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) bioanalytical guidelines. Pharmacokinetic analysis revealed rapid absorption of moscatilin in vivo, with a bioavailability of 85.82%. Tissue distribution studies indicated widespread presence across various organs, with elevated concentrations in the lungs and liver. RNA-seq and network pharmacology analysis identified signal transducer and activator of transcription 1 (STAT1) and serine protease inhibitor clade E member 1 (SERPINE1) as potential targets for moscatilin in liver disease treatment.

overall_design: Total RNA extraction, cDNA library construction, and mRNA transcriptome sequencing were conducted by Guangzhou IGE Biotechnology Co., Ltd. High-throughput paired-end sequencing was executed on the Illumina NovaseqTM 600 platform. The raw sequencing data underwent filtering to remove adapter sequences, low-quality reads, and reads containing 'N'. Clean reads were then aligned to the reference genome using HISAT2 software. Differential gene expression among samples was analyzed using DESeq2 software, followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and Gene Ontology (GO) function annotation for the differentially expressed genes.

first: Jianrong

last: Zhang

first: Weizhu

last: Wang

first: Minrui

first: Yaping

first: Yuanhao

first: Tingxuan

last: Zhou

first: Xuan

last: Lu

first: Jianli

first: Wei


title: Discovery of Competent Chromatin Regions in Human Embryonic Stem Cells [ATAC-seq]

first: Julian

last: Pulecio

first: Danwei

last: Huangfu


title: RNA-seq of DUS1L knockout and wild-type A375 human melanoma cells

summary: Transcriptome sequencing was performed on A375 human melanoma cells with CRISPR/Cas9-mediated knockout of DUS1L and wild-type control cells, with three biological replicates per group. Total RNA was extracted using TRIzol reagent, and RNA quality was assessed by NanoDrop spectrophotometer and Agilent 2100 Bioanalyzer. Indexed RNA-seq libraries were constructed and sequenced on the BGISEQ-500 platform to generate paired-end reads. Clean reads were aligned to the human reference genome (hg38), and gene-level read counts and differential expression profiles were obtained to investigate the impact of DUS1L knockout on global gene expression in A375 cells.

overall_design: Six RNA-seq libraries were generated from A375 cells: three biological replicates of wild-type cells (WT1–WT3) and three biological replicates of DUS1L knockout cells (DUS1LKO1–DUS1LKO3). Paired-end RNA-seq was performed on a BGISEQ-500 platform, and differential expression analysis between DUS1L knockout and wild-type cells was conducted.


title: tRF-seq of DUS1L knockout and wild-type A375 human melanoma cells

summary: Small RNA sequencing (tRF-seq) was performed on A375 human melanoma cells with CRISPR/Cas9-mediated knockout of DUS1L and wild-type control cells, with three biological replicates per group. Small RNA libraries were prepared using the NEBNext Small RNA Library Prep Set for Illumina and sequenced on an Illumina NextSeq 500 platform to generate single-end FASTQ files. Sequencing quality was assessed using FastQC, and 3' adaptor sequences were trimmed with cutadapt. Clean reads were aligned to the human tRNA genome using MINTmap to obtain read count-based expression profiles of tRNA-derived fragments (tRFs).

overall_design: Six small RNA-seq libraries were generated from A375 cells: three biological replicates of wild-type cells (WT1–WT3) and three biological replicates of DUS1L knockout cells (DUS1LKO1–DUS1LKO3). Small RNA libraries were prepared with the NEBNext Small RNA Library Prep Set and sequenced as single-end reads on an Illumina NextSeq 500 platform. MINTmap-based mapping to the human tRNA genome was used to quantify tRNA-derived fragments (tRFs), followed by DESeq2-based differential expression analysis between DUS1L knockout and wild-type samples.


title: Effect of cantharidin sodium treatment on gene expression in AGS and MKN1 gastric cancer cell lines (treated vs. untreated groups)

city: Gui Yang

first: Tai

last: Xie

address: No. 9 Beijing Road, Yunyan District, Guiyang City, Guizhou Province, P.R. China

institute: Guizhou Medical University

summary: Gastric cancer is a highly malignant gastrointestinal tumor, and cantharidin sodium has potential anti-tumor activity against gastric cancer cells. Here, we focus on AGS and MKN1 gastric cancer cell lines: each cell line was divided into cantharidin sodium treatment group and untreated control group, with 3 biological replicates set for each subgroup. We aim to reveal the differential gene expression profiles induced by cantharidin sodium in these two cell lines, and further clarify the molecular regulatory mechanism of cantharidin sodium in inhibiting gastric cancer cell progression through transcriptomic analysis.

overall_design: RNA-seq profiling of AGS and MKN1 gastric cancer cell lines (under cantharidin sodium treatment) and their corresponding untreated control groups, with 3 biological replicates per subgroup

first: Dan

last: Ma

first: Yi juan

first: Jun Zhao

last: Wan

first: Ming


title: Differential Roles of Type I Topoisomerases in Regulating HPV Pathogenesis

city: Chiacgo

first: Arushi

last: Vats

state: Illinois

address: 320E Superior street

department: Microbiology-Immunology

institute: Northwestern University

summary: High-risk human papillomaviruses (HPVs) activate the ATM and ATR pathways by inducing DNA breaks through the action of viral oncoproteins E6 and E7, which target factors such as topoisomerases. Type I topoisomerases cleave and relegate a single strand of DNA, and little is known about how they regulate HPV pathogenesis. The levels of type I topoisomerases, TOP1A, TOP3A, and TOP3B, were all elevated in cells maintaining high-risk HPV genomes, as well as in squamous cell carcinomas. Only TOP1A and TOP3B, but not TOP3A, bound to HPV genomes and were critical for regulating viral gene transcription and replication with little effect on cell growth. Furthermore, the knockdown of TOP1A or TOP3B reduced levels of DNA breaks and differentially altered the expression of genes in key pathways. TOP1A knockdown reduced the expression of IL6 and activation of the pro-cytokine signaling pathway. In contrast, TOP3B targeted EGR3, which regulates growth and differentiation. Finally, TOP1A and TOP3B differentially regulate the formation of R-loops, which are critical for viral replication. These findings demonstrate the differential roles of type I topoisomerases in HPV pathogenesis.

overall_design: CIN 612 cells (keratinocytes isolated from a cervical biopsy that stably maintain the HPV 31 genome) were transduced with lentivirus to express either a scrambled sequence (Samples 1/2), shRNA targeting TOP1A (Samples 3/4), or TOP3B (Samples 5/6). RNA sequencing was performed on each of these samples and the scramble control CIN 612 cells were compared to CIN 612 cells depleted with TOP1A or TOP3B

first: Conor

middle: W

last: Templeton

first: Laimonis

last: Laimins


title: Chromatin accessibility profiling of adult C. elegans in response to heat hormesis regimen

city: Ithaca

first: Siu Sylvia

last: Lee

address: 526 Campus Rd

department: Molecular Biology and Genetics

institute: Cornell University

summary: To understand the molecular basis of heat hormesis, we adapted a regimen in which C. elegans that had been cultured at 20°C were exposed to 30°C for 6 hours during early adulthood (referred to as ‘priming’), allowed to recover for 12 hours at 20°C, and then challenged with heat shock (HS) at 35°C. We applied this hormesis regimen to wild-type (N2) or germline-less glp-1(e2144) worms.

overall_design: Chromatin accessibility profiling was conducted on nuclei isolated from Naïve (N) and Primed (P) groups of glp-1(ts) mutant or wild-type (N2) worms at Timepoints 1 (after Priming), 2 (Recovery), 3.3 (upon 3-hour HS), 3.6 (upon 6-hour HS), and NC (negative control-before priming) across the heat hormesis regimen. For the glp-1 mutant, four biological replicates were included for each condition, except for Timepoint 3.6, which had two biological replicates. For N2, there were two biological replicates per condition.

