title: Prostate Tumor Immune Microenvironment Changes Following Immunotherapy Shared by Patients who Developed Anti-Tumor Response or Immune-Related Adverse Events

city: Madison

first: Douglas

last: McNeel

state: WI

address: 1111 highland ave, 7007

department: UW Carbone Cancer Center

institute: University of Wisconsin Madison

summary: Here, we evaluated the changes within the tumor immune microenvironment in patients who had any PSA decline (clinical responder) while on treatment, or developed irAEs, with the goal of identifying mechanisms of response or adaptive mechanisms of treatment resistance. Specifically, we performed gene expression analysis of tumor biopsies by isolating total RNA from the FFPE tissue and also performed protein expression analysis in spatial context for regions populated with CD8 T cells and regions where CD8 T cells were absent.

overall_design: tumor biopsies obtained pre- and post-treatment from 12 patients were evaluated by gene expression profiling and spatial protein analysis

first: Ichwaku

last: Rastogi


title: Reduced PRC2 function causes asparaginase resistance in T-ALL by decreasing WNT pathway activity

status: Public on Dec 23 2025

city: Dublin

first: Jonathan

last: Bond

address: Belfield

country: Ireland

institute: Systems Biology Ireland, UCD

type: Expression profiling by high throughput sequencing

summary: Loss-of-function mutations and deletions in core components of the epigenetic Polycomb Repressive Complex 2 (PRC2) are associated with poor initial treatment response in T-acute lymphoblastic leukemia (T-ALL), but the mechanisms that underpin resistance to individual therapies are unknown. We leveraged an isogenic T-ALL cellular model and primary patient data to investigate how PRC2 alterations affect signaling pathway activity in leukemia cells, and whether these changes may influence therapy response. Integration of transcriptomic, proteomic and phosphoproteomic results revealed markedly reduced activity of the WNT-dependent stabilization of proteins (WNT/STOP) pathway in leukemia cells lacking core PRC2 factor EZH2. Importantly, these results closely matched transcriptional readouts from T-ALL patient samples with PRC2 mutations and deletions. We discovered that PRC2 loss significantly reduced sensitivity to key T-ALL treatment asparaginase, and that this was mechanistically linked to increased cellular ubiquitination levels due to WNT/STOP suppression that bolstered leukemia cell asparagine reserves. These results also strongly correlated with transcriptional profiles of asparaginase resistance in an independent T-ALL patient cohort. We further found that asparaginase resistance in PRC2-depleted leukemic blasts could be mitigated by pharmaceutical proteasome inhibition, thereby providing a potential avenue to tackle induction treatment failure in these cases.

overall_design: We performed RNA-Seq on Jurkat EZH2-WT (biological triplicates) and two Jurkat CRISPR/Cas9-edited-EZH2-KO: A7 and C9 (biological triplicates for each).


title: NKG2A+ NK Cell Cytotoxicity of Epstein-Barr Virus Infected B Cells is Mediated Through the NKG2D and NKp30 Activating Receptors

status: Public on Dec 22 2025

city: Stanford

first: Josselyn

middle: K

last: Pena

state: CA

address: 1201 Welch Rd

department: Surgery

country: USA

institute: Stanford University

summary: Epstein–Barr virus (EBV) is a ubiquitous gamma-herpesvirus that persists as a chronic, asymptomatic infection in over 90% of the adult human population. EBV infections are mainly asymptomatic and subside due to a vigorous host T cell response with the virus transitioning to latency in a subset of memory B cells. Failure to control latent EBV infection can result in a variety of EBV-associated malignancies, including lymphoproliferative diseases in immunocompromised people. Studies in both experimental models and humans suggest that NK cells are critical in the host defense against EBV. In this study we utulized scRNA-seq to better understand the phenotype of NK cells that respond to EBV-infected B cells. We generated a panel of EBV+ lymphoblastoid cell lines (EBV+ LCL) and performed coculture experiments with primary NK cells and their autologous EBV+ LCL  using 10x Genomics scRNA-seq. Subsequent analyses following alignment and data processing were completed in Python.