first: Hsin-Yun

last: Chang

first: Siu


title: Single-nuclei multiome and spatial analyses of early, untreated SSc skin identify a proinflammatory vascular niche of macrophage-fibroblast signaling

first: Helen

last: Jarnagin

overall_design: Single nuclei RNA and ATAC sequencing and with paired spatial sequencing on 10 human scleroderma skin tissues and 4 control samples.

first: Michael

last: Whitfield


title: Single-cell RNA-seq of colonic tumors from epithelial HO-1 knockout and control mice in a colitis-associated cancer model

first: Joseph

last: Onyiah

summary: This dataset contains single-cell RNA sequencing profiles of colonic tumor cells isolated from mice with intestinal epithelial-specific deletion of Hmox1 and littermate controls. Tumors were generated using the azoxymethane-dextran sodium sulfate (AOM-DSS) model of colitis-associated cancer. Following enzymatic dissociation and live cell sorting, single-cell libraries were prepared using the 10x Genomics Chromium platform and sequenced to characterize transcriptional programs within the tumor microenvironment. The dataset was designed to examine genotype-dependent shifts in epithelial transcriptional programs within the tumor microenvironment, with a focus on stress-adaptive responses, iron metabolism, and oxidative stress regulation.

overall_design: Colonic tumors were harvested from Hmox1fl/fl (control) and Hmox1ΔIEC (epithelial HO-1 knockout) mice following AOM-DSS treatment (day 80). Tumors >2 mm were pooled by genotype, dissociated, and sorted for live cells (7AAD). Single-cell libraries were generated using the 10x Genomics Chromium platform and sequenced. Data were processed using Seurat with standard filtering, normalization, and clustering workflows.


title: Circulating transcriptome analysis detects gene signatures, immune cell activation and abundance to predict response to immunotherapy in bladder cancer

city: Dijon

first: Romain

last: Boidot

address: 1, rue Pr Marion

country: France

institute: CGFL

summary: Background. Immune Checkpoint Blockers (ICB) have revolutionized oncology by achieving durable tumor responses in advanced cancers. Nevertheless, currently approved biomarkers (PD-L1, MSI, TMB) have suboptimal positive and negative predictive values for tumor response and survival. Patients and Methods. We aimed to assess the value of RNA sequencing from circulating blood, a new type of liquid biopsy, to predict responses to ICB. We performed total paired-end RNA sequencing at 20 million reads and analyzed differential gene expression, signaling pathway activation, and immune cell abundances according to cancer outcomes. The analysis was conducted on baseline frozen whole blood samples from 164 patients prospectively enrolled in the IOPREDI study. Results. We found that some immune-related genes and signaling pathways were highly expressed in patients who achieved a durable clinical benefit. Furthermore, analyses of both progression-free survival (PFS) and overall survival (OS) confirmed significantly higher expression levels of immune-related signaling pathways in long-term survivors. Gene expression signatures capable of classifying patients based on clinical response or PFS were also identified. Interestingly, deconvolution analysis revealed a significant higher abundance of resting NK cells in patients with prolonged PFS or OS, in contrast to other cytotoxic cell types. Finally, high expression of the CST7 gene and increased abundance of naïve B lymphocytes were associated with immune-related adverse events (irAEs). Conclusion. Total RNA sequencing from whole blood provides high-quality data to predict clinical response, survival, and occurrence of irAEs. The use of this liquid biopsy prior to immune checkpoint blockage could improve treatment efficacy and irAEs management.

overall_design: Whole bloods from 164 patients with urinary tract cancer were collected on EDTA tubes. After RNA extraction, total RNAseq was performed and obtained results were used for statistical analysis with different clinical parameters such as clinical response, progression free and overall survivals, and immune related adverse events.

first: Sandy

last: Chevrier

first: Corentin

last: Richard


title: scRNA-seq of a knockout village during hPSC-derived islet differentiation

city: Washington

first: Bicna

last: Song

state: DC

address: 7144 13th Pl NW

institute: Children's National Hospital

summary: This submission consists of processed single-cell RNA-seq data of knockout village samples, provided as filtered feature–barcode matrices generated using Cell Ranger v7.1.0 with default parameters. We previously deposited the same biological samples and data processed by STAR under GEO accession GSE288587. The corresponding raw FASTQ files are available in ENA under accession ERP168459. The supplementary file (reanalysis_samples.xlsx) provides a reference table linking each sample in this submission to its corresponding GSM in GSE288587, which contains the detailed experimental descriptions of culture information, genotypes, and differentiation stages.

relation:
Reanalysis of: GSM8770302
Reanalysis of: GSM8770313
Reanalysis of: GSM8770303
Reanalysis of: GSM8770314
Reanalysis of: GSM8770304
Reanalysis of: GSM8770315
Reanalysis of: GSM8770305
Reanalysis of: GSM8770316
Reanalysis of: GSM8770306
Reanalysis of: GSM8770317
Reanalysis of: GSM8770307
Reanalysis of: GSM8770318
Reanalysis of: GSM8770308
Reanalysis of: GSM8770319
Reanalysis of: GSM8770309
Reanalysis of: GSM8770320
Reanalysis of: GSM8770310
Reanalysis of: GSM8770321
Reanalysis of: GSM8770311
Reanalysis of: GSM8770322
Reanalysis of: GSM8770312
Reanalysis of: GSM8770323

overall_design: The knockout village consist of 3 WT and 76 knockout lines targeting 30 pancreas developmental regulators and diabetes genes, including GATA6, PDX1, GATA4, FOXA1, FOXA2, MNX1, NEUROD1, NKX2-2, RFX6, GSC, HHEX, HNF4A, PAX6, PBX1, GLIS3, NEUROG3, ARX, OTUD5, BMPR1A, TLE3, BCOR, QSER1, TET1, TET2, TET3, TADA2B, KDM2B, PROSER1, ONECUT1, and NANOG. Edited lines were generated in two hPSC backgrounds (HUES8 and H1). Individual clonal lines were uniquely barcoded by lentiviral infection with GFP-barcode plasmids from the LARRY library and pooled to form a knockout village for differentiation. Samples were collected at five differentiation stages: day -1 (hPSC, 1 day prior differentiation), day 3 (definitive endoderm, DE), day 7 (posterior foregut, PFG), day 11 (pancreatic progenitor, PP) and day 18 (early SC-islet). In addition, WT cells in H1 and HUES8 backgrounds were differentiated and sequenced in parallel at 17 time points: day -1 (hPSC), 3, 5, 7, 9, 10, 11, 12, 13, 14, 15, 16, 18, 21, 25, 26, 35.

first: Dingyu

last: Liu

first: Dapeng

last: Yang

first: Renhe

last: Luo

first: Wei

last: Li

first: Danwei


title: Spatial transcriptome sequencing of chicken and duck beaks

city: Hangzhou

last: Project

address: YuhangTang

summary: The power of any genotype-phenotype mapping approach ultimately depends on whether the identified genetic variants correspond to genes with demonstrable roles in the developmental processes that shape the target traits. We generated bulk RNAseq, single cell and spatial transcriptomic data from multiple embryonic stages (HH29, HH30, HH36 and HH38) for beak buds and whole embryos to evaluate whether candidate genes are functionally enriched in the cellular and molecular programs regulating beak morphogenesis.