overall_design: Following coculture with an autologous EBV+ LCL, cells were sorted for CD107a+ (responsive) and CD107a- (nonresponsive) NK cells using scRNA-seq.

first: Joshua

middle: S

last: Badshah

first: Sheri

middle: M

last: Krams


title: Transcriptomics of PLAG1 VC and KD in K562 imatinib resistant cell using RNA-sequencing.

city: Kharghar

first: Rukmini

last: Govekar

state: Maharashtra

address: Sector 22, Utsav Chowk-CISF Rd, Owe camp

department: Cancer Research Institute

country: India

institute: Advanced centre for Treatment, Research and Education in Cancer

summary: CML-CP Patients showing resistance to Imatinib, due to Bcr-Abl mechanisms of resistance  are treated with 2nd and 3rd generation TKIs but some non-responders shown Bcr-Abl independant mechanism of resistance. We hypothesize that this could be due to activation of novel pathway, or activation of downstream molecules of Bcr-Abl pathway. In this study we have performed Transcriptomics and epigenomics analysis of K562 and KU812 CML-BC cell sensitive and resistant to imatinib, ITGB3  showed high transcript levels, and was also  hypomethylated in K562/R cells as compared to sensitive counterparts and showed higher protein levels  in both K562 and KU812 resistant cell lines. Previous studies in the lab also showed  ITGB1 as a differentiator in proteomic analysis and further on-going studies indicate its role in resistance. ITGB1 was however not modulated at transcriptional level.  The protein levels of ITGB3 were significantly increased in resistant cells (K562; KU812: while those of ITGB1 were significantly decreased in resistant cells (K562; KU812. With the known switching of integrins, whether higher expression of ITGB3 had a role in modulating the levels of transcriptionally non-regulated ITGB1 was investigated in K562/S cells with ITGB1 knocked down (KD)  protein levels of ITGB3 were upregulated as compared to VC. Similarly,  knock down of ITGB3 in K562/R resulted in increased level of  ITGB1 protein. These striking results confirmed switching of integrins and explained reduced levels of ITGB1 despite no alteration at genomic or transcriptomic level in resistant cells.

overall_design: RNA-Sequencing of PLAG1 VC and KD in K562 cells resistant to imatinib.

first: Shashi

last: Kumari

first: Dr. Rukmini

first: Dr. Rahul

last: Mojidra

first: Dr. Mythreyi

last: Narasimhan


title: Transcriptional Profiling of Synovial Macrophages using Minimally Invasive Ultrasound-Guided Synovial Biopsies in Rheumatoid Arthritis [dataset1]

city: Chicago

first: Deborah

middle: R

last: Winter

state: IL

address: 240 E. Huron Street, M300

institute: Northwestern University

summary: Pilot study of the feasibility and clinical significance of whole tissue and cell-type-specific transcriptional profiling of cells from synovial biopsy in Rheumatoid Arthritis (RA)

overall_design:
RNA-seq of whole tissue from synovial biopsy of wrists in adults with RA.  Osteoarthritis (OA) tissue from whole joint replacement is processed analalagously for comparison.

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

first: Harris

last: Perlman


title: Epigenetic Signatures of Brain Aging by Single Nucleus Methylation Sequencing [FC-18mo]

city: La Jolla

first: Joseph

last: Ecker

address: 10010 North Torrey Pines Road

department: Genomic Analysis Laboratory

institute: HHMI-Salk-Institute

type: Methylation profiling by high throughput sequencing

summary: Illustrating aging effects on the epigenomic features of brain is critical to understand brain functional decline and neurodegeneration. Single nucleus methylation sequencing was applied to brain regions of both male and female mouse at the age of 2, 9 and 18 months. We use these data to identify the epigenomic changes with aging in mouse brains.