sample_organism:
Anas platyrhynchos
Gallus gallus

platform_organism:
Anas platyrhynchos
Gallus gallus

overall_design: Spatial transcriptomics was performed on cryosectioned chicken and duck embryonic beaks at stages (HH29 and HH36) using Stereo-seq (BGI). Sections were mounted onto Stereo-seq chips, fixed, and permeabilized to capture poly(A)+ mRNAs on DNA nanoball arrays. cDNA synthesis and library preparation were carried out following manufacturer protocols, and libraries were sequenced on the DNBSEQ platform. Spatial barcodes and unique molecular identifiers (UMIs) were decoded to generate expression matrices with positional information.


title: Development of antibody-drug conjugates targeting L1CAM to treat metastatic cancer

city: New York

first: Jin Suk

last: Park

address: 417 East 68th St

institute: MSKCC

summary: Effective treatments for metastatic disease have eluded precision oncology efforts and represent a major unmet need. The L1 cell adhesion molecule (L1CAM) is a transmembrane protein expressed by populations of Metastasis Stem cells (MetSC), that are enriched for the ability to reinitiate and propel metastatic growth and display phenotypic plasticity and resistance to conventional chemotherapies. Notably, L1CAM+ MetSC have been detected in numerous human solid tumor types and in multiple disseminated organ sites. As a selective marker of MetSC, L1CAM is a promising candidate for molecularly targeted drugs aimed at eliminating metastases, yet strategies to date have not achieved clinical success. Here, we develop antibody-drug conjugates to deliver highly toxic PNU-159682 payloads to L1CAM-expressing cells. We report the generation of monoclonal antibodies (mAb) with high binding affinity, specificity and selectivity for the human L1CAM extracellular domain. After further optimization of the L1CAM mAbs, PNU-159682 was conjugated to generate ADC variants with both cleavable and non-cleavable linkers, with an average drug-antibody-ratio (DAR) of four. The three ADCs (Y-004, Y-005 and Y-006) potently killed cells with varying levels of surface L1CAM expression. L1CAM ADCs given as monotherapy resulted in robust tumor growth control and extended survival in mice harboring subcutaneous L1CAM+ xenografts or L1CAM+ lung metastases, with safety analyses indicating a feasible therapeutic window. Our findings provide strong proof-of-concept to extend preclinical development of these novel L1CAM ADCs as therapeutic agents for advanced cancer.

overall_design: LUAD PDX tumoroids were generated, dissociated as single cells, FACS-sorted and submitted for single-cell sequencing.


title: Proximal ganglionic intestine in Hirschsprung Disease is fibrotic and stiff

city: Charlottesville

first: Lily

middle: S.

last: Cheng

state: VA

address: 409 Lane Rd

department: Surgery

institute: University of Virginia

summary: Hirschsprung disease (HSCR) is a congenital intestinal disorder characterized by the absence of ganglia in the distal intestine. Despite surgical resection of the aganglionic intestine and pull-through surgery, HSCR patients still experience bowel dysfunction, indicating that latent abnormalities may also exist in the proximal ganglionic intestine. To elucidate possible causes of postoperative bowel dysfunction in HSCR, we investigated differences in the proximal ganglionic intestine using an animal model of HSCR (Ednrb-null mice) and validated our findings in tissue from human HSCR patients. We found that the proximal ganglionic colon of HSCR mice exhibited greater stiffness and fibrosis than their wild-type littermates. Similarly, submucosal fibrosis was significantly greater in the proximal ganglionic intestine of HSCR patients than in intestinal tissue from age and site-matched controls. Furthermore, we observed dysregulated expression of extracellular matrix (ECM)-related genes in the proximal ganglionic intestine of HSCR mice compared to controls. We conclude that increased fibrosis, stiffness, and alterations in ECM composition may contribute to persistent dysfunction of the ganglionic intestine in HSCR. These findings add to the growing body of literature that describe abnormalities in the proximal ganglionic intestine of HSCR and suggest that HSCR is not limited to the aganglionic intestine alone.

overall_design: bulk RNA-sequencing of the proximal ganglionic colon from Hirschsprung Disease mouse model (Ednrb -/-) and equivalent segments  from wildtype littermates to investigate differences in the expression levels of extracellular matrix-related genes.

first: Prisca

last: Obidike

first: Britney

last: Hsu

first: Chioma

last: Moneme

first: Oluyinka

last: Olutoye

first: Walker

middle: D

last: Short

first: Hui

first: Swathi

last: Balaji

first: Yuwen

last: Zhang

first: Sundeep

middle: G

last: Keswani


title: Molecular indicators of hepatocellular carcinoma resistance to Y-90 microspheres revealed through high-throughput screening of cell lines and transcriptomics

city: St. Louis

first: Alexander

last: Zheleznyak

address: 660 S. Euclid

institute: WUSM

summary: Molecular signatures predict prognosis in hepatocellular carcinoma; however, their relevance to Y-90 radioembolization remains unknown. Here, we perform Y-90 microsphere screens in a panel of genetically diverse human HCC cell lines. Ten transcriptomically diverse human HCC cell lines were exposed to 20 MBq of Y-90 glass beads. Normalized AUC values qualified sensitivity. Whole-transcriptome  RNA sequencing at baseline and post-treatment underwent differential expression analysis and gene set enrichment analysis. The response of HCC cell lines to Y-90 was heterogeneous, with SK-Hep1 and SNU-449 being the most resistant and PLC/PRF/5 being the most sensitive.

overall_design: RNA-seq profiling of genetically diverse human HCC cell lines SK-Hep1, Hep-3B, Hep-G2, MHCC-97H, SNU-449, SNU-423, SNU-475, SNU-387, PLC/PRF/5, and SNU-398 before and after treatment with 20 MBq of Y-90.

first: Dhanusha

last: Duraiyan

first: Jessica

last: Silva-Fisher

first: Christopher

last: Malone


title: SCD1 knockdown led to differential gene expression in UMUC3 and SW780 cells (RNA-seq)

city: Ningbo

first: Ye

last: Shazhou

state: Zhejiang Province

address: 59 Liuting street

institute: The Affiliated First Hospital of Ningbo University

summary: The goal of this study was to define the cohort of genes whose expression is regulated by SCD1 function in bladder cancer. Towards this goal, UMUC3 and SW780 cells stably knockdown SCD1  were established. In addition, a control cells was generated by transfection with empty vector. Subsequently, total RNA was isolated and then performed transcriptome RNA sequence.

overall_design: We had four groups: (1) UMUC3-shNT, (2) UMUC3-shSCD1, (3) SW780-shNT, and (4) SW780-shSCD1

first: Shazhou

last: Ye

first: Yanping


title: Evaluation of cellular distribution of intravenously injected LNP in the skeletal muscle

city: Muraoka-Higashi 2-chome Fujisawa, Kanagawa

first: Taisuke

last: Mochida

country: Japan

institute: Takeda Pharmaceutical Company Limited

summary: Muscle satellite cells are essential for the regenerative ability of skeletal muscles and are an attractive therapeutic target for gene transduction in Duchenne muscular dystrophy (DMD). However, there is currently no efficient delivery technology to transduce satellite cells in vivo. This study showed that a lipid nanoparticle (LNP)-mediated delivery of CRISPR-Cas9 mRNA and gRNA (LNP-CRISPR) induces exon skipping in satellite cells more efficiently when injected into a DMD mouse model.

overall_design: Single-cell mRNA sequencing of cells isolated from skeletal muscles in DMD mouse model.

first: Shinya

last: Asano

first: Shinsuke

last: Araki

first: Naoto

last: Inuaki

first: Akitsu

last: Hotta


title: Gene expression profiling of adult C. elegans in response to heat hormesis regimen

first: Siu Sylvia

last: Lee

overall_design: Gene expression profiling was conducted using 3' mRNA sequencing of Naïve (N) and Primed (P) groups in glp-1(ts) mutants or wild-type (N2) worms at Timepoints 1 (after Priming), 2 (Recovery), 3.3 (upon 3-hour HS), 3.6 (upon 6-hour HS), and NC (negative control-before priming) across the heat hormesis regimen. For the glp-1 mutant, five biological replicates were included for each condition, except for Timepoint 3.6, which had three biological replicates. For N2, there were two biological replicates per condition.