overall_design: Apply snmC-seq3 to mouse brain regions to identify aging effects on epigenomics

first: Wei

last: Tian

first: Anna

last: Bartlett

first: Jordan

last: Altshul

first: Rosa

last: Gomez Castanon

last: Nery

first: Jacinta

middle: D

last: Lucero

first: Julia

last: Osteen

first: Huaming

last: Chen

first: M

last: Behrens


title: Single-cell and isoform-specific translational profiling of the mouse brain with long-read sequencing [scRNA-Seq, longread]

first: Brian

last: Yee

address: 9500 Gilman Dr

department: Health

institute: UCSD

summary: The brain displays the richest repertoire of post-transcriptional mechanisms regulating mRNA translation. Among these, alternative splicing has been shown to drive cell type specificity and, when disrupted, is strongly linked to neurological disorders. However, genome-wide measurements of mRNA translation with isoform-sensitivity at single-cell (sc) resolution have not been achieved. To address this, we deployed Ribo-STAMP (Surveying Ribosomal Targets by APOBEC-Mediated Profiling) coupled with long-read scRNA-sequencing (scRNA-seq) in the brain. We generated the first isoform-sensitive sc translatomes of the mouse hippocampus at postnatal day 25, discovering cell type-specific translation of 3,857 alternative transcripts across 1,641 genes, and identifying isoforms of the same genes undergoing differential translation within and across eight different cell types. We defined high and low translational states in CA1 and CA3 neurons, with synaptic and metabolic genes enriched in high states. Surprisingly, we found that CA3 exhibited higher basal translation compared to CA1, as confirmed by metabolic labeling of newly synthesized proteins and immunohistochemistry of translational machinery components. This accessible platform will expand our understanding of how cell type- and isoform-specific translation drives brain physiology and disease.

overall_design: Single-cell suspensions from hippocampi were obtained from three male P22 C57BL/6J mice which were stereotactically injected in the dHC with 800nl EF1a-Ribo-STAMP virus. Results were analyzed using long-read scRNA-seq on PacBio Revio.

first: Samantha

middle: L

last: Sison

first: Federico

last: Zampa

first: Eric

last: Kofman

first: Su Yeun

last: Choi

first: Pratibha

last: Jagannatha

first: Gene

middle: W

last: Yeo

first: Giordano

last: Lippi


title: Single-cell and isoform-specific translational profiling of the mouse brain [scRNA-seq]

summary: The brain displays the richest repertoire of post-transcriptional mechanisms regulating mRNA translation. Among these, alternative splicing has been shown to drive cell type specificity and, when disrupted, is strongly linked to neurological disorders. However, genome-wide measurements of mRNA translation with isoform-sensitivity at single-cell (sc) resolution have not been achieved. To address this, we deployed Ribo-STAMP (Surveying Ribosomal Targets by APOBEC-Mediated Profiling) coupled with short- and long-read scRNA-sequencing (scRNA-seq) in the brain20. We generated the first isoform-sensitive sc translatomes of the mouse hippocampus at postnatal day 25, discovering cell type-specific translation of 3,857 alternative transcripts across 1,641 genes, and identifying isoforms of the same genes undergoing differential translation within and across eight different cell types. We defined high and low translational states in CA1 and CA3 neurons, with synaptic and metabolic genes enriched in high states. Surprisingly, we found that CA3 exhibited higher basal translation compared to CA1, as confirmed by metabolic labeling of newly synthesized proteins and immunohistochemistry of translational machinery components. This accessible platform will expand our understanding of how cell type- and isoform-specific translation drives brain physiology and disease.

overall_design: Single-cell suspensions from hippocampi were obtained from three male P22 C57BL/6J mice which were stereotactically injected in the dHC with 800nl EF1a-Ribo-STAMP virus. Results were analyzed using scRNA-seq.


title: Impacts on the Placenta-Brain Axis in Pregnant Mice Devoid of Gut Microbiota

city: Columbia

first: Trupti

last: Joshi

state: Missouri

address: 103 Math Science Building

department: Department of Biomedical Informatics, Biostatistics and Medical Epidemiology (BBME)

institute: University of Missouri-Columbia

summary: Gut microbiota play an important role in regulating individual health. It is also increasingly apparent that changes in maternal gut microbiota result can render her offspring more susceptible to diseases later in life. Such bacterial induced changes likely originate in the womb. As the placenta is the primary communication organ between mother and conceptus, it is vulnerable to in utero environmental changes, including those associated with maternal gut microbiota. The placenta also relays nutritional and other factors, including serotonin, to the developing fetal brain that help guide development of this organ. Thus, placental disruptions can influence the placenta-brain axis and subsequently neurobehavioral programming with potential long-term consequences. One mechanism by which changes in maternal gut microbiota might effect the placenta are through alterations in bacterial short chained fatty acids (SCFA). The hypothesis, thus, tested in the current studies is that absence of a maternal gut microbiota, as occurs in germ-free (GF) mice, would impact bacterial SCFA in her fecal samples and in the fetal placenta and brain. Secondarily, we tested whether transcriptomic changes would be evident in the placenta and fetal brain from conceptuses derived from GF relative to multi-pathogen free (MPF) pregnant females.

overall_design: RNA was isolated from frozen placenta and fetal brain samples from conceptuses derived from pregnant germ-free (GF) and multi-pathogen free (MPF) females. We previously determined the sex of the conceptus by usage of a PCR based on the Y-chromosome located Sry gene. This gene identified those conceptuses that were male from those who were female. Quality and quantity of RNA was determined by fragment analyzer. RNA was then subjected to RNAseq. Resultant libraries were pooled and run on an Illumina NovaSeq X sequencer with a paired end 100 bp read format on a 10B flow cell to generate ~100 million paired reads per sample. The RNA-seq data analysis pipeline commenced with quality assessment of the raw Illumina sequencing reads using FastQC. High-quality reads were then aligned to the mouse reference genome (GRCm39) by using the Hisat2 aligner with its index-building functionality. Following alignment, transcript assembly and quantification were carried out with Cufflinks, which estimates gene expression levels in terms of FPKM (Fragments Per Kilobase of transcript per Million mapped reads). To identify differentially expressed genes (DEGs), we used Cuffdiff. For both placenta and brain samples, DEGs were defined by using a log₂ fold change (log2FC) threshold of 1 and a q-value < 0.05. Visualization of differential expression results is performed using Python. Volcano plots were generated for each comparison with the matplotlib package to highlight significantly upregulated and downregulated genes. Principal Component Analysis (PCA) was conducted on the gene expression profiles (FPKM values) with the scikit-learn library to assess sample clustering, and the resulting components were visualized with seaborn package for clear and informative plotting.

first: Rosalind

middle: T

last: Herrington

first: Zhen

last: Lyu

first: Ragavi

last: Raman

first: Sarah

middle: E

last: Seda

first: Claire

last: Snyder

first: Nathan

middle: J

last: Bivens

first: Zhentian

last: Lei

first: Tanhaul

last: Islam

first: Lloyd

last: Sumner

first: Cheryl

last: Rosenfeld


title: Crosstalk between the Ino80 complex and TOR signaling drives fungal adaptation to hypoxia through chromatin remodeling