first: Hsin-Yun

last: Chang

first: Siu


title: Transcriptomic changes in human SCC-25 Parental and RARG knockout cells in response to 6h and 48h treatments with RARγ agonists CD1530 or all-trans retinoic acid (RA).

first: Helen

last: Hoxie

address: 1300 York Avenue

department: Pharmacology

institute: Weill Cornell Medical College

summary: Vitamin A (retinol) metabolism and signaling through nuclear retinoic acid receptors (RARs α,β,γ) regulate embryonic development, immune functions, and cell differentiation in most cell types.  RAR gamma (RARγ) is highly expressed in stratified squamous epithelial cells of the tongue, esophagus, and skin.  While data indicate that RARγ agonism is anti-tumorigenic in oral cavity squamous cell carcinoma (OCSCC), the specific, primary gene targets of RARγ remain poorly characterized. Here, we define the transcriptomic consequences of RARγ activity in OCSCC. Using a CRISPR-Cas9-mediated knockout (KO) of RARγ and pharmacological treatments with RAR agonists, we identified RARγ-specific gene targets and pathways, as well as targets that overlap with other RA-responsive nuclear receptors.

overall_design: RNA-seq profiling of parental SCC-25 cells and their CRISPR-mediated RARG knockout derivative (RARGKO) with and without added ligand (1μM synthetic RARγ agonist CD1530 or 1μM endogeous ligand retinoic acid) at 6h and 48h timepoints.

first: Xiao-Han

last: Tang

first: Lorraine

middle: J

last: Gudas


title: DNA methylation and lncRNA expression control asynchronous DNA replication at specific imprinted gene domains [RNA-seq]

city: Gif-sur-Yvette

first: Benoit

last: Moindrot

address: 1, avenue de la terrasse

institute: Institute for Integrative Biology of the Cell

summary: Besides genome-wide patterns of replication timing (RT), some genes display allelic replication asynchrony in stem cells, brought about by stochastic events and genetic polymorphisms. Whether epigenetic modifications control asynchronous replication remains unclear. We explored mammalian imprinted domains, where parental DNA methylation imprints mediate allele-specific gene expression. Our genome-wide and locus-specific assays in mono-parental and hybrid mouse ESCs reveal pronounced RT asynchrony—which is parent-of-origin dependent and lost upon neural differentiation—at the Dlk1-Dio3 and Snrpn domains, which both comprise lncRNA polycistrons.  Generating a range of mutant lines, we find that asynchronous replication at Dlk1-Dio3 is mediated by differential DNA methylation, and that the lncRNA Meg3 controls early replication across parts of the domain on the maternal chromosome.  RT thereby becomes unlinked from 3D chromatin architecture as assayed by Hi-C.  The combined replication timing, DNA methylation, 3D chromatin structure and gene expression data highlight how parental methylation imprints and lncRNA expression control replication and can override RT domain organisation.

overall_design: RNA-seq of mESC cells (BJ1 and JB1 genetic background). Delta-Meg3-C1, Zfp57-/- and Zfp57-/-Meg3pro-/- cell lines were obtained by CRISPR-Cas9 deletion.

first: Yui

last: Imaizumi

first: Pol

last: Arnaud-Romero

first: Jean-Christophe

last: Andrau

first: Robert

last: Feil


title: DNA methylation and lncRNA expression control asynchronous DNA replication at specific imprinted gene domains [Hi-C]

overall_design: Hi-C in monoparental mESC cells (AK2 and PR8 cells).

first: François

last: Charon

first: Daan

last: Noordermeer


title: Single cell profiling of CD11b+ cells from mouse cortex following astrocyte overexpression of mCherry-CAAX or Hevin

city: Durham

first: Cagla

last: Eroglu

state: NC

address: 307 Research Dr

institute: Duke University

summary: Astrocyte derived Hevin regulates formation and maintenance of excitatory thalamocortical synapses in the developing mouse cortex. However, the role of astrocyte secreted factors on other cell types is still understudied. To interrogate the response of microglia to the release of astrocye derived Hevin, astrocytes were virally transduced with AAVs to overexpress Hevin or mCherry-CAAX.

overall_design: P0-P1 mice were intracortically injected with AAVs to overexpress Heivn or mCherry-CAAX under the control of the GfaABC1D promoter to restrict expression to astrocytes. Microglia were then isolated from cortex of these mice at P15 using Magnetic Activated Cell Sorting (MACS) for CD11b+ cells. These CD11b+ cells were then used as input for single cell library preparation using the PIPseq v4.0PLUS kit from FluentBio. Libraries were then sequenced using Illumina NovaSeq 6000.

first: Juan J.

last: Ramirez


title: CUT&RUN in SCC-25 Parental and RARG knockout cells maps RARγ binding sites and genome wide changes in H3K4 & H3K27 trimethylation

overall_design: CUT&RUN profiling of SCC-25 Parental (PAR) & RARG knockout (RARGKO) cell lines using antibodies targeting RARγ, H3K4me3, H3K27me3, and IgG. IgG is used as a negative control.


title: ChIP-seq analysis in HepG2-TAF2-V5 clone

city: Richmond

first: Mikhail

last: Dozmorov

address: 830 E Main St

department: Biostatistics

institute: Virginia Commonwealth University

summary: Taf2 is a transcription co-factor and a member of TFIID complex. To better understand gene regulation by Taf2 we generated HepG2 cells stably overexpressing TAF2-V5. This clone was used for ChIP using control IgG or V5 antibody. We identified TAF2-V5 binding near the transcription start sites of 37 genes.

overall_design: Cells were subjected to IP using IgG or V5 antibody following which DNA was isolated for library preparation and sequencing.

first: Devanand

last: Sarkar


title: Gene expression analysis of hepatocyte-specific Taf2 conditional knockout mice

summary: Taf2 is a transcription co-factor and a member of TFIID complex. To better understand gene regulation of Taf2 we generated a hepatocyte-specific Taf2 conditional knockout mouse. RNA-seq was performed in hepatocytes isolated from these mice as well as WT mice. It shows that Taf2 deletion in hepatocytes induces cell death and compensatory proliferation which contributes to inflammation and fibrosis in the liver.

overall_design: Hepatocytes were isolated from WT and hepatocyte-specific Taf2 conditional knockout mice. Two days after plating the cells were harvested, RNA was extracted and subjected to RNA-seq analysis.


title: snATAC-seq profiling of immune cells in human mucosal tissues

first: Peter

last: Sims

address: 3960 Broadway, Lasker 203AC

institute: Columbia University

summary: We performed single-nucleus ATAC-seq (snATAC-seq) to identify cell type- and tissue-specific transcriptional regulatory programs, including transcription factors and cis-regulatory elements, across multiple immune cell lineages from human jejunum and lung.

overall_design: Immune cells were isolated from fresh jejunum or lung tissue specimens procured from human organ donors. Nuclei were isolated from the cells and profiled with snATAC-seq.

first: Daniel

middle: P

last: Caron

first: Steven

middle: B

last: Wells

last: Szabo

first: Jennifer

last: Hwu

first: Siddhi

last: Nargund

first: David

last: Chen

first: Donna

last: Farber


title: DNA methylation and lncRNA expression control asynchronous DNA replication at specific imprinted gene domains    [Capture Hi-C]

first: Benoit

last: Moindrot

overall_design: Capture Hi-C in wild-type and mutant mESC cells (BJ and JB genetic background). NPC samples were obtained by in-vitro differentiation of mESCs.