city: Montreal

first: Adnane

last: Sellam

address: 5000, rue Bélanger

country: Canada

institute: Universite de Montreal

type: Genome binding/occupancy profiling by high throughput sequencing

summary: In the present study, we undertook a genetic screen of the GRACE (Gene Replacement and Conditional Expression) collection to identify genes required for hypoxic (5% O2) adaptation. Through this comprehensive screen, we identified many genes spanning diverse functional categories that are required for optimal growth under hypoxia, revealing new pathways likely involved in O2 sensing, signaling, and adaptation to O2 depletion. We identified two subunits of the Ino80 chromatin-remodeling complex and one subunit of the TOR (Target Of Rapamycin) pathway as critical for sustaining fungal growth under O2-depleted conditions. Integration of ino80 mutant transcriptomic data with Ino80 DNA-binding and nucleosome-occupancy profiles under conditional INO80 depletion revealed that this chromatin remodeler controls the cellular phosphate demand accompanying hypoxic adaptation. The similarity between chromatin accessibility changes upon TOR inhibition and INO80 depletion suggests that Ino80 functions in concert with the TOR pathway to promote growth and maintain phosphate homeostasis under O2-limiting conditions. These findings provide new insight into how nutrient and O2 signaling converge on chromatin remodeling mechanisms, offering a foundation for understanding the regulatory logic that underlies hypoxic adaptation in pathogenic fungi.

overall_design: In this study, we identified transcripts that were differentially regulated in the ino80 mutant, which lacks the chromatin remodeler Ino80. The transcriptional profile of ino80 cells grown under hypoxic conditions (5% O₂) was compared to that of the wild-type (WT) strain grown under the same conditions. Hypoxia-responsive genes were determined by comparing the transcriptome of WT cells grown under hypoxia (5% O₂) to that of cells maintained under normoxia (21% O₂). For ChEC-seq, DNA cleavage profiles from Ino80-MNase cells (C-terminal MNase-tagged INO80 at the native locus) were normalized to those from MNase-free control cells (WT cells with ectopic MNase expression). For MNase-seq, DNA counts from doxycycline-treated pTET-INO80 or pTET-TOR1 strains (INO80- and TOR1-depleted cells) were compared to those of their untreated counterparts.

first: Antony T.

last: Vincent

first: Faiza

last: Tebbji

first: Manjari

last: Shrivastava

first: Morgane

last: Dubé


title: Long-term Atrazine exposure promotes ovarian dysfunction by enhancing the inflammatory microenvironment and inducing fibrosis

city: Changchun, China

first: Hao

last: Wu

address: Xinjiang Street, Changchun, Jilin

country: China

institute: Jilin University

summary: This study investigated the effects of long-term exposure to the herbicide Atrazine (ATZ) on ovarian function in rats and its underlying mechanisms. Following 180-day exposure to 2 μM and 10 μM ATZ via drinking water, ATZ reduced ovarian weight and index, disrupted follicular development, increased atretic follicles, and altered serum hormone levels, including decreased estradiol (E2) and increased follicle-stimulating hormone (FSH), indicating diminished ovarian reserve (DOR). Histological analysis revealed that ATZ exposure induced ovarian fibrosis, characterized by increased collagen deposition and upregulation of fibrosis-related proteins (collagen I/III, fibronectin, α-SMA). Mechanistically, ATZ activated the Wnt/β-catenin signaling pathway, upregulating the expression of Wnt1, MMP2, MMP9, and Cyclin D1 in ovarian granulosa cells. Additionally, ATZ promoted M2 macrophage polarization in the ovarian microenvironment, accompanied by increased anti-inflammatory cytokine IL-4 and decreased pro-inflammatory cytokine IL-6, suggesting that ATZ induces fibrosis through inflammatory regulation of the Wnt pathway. These findings reveal a novel mechanism by which long-term low-dose ATZ exposure impairs ovarian function via the inflammation-fibrosis axis, providing experimental evidence for the reproductive toxicity of environmental pollutants.

overall_design: The SD rats (21 days old) were intraperitoneally injected with 60 IU of pregnant mare serum gonadotropin (PMSG), after 48 h the rats were euthanized by cervical dislocation under anesthesia.   Bilateral ovaries were removed from abdominal cavity of rats and washed with precooled PBS for 3 times.   GCs were isolated using sterile scissors to cut the ovaries into 2 mm pieces, placed in the precooled medium [DMEM/F12 (HyClone)+10% fetal bovine serum (FBS, Gibico)] and centrifuged at 1200 rpm for 5 min.   The precipitation was resuspended with 0.25% pancreatic enzyme containing 0.02% EDTA and placed in an incubator at 37 ℃ for 1 hour.   After 40 μm sterile cell sieve filtration, the suspension was added into complete medium [DMEM/F12+10% FBS+1% Insulin-Transferrin-Sodium Selenite (100×, Procell)+1% MEM Non-Essential Amino Acids, NEAA (100×, Procell)+1% Penicillins-streptomyci (100×)] for culture. In order to identify the GCs, its positive marker FSHR was detected by immunofluorescence.