title: Disambiguating Transcriptional Effects from Post-Transcriptional Effects of Genetic Variants with an Inferred Stability Optimized Massively Parallel Reporter Assay (ISOMPRA).

first: Joseph

last: Dougherty

address: 4370 Duncan Ave

department: Genetics

institute: Washington University in St. Louis

summary: Some widely used methods to study functional effects of DNA sequence on transcription cannot readily disambiguate these from post-transcriptional impacts of the same element on RNA. Therefore, we developed an approach enabling assessment of DNA/RNA regulatory elements in parallel: Inferred Stability Optimized Massively Parallel Reporter Assay (ISOMPRA). ISOMPRA quantifies pre-spliced RNA to more directly assess transcription rate, and spliced RNA, corrected for transcription rate, to correctly identify RNA stability-altering 3’ UTR elements. We benchmark ISOMPRA with both predicted enhancers and 3’ UTR elements, selected regions containing autism patient mutations. We identify numerous functional enhancers/UTRs, and discover a subset with high impact mutations. We further show failing to consider both potential transcriptional and post transcriptional effects results in numerous false-positives and false-negatives. Finally, we adapt ISOMPRA for in vivo analysis of element effects via AAV transduction. Combined with Cre-dependent priming sites, this permits analyses of both transcriptional and post-transcriptional effects within specific cell types of the brain, and further enables measurement of regulatory impacts of in vivo traits, such as sex.

sample_organism:
Escherichia coli
Homo sapiens
Mus musculus
Adeno-associated virus 9

overall_design: RNA sequencing libraries prepared from mouse RNA and HEK293 cell RNA alongside plasmid and AAV sequenced DNA

first: Anthony

last: Fischer

first: Din

last: Selmanovic

first: Colin

last: Florian

first: Tomas

last: Lagunas

first: Stephen

last: Plassmeyer

first: Lily

last: Meyer


title: Tumor-associated macrophages (TAMs) with or without HRS knockdown (KD)

city: PHILADELPHIA

first: Wei

last: Guo

state: PENNSYLVANIA

address: 433 S. University Avenue

institute: University of Pennsylvania

summary: To study the mechanism by which HRS regulates macrophage polarization, we performed bulk RNA sequencing on TAMs with or without HRS KD.

overall_design: Enriched human monocytes were obtained from healthy volunteers by Human Immunology Core at the University of Pennsylvania.  Human macrophages (Mφ) were generated by incubating human monocytes with GM-CSF (10 ng/mL) for 5 days. Briefly, to produce conditioned medium (CM), WM9 were seeded at 70-80% confluence in 15-cm plates and cultured in RPMI1640 medium supplemented with 100 IU penicillin, 100 μg/ml streptomycin and 2% FBS for 3 days. The supernatant was then collected and concentrated 40-fold using centricons (Millipore, Inc.). To make the final CM, concentrated media were diluted at a 1:80 in complete RPMI 1640 or DMEM. TAMs were subsequently induced by incubating with CM for 48 hrs. TAMs with or without HRS KD were collected, and the total RNA was extracted using a PureLink RNA mini kit (Invitrogen) and the RNA-seq was performed by Novogene. The ClusterProfiler package (version 3.18) was then used for GO enrichment analysis.

first: Wenqun

last: Zhong


title: Small-RNA sequencing of human urinary extracellular vesicles reveals association of high sodium diet with renal pro-inflammatory pathways

city: Torino

first: Giovanni

last: Birolo

state: TO

address: Via Santena 19

department: Medical Sciences

country: Italy

institute: University of Turin

summary: Background High sodium intake is associated with arterial hypertension and cardiovascular disease, through mechanisms that go beyond hemodynamic changes, including endothelial dysfunction, oxidative stress, and induction of a pro-inflammatory milieu. The aim of this study was to assess the role of dietary sodium modulation on renal pathophysiology through evaluation of small RNA cargos on urinary extracellular vesicles (uEVs). Methods Fourteen high-risk normotensive subjects with normal kidney function were prospectively enrolled to undergo a low sodium diet (LSD) followed by high sodium diet (HSD). The uEVs were isolated from a 24h urine collection at the end of each diet phase and profiled by small RNA sequencing. Selected differentially expressed microRNAs (miRNAs) were validated in human proximal tubular cell line (HK-2 cells) to assess miRNA-mRNA target interactions. Results We identified 111 small RNA species of which 30 were significantly different between LSD and HSD. Bioinformatic network analysis showed that pathways related to the innate and adaptive immune system, interleukin and interferon signalling were enriched in HSD, whereas pathways related to peroxisome proliferator-activated receptor alpha (PPARα) regulation were enriched in LSD. In HK-2 cells, the inhibition of miR-320b, down-regulated in HSD, increased intercellular adhesion molecule 1 (ICAM-1), with renal pro-inflammatory effects. The inhibition of miR-10b-5p, down-regulated in LSD, increased PPARα, which has an anti-fibrotic and anti-inflammatory role in the kidney. Conclusions Small RNA characterization from uEVs revealed that HSD is associated with pro-inflammatory changes, potentially contributing to sodium-induced low-grade renal inflammation.

first: Fabrizio

last: Buffolo

first: Jacopo

last: Burrello

first: Barbara

last: Pardini

first: Sonia

last: Tarallo

first: Elisabetta

last: Casalone

first: Margherita

last: Alba

first: Carlotta

last: Pomatto

first: Brooke

last: Honzel

first: Laura

last: Tsai

first: Paolo

last: Mulatero

first: Anand

last: Vaidya

first: Silvia

last: Monticone


title: Epithelial RXRα activity induced by Westernization of diet instigates enteritis in Crohn’s disease

city: Innsbruck

first: Christina

last: Plattner

address: Innrain 80

department: Institute of Bioinformatics

country: Austria

institute: Medical University of Innsbruck

summary: Westernization of diet, partly characterised by excess of long-chain fatty acids, provides a risk for developing Crohn’s disease (CD), and perturbs the function of specialized intestinal epithelium termed Paneth cells. The lack of a cellular and molecular framework of lipid sensing in intestinal inflammation poses a constraint to understanding detrimental effects of Western diets. Here, we report how Paneth cell sensing of lipid excess in a Western diet is translated into CD-like intestinal inflammation. Small intestinal transcriptional profiling of CD patients from three independent cohorts identified increased activity of the transcription factor retinoid x receptor alpha (RXRα) specifically in intestinal epithelium. Intestinal epithelial RXRα activity was induced by polyunsaturated fatty acid (PUFA) excess in a Western diet in mice with CD-like enteritis. PUFA-induced RXRα activity in Paneth cells governed chronic transmural enteritis by enabling the expression of the interleukin 8 homologue CXCL1. Blockade of PUFA-induced RXRα activity by 9-cis retinoic acid ameliorated CXCL1 production and enteritis, and patients receiving isotretinoin therapy (a precursor for 9-cis retinoic acid) displayed reduced odds of developing CD. Collectively, we identify Paneth cells as gatekeepers of lipid stress that exploit RXRα to sense and translate PUFA excess into enteritis, which could be targeted to prevent or treat CD.

overall_design: Bulk RNA-seq profiling of siGpx4 (n=3) vs siCtrl MODE-K IECs (n=3) stimulated with arachidonic acid (AA) for 8 hours or with the omega-3 PUFA docosahexaenoic acid (DHA), and single-cell RNA-seq profiling of WT (n=3) and Gpx4+/-IEC mice (n=4) fed with a PUFA enriched Western diet for 3 months.

first: Moritz

first: Felix

last: Grabherr

first: Valentin

last: Marteau

first: Dietmar

last: Rieder

first: Zlatko

last: Trajanoski

first: Timon

last: Adolph


title: Transcriptomic Profiling of anti-CD276/NKG2DLs CAR-T Cells Stimulated by A549 Cells

city: Shanghai

first: Lina

last: Wang

email: qdwln95@163.com, lina-wang@sjtu.edu.cn

address: 639 Zhizaoju Road

institute: Shanghai JiaoTong University School of Medicine

summary: 1. we performed a bulk RNA-seq analysis after 24-hour co-culture with A549 cells. Compared with untransduced T cells, CAR-T cells presented broad transcriptomic differences. Pathway enrichment analysis of the differentially expressed genes (DEGs) revealed significantly upregulated cytokine-receptor cross-talk pathway, as well as TNF, JAK-STAT, NF-κB, and IL-17 signaling pathways in CAR-T cells compared with that in UTD. All these pathways are critical for T cell activation, proliferation, differentiation, and effector functions. 2. We also carried out continuous antigen exposure (CAE) wherein CAR-T cells were induced into an exhaustion state. We observed substantial transcriptomic differences and thousands of DEGs among distinct CAR-T cell groups and the UTD group. To better understand the specific advantages of Bicephali CAR-T cells over two single-targeting CAR-T cells, we employed Venn diagrams to visualize the DEGs among the three groups and revealed 346 unique DEGs of multi-targeting Bicephali CAR-T cells. We further subjected these 346 DEGs to Gene Ontology (GO) pathway enrichment analysis and found that positive regulation of T cell function and oxidative stress were among the top enriched pathways.

overall_design: CAR-T cells were incubated with A549 tumor cells for 24h at a 1:1 E/T ratio, or  incubated with A549 cells (E:T = 1:2) for  five cycles (CAE). Then the cell suspensions were harvested, and the CAR-T cells were sorted using biotin-conjugated anti-BCMA mAbs (Cat# 357514, BioLegend) and anti-biotin magnetic beads (Cat# 480016, BioLegend). Total RNA was then extracted using RNA Extraction Reagent (Cat# R401-01, Vazyme) following the manufacturer’s instructions. RNA quality assessment and transcriptome sequencing were conducted by Oebiotech (Shanghai, China).

first: Yanlei

last: Zhang

first: Shuixiu

last: Peng

first: Chenchen

last: Hou

first: Huaqi

last: Guo

first: Tianyu

last: Zhou

first: Alex

last: H. Chang

first: Lifeng

last: Yan

first: Weining

last: Xiong


title: Alteration of skin fibroblast steady state contributes to healing outcomes [bulk RNA-seq]

city: New Haven

first: Yaqing

last: Huang

state: CT

address: 10 Amistad Room 304A

department: Pathology

institute: Yale University

summary: Fibroblasts display complex functions associated with distinct gene expression profiles that influence matrix production and cell communications and the autonomy of tissue development and repair. Thrombospondin-2 (TSP-2), produced by fibroblasts, is a potent angiogenesis inhibitor and negatively associated with tissue repair. Single-cell (sc) sequencing analysis on WT and TSP2KO skin fibroblast demonstrate distinct cell heterogeneity. Specifically, we found an enrichment of Sox10+ multipotent progenitor cells, identified as Schwann precursor cells, in TSP2KO fibroblasts, while fibrosis-related subpopulations decreased. Immunostaining of tissue and cells validated the increase of this Sox10+ population in KO fibroblasts. Furthermore, in silico analysis suggested enhanced pro-survival signaling, including WNT, TGF-β, and PDGF-β, alongside a reduced BMP4 response. Additionally, the creation of two TSP2KO NIH3T3 cell lines using the CRISPR/Cas9 technique allowed functional and signaling validation in a less complex system. Moreover, KO 3T3 cells exhibited enhanced migration and proliferation, with elevated levels of pro-regenerative molecules including TGF-β3 and Wnt4, and enrichment of nuclear β-catenin. These functional and molecular alterations likely contribute to improved healing and increased neurogenesis in TSP2-deficient wounds. Overall, our findings describe the heterogeneity of dermal fibroblasts and identify pro-regenerative features of TSP2KO fibroblasts.

overall_design: we applied bulk RNA sequencing on WT and TSP2KO mouse dermal fibroblasts (n=3).

first: Nuoya

first: Hao

last: Xing

first: Jingru

last: Tian

first: Dingyao

first: Daqian

last: Gao

first: Henry

last: Hsia

first: Jun

last: Lu

first: Sam

last: Raredon

first: Themis

last: Kyriakides


title: Gene expression analysis of HepG2 cells overexpressing TAF2

first: Mikhail

last: Dozmorov

summary: Taf2 is a transcription co-factor and a member of TFIID complex. To better understand gene regulation of Taf2 we generated stable clones of HepG2 cells overexpressing either eGFP or TAF2-V5. The analysis shows that overexpression of TAF2 activates several pro-tumorigenic pathways and inhibits tumor suppressor pathways.

overall_design: RNA was isolated from exponentially growing cells and subjected to RNA-seq analysis.

first: Devanand

last: Sarkar


title: Post-transcriptional modifications on tRNA fragments confer functional changes to high-density lipoproteins in atherosclerosis

city: Nashville

first: Kasey

last: Vickers

address: 2220 Pierce Ave, PRB 312

institute: VUMC

summary: Epitranscriptomic modifications on RNA play critical roles in stability, processing, and function, partly by influencing interactions with RNA-binding proteins and receptors. The diverse role(s) of post-transcriptional RNA modifications on cell-free small non-coding RNAs (sRNA) remain poorly understood, particularly in disease contexts. Although HDL confer many beneficial properties to health, HDL particles lose these features during atherosclerotic cardiovascular disease (ASCVD). As HDL transport sRNA fragments from classes of RNA known to harbor extensive modifications, we predicted that changes to regulatory modifications on HDL-sRNAs may contribute to HDL functionality changes in ASCVD. To assess HDL-RNA modification levels in ASCVD, total RNA were isolated from HDL of healthy subjects (CAC=0) and those with advanced atherosclerosis, as determined by coronary artery calcium (CAC) scores >21 Agatston units. HDL-RNA were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and AlkB-facilitated RNA (de)Methylation Sequencing (ARM-seq). LC-MS/MS revealed elevated levels of modified nucleosides in HDL-sRNAs from ASCVD subjects, while ARM-seq detected an enrichment of m1A-modified sRNAs-derived from parent tRNAs (tDR), particularly tDR-ArgACG-1. To assess the impact of disease HDL on immune cells, primary macrophages were treated with HDL isolated from ASCVD (CAC+) and control subjects, and results showed that ASCVD-HDL directly induced expression of genes associated with immune signaling and cell adhesion, including TMEM123, a transmembrane protein linked to immune cell migration, adhesion, and cytoskeletal organization. In addition, recombinant HDL particles loaded with m1A-tDR-ArgACG-1 were found to significantly increase TMEM123 expression in treated primary macrophages. Together, these findings propose a model in which HDL-delivered m1A-HDL-tDRs directly stimulate immune signaling, macrophage activation, and pro-inflammatory sub-phenotypes within the atherosclerotic lesion. This study highlights HDL-associated RNA modifications as previously unrecognized contributors to immune modulation, with direct implications for targeting modified HDL-tDRs as a new approach to therapy for ASCVD.

overall_design: Total RNA sequencing of human monocyte derived macrophages treated with reconstituted HDL (rHDL) complexed with synthetic tRNA fragment that contain a m1A methylation

first: Elizabeth

last: Semler

first: Danielle

last: Michell


title: GSDME-controlled pyroptosis promotes muscle repair by coordinating tissue resident macrophages and fibro-adipogenic progenitors

city: shanghai

first: Qi

last: Cao

address: NO.325 Guohe Road

institute: Second Military Medical University

summary: GSDME (Gasdermin E) is a critical protein known for its role in pyroptosis, a form of programmed cell death associated with inflammation. We demonstrated that GSDME as a crucial mediator in muscle repair, particularly in the context of muscle damage induced by cardiotoxin (CTX). Specifically, we found that GSDME-controlled pyroptosis promotes muscle repair by coordinating the activities of tissue-resident macrophages and fibro-adipogenic progenitors (FAPs).

overall_design:
Prepare mouse gastrocnemius as a single-cell suspension. 10x genomic was used to capture the transcriptomic information of the single cells. All libraries were sequenced by Novaseq on a 150 bp paired-end run.
data colum header nnnnn-1 = GSDME-KO-CTX
data colum header nnnnn-2 = WT-CTX

first: Q

first: P

last: Wang


title: Evaluation of cellular distribution of LNP-CRISPR in the skeletal muscle

first: Taisuke

last: Mochida

summary: Muscle satellite cells are essential for the regenerative ability of skeletal muscles and are an attractive therapeutic target for gene transduction in Duchenne muscular dystrophy (DMD). However, there is currently no efficient delivery technology to transduce satellite cells in vivo. This study showed that a lipid nanoparticle (LNP)-mediated delivery of CRISPR-Cas9 mRNA and gRNA (LNP-CRISPR) induces exon skipping in satellite cells more efficiently when injected intramuscularly into a DMD mouse model.

overall_design: Single-cell mRNA sequencing of cells isolated from skeletal muscles in DMD mouse model.

first: Shinya

last: Asano

first: Shinsuke

last: Araki

first: Naoto

last: Inuaki

first: Akitsu

last: Hotta


title: EPOP Restricts PRC2.1 Targeting to Chromatin by Directly Modulating Enzyme Complex Dimerization (RNA-Seq)

city: Dallas

first: Xin

last: Liu

state: TX

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center

summary: Polycomb repressive complex 2 (PRC2) mediates developmental gene repression as two classes of holocomplexes, PRC2.1 and PRC2.2.  EPOP is an accessory subunit specific to PRC2.1, which also contains PCL proteins.  Unlike other accessory subunits that collectively facilitate PRC2 targeting, EPOP was implicated in an enigmatic inhibitory role, together with its interactor Elongin BC.  We report an unusual molecular mechanism whereby EPOP regulates PRC2.1 by directly modulating its oligomerization state.  EPOP disrupts the PRC2.1 dimer and weakens its chromatin association, likely by disabling the avidity effect conferred by the dimeric complex.  Congruently, an EPOP mutant specifically defective in PRC2 binding enhances genome-wide enrichments of MTF2 and H3K27me3 in mouse epiblast-like cells (EpiLCs).  Elongin BC is largely dispensable for the EPOP-mediated inhibition of PRC2.1.  EPOP defines a distinct subclass of PRC2.1, which uniquely maintains an epigenetic program by preventing the over-repression of key gene regulators in the continuum of pluripotency and differentiation.

overall_design: Total RNA sequencing (RNA-seq) for mESCs and EpiLCs expressing WT or mutant EPOP.

first: Xiuli

first: Ze

last: Yu

first: Chao

last: Xing


title: Prostate Cancer In Situ Autovaccination With the Intratumoral Viral Mimic Poly-ICLC: Making a Cold Tumor Hot

city: San Diego

first: Elai

last: Davicioni

state: California

address: 9725 Lusk Blvd

institute: Veracyte, Inc

summary: Gene expression study of the poly-ICLC Phase II clinical trial

overall_design: Retrospective analysis of 24 tumor transcriptomes from 12 subjects in the poly ICLC trial

first: S

last: Nair

first: D

last: Chakravaty

first: E

first: A

last: Tewari


title: The hegemonic EWSR1::ETS oncogene overrules core regulatory circuitry mechanisms in Ewing sarcoma.

city: Paris

first: Olivier

last: Delattre

address: 26 rue d'ulm

institute: Institut Curie

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

overall_design: Refer to individual Series


title: EPOP Restricts PRC2.1 Targeting to Chromatin by Directly Modulating Enzyme Complex Dimerization (ChIP-Seq)

overall_design: Chromatin immunoprecipitation followed by sequencing (ChIP-seq) for MTF2, JARID2, and H3K27me3 in mouse epiblast-like cells (EpiLCs) containing WT or mutant EPOP.


title: Effects of active-state RAS inhibition on the colorectal cancer cell transcriptome

city: Berlin

first: Nils

last: Blüthgen

address: Chariteplatz 1

department: Institute of Pathology

country: Germany

institute: Charite Universitätsmedizin Berlin

summary: Here, we identified and categorized mechanisms of resistance to broad-spectrum active-state RAS inhibition in colorectal cancer cell lines. Emerging resistant cell populations after long-term dose escalation revealed distinct patterns of genomic mutations and phospho-protein states. To assess transcriptomic footprints associated with RASi resistance, we employed single cell RNA sequencing. We compared parental DLD1 and SW480 cells, and RASi-resistant sublines termed DLD1-FIREX-high, DLD1-FIREX-low, DLD1-cFIREX-high and SW480-resistant.  Cells were cultured in the absence or presence of ERK or RAS inhibitors as indicated. This experimental design enables the identification of transcriptomic shifts induced by RAS and ERK inhibition, thereby facilitating assessment of their reactivation upon emergence of secondary resistance. Finally, we used SW480-et cells expressing a murine Eco receptor. These cells were transfected with lentiviral oncogene vectors and subsequently cultured for 72h in the absence or presence of RAS inhibitor. We find that the subpopulations show different levels of pathway activities, including MAPK. Our findings endorse single-cell analyses as a powerful approach for dissecting the complex landscape of therapy resistance.

overall_design: We cultured parental DLD1 and SW480 cells, and RAS inhibitor-resistant sublines DLD1-FIREX-high, DLD1-FIREX-low, DLD1-cFIREX-high and SW480-resistant, in the absence or presence of RAS inhibitor or ERK inhibitor. In addition, we cultured  SW480-et cells transfected with viral oncogenes cultured in the absence or presence of RAS inhibitor. After 72 h, we harvested cells and subjected cells to scRNA-seq.

first: Oleksandra

last: Aust

first: Alexandra

last: Trinks

first: Viola

last: Hollek

first: Markus

last: Morkel


title: The role of chromosome-specific aneuploidy in Bub3 deficient cells

city: Hanover

first: Soni

last: Lacefield

state: New Hampshire

address: 74 College St

department: Biochemistry and Cell Biology

institute: Geisel School of medicine at Dartmouth

summary: The study aims to check how and which chromosomes are missegregated when Bub3, a spindle checkpoint gene, is deleted in budding yeast, and whether cells maintain the aneuploidy.

overall_design: Illumina sequencing libraries were generated using tagmentation workflow, normalized, pooled for NextSeq2000 Illumina sequencing, targeting 10 million 150bp paired-end reads per sample.

first: Pallavi

last: Gadgil

first: Olivia

last: Ballew

first: Timothy

last: Sullivan


title: Single-cell transcriptome comparison of myeloid cells’ in the retina in response to transplantation of human stem-cell derived neurons and to ocular damage reveals the reversibility of the microglia activation.

city: Boston

first: Emil

last: Kriukov

address: 20 Staniford St

institute: Schepens Eye Research Institute

summary: Retinal ganglion cells (RGCs) play a critical role in the transmission of visual signals from the retina to the brain required for proper vision. The optic neuropathies, including glaucoma, NAION,  LHON, traumatic optic neuropathy, optic pathway glioma result in the reversible and irreversible changes in RGCs, ultimately leading to their death.  RGC loss is permanent given that these cells cannot regenerate in mammals. Neuroregenerative strategies for managing optic neuropathies have mainly focused on restoring the lost RGC cell population in a glaucomatous retina. This treatment approach not only aims to halt disease progression, but also to restore the lost vision by replacing the damaged/lost RGCs through RGC transplantation. Significant strides have been made in this field of RGC transplantation and regeneration. As a first step of RGC transplantation, several protocols now exist to differentiate cells into primary RGCs, and several sources of donor RGC have been established including human ES and iPSC-derived RGCs. To achieve functional regeneration, the transplanted RGCs must survive, integrate, and extend their axons and establish connections within the retina and the brain. Of the many factors influencing initial donor RGCs survival, the innate immunity [ref] and primarily microglia reactivity is of particular interest. In ocular models of glaucoma, microglia have been heavily implicated in driving disease progression, promoting a pro-inflammatory environment. In some instances, activated microglia have been directly implicated in RGC loss by mediating phagocytosis of the damaged or injured RGCs. In line with this, our recent work, shows that activated host microglia are detrimental to donor RGCs, presumably via phagocytosis of the donor RGCs. We observed that pre-treatment of donor RGCs with FasL and annexin V to block the exposed phosphatidylserine residues of the stressed donor RGCs prior to transplantation reduced activation of host microglia, and better RGC survival rates post transplantation. Studies like these and others underscore the importance of modulating host microglia reactivity for better transplantation outcomes. To further improve donor RGC survival post transplantation, a better understanding of the host myeloid cell reactivity following donor RGC transplantation is paramount. We and others have previously shown the importance of modulating the retinal microenvironment to promote RGC survival. In the current study, we profiled the transcriptome of the host myeloid cell population (CX3CR1-GFP) using a single cell RNA sequencing approach, to delineate the myeloid cell population most relevant for donor RGC survival post transplantation. A focused analysis on the microglia cell population was performed, given their established role in glaucoma disease progression. In addition to profiling the expression pattern of the known classic microglia disease associated signatures such as ApoE, Spp1, and Lgals, we identified several other genes associated with, and or driving microglia activation, including Cybb, Atf3, Aurka and Kif22. Given the known function of these genes in other disease contexts, we believe that these genes are important for modulating donor RGC survival post transplantation, thus warrant further investigation in this context. Taken together, with reference to our established adult mouse retina cell atlas, we present a comprehensive analysis of the host myeloid cell population, specifically focusing on microglia reactivity status, at a single cell resolution level, providing insights into the regulatory mechanisms that may be important for donor RGC survival and integration into glaucomatous retina post transplantation.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Three days following RGC transplantation, mice were euthanized, eyes enucleated and retinal tissue isolated and transferred into a tube containing PBS with 0.04% BSA on ice. Eight mouse retinas were pooled for each sample. The tissue was dissociated by mechanical and enzymatic methods using Gentle MACS protocols. Briefly, retinas were allowed to settle to the bottom of the tube and excess PBS carefully removed. Tissues were then rinsed with HBSS and transferred to a MACS M-tube and let to settle to the bottom of the tube before again removing excess HBSS. Tissues were then resuspended in 2ml of dissociation solution containing DNAse 1 (1mg/ml). The mixture was incubated at 37°C for 5min in a heating bath. The M-tube was then inverted ensuring that all retina tissues remained in solution, and the tube placed into the MACS tissue dissociator and the brain 1 program run. At the end of brain 1 program, the M-tube was removed from the MACS dissociator and incubated at 37°C for 5mins in a heating bath. The tube was then inverted again and placed back into the MACS dissociator and program brain 2 initiated, after which 10mL of isolation buffer was added to the dissociate and the mixture triturated 7-10 times with a 1000µl pipette tip. Then 12ml of the homogenate was transferred into a 15ml tube and cells pelleted at 200g for 5 mins at 4°C. The cell pellet was resuspended in 500µl of isolation buffer and filtered using a 70um cell strainer into a FACS tube for sortingAll tubes for this procedure were pre-blocked with 2% BSA in HBSS. Separate single stain samples for TdTomato+ and GFP+ were prepared and used to set the gates. The harvested cells were sorted separately for TdTomato +ve, and GFP +ve, and collected into separate collection tubes each containing 200μl of PBS with 0.04% BSA. The sorted cells were taken for single cell RNA sequencing library preparation. Of the sorted cells, up to 10,000 viable cells per sample were used for single cell RNA library preparation following the 10x genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 protocol. Briefly, the droplet-based encapsulation method was used to encapsulate single cells with gel beads, partitioning oil, and a reverse transcription master mix generating GEMs. The generated GEMs were cleaned, gene expression libraries constructed and sequenced at a sequencing depth of 20,000 read pairs per cell, paired-end dual indexing (See manufactures instructions for detail).

first: Anthony

last: Mukwaya

first: Milica

last: Margeta

first: Petr

last: Baranov


title: Transcriptome Analysis of Myometrium (normal and at-risk) and Fibroid Tissues in Black and White Women

city: Chicago

first: Ayman

last: Al-Hendy

address: 5841 S Maryland Ave

department: OBGyN

institute: University of Chicago

summary: Uterine fibroids are benign tumors of the uterus that disproportionately affect women, with significant racial disparities observed in their incidence, severity, and clinical outcomes. Despite these disparities, the molecular mechanisms underlying these racial differences remain poorly understood. This study aims to examine the transcriptome of whole human normal and at-risk myometrium as well as uterine fibroid tissues obtained from both Black and White women to gain insights into the genes and pathways contributing to this disparity.

overall_design: Examine the transcriptome of whole human normal myometrium (MyoN), myometrium at-risk (MyoF, adjacent to fibroids), and uterine fibroid tissues obtained from both Black and White women

first: Qiwei

last: Yang

first: Mohamed

last: Ali

first: Maria

middle: V

last: Bariani

first: Cheryl

last: Walker

first: Sandra

last: Grimm

first: Cristian

last: Coarfa


title: Gene expression profiles of human iPSC-derived microglia in different culture conditions [Bulk RNAseq]

city: Cambridge

first: Emile

last: Wogram

state: MA

address: 455 Main St

institute: Whitehead Institute for Biomedical Research

summary: iPSC-derived microglia precursor cells were integrated into the cerebral organoid slice cultures, FACS sorted based on expression of green fluorescent protein after 10 and 60 days, and compared to iPSC-derived microglia in monoculture. The expression profiles were further compared to published RNAseq data from primary human microglia and iPSC-derived human microglia transplanted in the mouse brains.

overall_design: Microglial precursor cells were cultured as monocultures or added to cerebral organoid slice cultures. For bulk RNA sequencing, the cells were FACS sorted based on their green fluorescent protein expression. The samples originated from different batches of differentiations from an iPSC line obtained from human foreskin fibroblasts from a healthy 2-year-old male from the Coriell Institute (AG07095) and from an already established and characterized embryonic stem cell line H1. From the iPSC line, microglia precursor cells were analyzed, microglia in monoculture (after 10 and 60 days), and microglia in cerebral organoids (after 10 and 60 days). In addition, microglia in cerebral organoids were treated with levetiracetam or PBS-control and analyzed. From the H1 line, microglia cultured for 60 days in cerebral organoid slice cultures were analyzed. An n=6 was used for each sample set, except for microglia in cerebral organoid slice cultures treated with levetiracetam (n=2) and treated with PBS-control (n=3). One n corresponds to all collected GFP+ cells pooled from 4-6 organoid sections for cells isolated from organoid sections. For microglia monoculture and microglia precursor samples, one n corresponds to two pooled wells of a 6-well plate, with one plate generating three samples. The microglia precursor cells were floating cells.

first: George

last: Bell

first: Rudolf

last: Jaenisch