first: Wei

last: Liu #

last: Wu#

first: Kai

last: Wang

first: Yue

first: Bowei

last: Zhao

first: Zhuo

last: Meng

first: Binyang

last: Xie

first: Yueshi

last: Zhuang

last: Su

first: Junyu

last: Chen

first: Lijing


title: Differential retinoic acid responses across testicular development in vitro

city: Seattle

first: Edward

last: Kelly

state: Washington

address: 1959 NE Pacific St

institute: University of Washington

summary: Spermatogonial differentiation is controlled by distinct spatial and temporal activities of metabolizing enzymes and signaling molecule formation. In vitro models are reductionist ways to examine the interactions between Sertoli cells and spermatogonial stem cells, which may be therapeutic targets for infertility that cause non-obstructive azoospermia, due to either Sertoli-cell only  syndrome, hypospermatogenesis or maturation arrest.  Here, we find that nanomolar doses of isotretinoin are sufficient to drive Stra8 expression in vitro, an interaction that is both dose-dependent and inhibitable. We compare complex in vitro models (CIVMs) seeded from cells isolated post-natal day 5 (PND 5) and post-natal day 10 (PND 10) Sprague Dawley rat testis.  The CIVMs maintain metabolic capacity to produce bioactive retinoids form retinol, . We also investigate the impact of common media supplementations on spermatogonial phenotype and find that  they can impact  the expression of Stra8 and Plzf as markers of early differentiating and undifferentiated spermatogonia, respectively. These results highlight the power of in vitro models to investigate the dynamics of the spermatogonial niche.

overall_design: RNA-seq of in vitro cultures of PND 5 and PND 10 testis from three different litters each.

first: Brad

middle: C

last: Hansen

first: Sarah

middle: A

last: Hatem

first: Lucas

last: Huang

first: John

last: Amory

first: Elaine

last: Faustman


title: Differential Cellular Mechanisms Underlie Language and Executive Decline in Amyotrophic Lateral Sclerosis [Visium spatial transcriptomics]

city: New York

first: Christopher

last: Jackson

state: NY

address: 101 Avenue of the Americas

department: Center for the Genomics of Neurodegenerative Disease

institute: New York Genome Center

summary: Half of all amyotrophic lateral sclerosis (ALS) patients demonstrate a spectrum of cognitive and behavioral changes over the course of the disease, but the mechanisms underlying this heterogeneity remain unclear. We assemble a high-resolution cellular map of prefrontal cortex regions of the ALS brain by integrating spatial and single-nucleus transcriptomic profiles of a cognitively stratified ALS patient cohort that includes non-neuropathological controls. We find cellular programs characteristic of ALS, including a frequent gliotic response. We also find that executive and language cognitive impairments differ from ALS without cognitive impairment, and from each other, in the extent and pattern of neuronal dysregulation and neuron-glial interactions across different brain regions. These findings reveal a relationship between cognitive phenotype and prefrontal cortex dysfunction in ALS.

overall_design: 246 10x Visium spatial transcriptomic libraries generated from 64 human donor prefrontal cortex samples (BA44/45 and BA46), across a range of non-neuropathological sudden-death controls, ALS patients, and ALS patients who exhibit cognitive impairment on a standardized screening exam

first: C

first: A

last: Daly

last: Roque

first: J

last: Petrescu

first: H

last: Phatnani


title: Hybrid capture RNA-seq defines temporal gene expression in Rickettsia

city: Cambridge

first: Allison

last: Scott

state: MA

address: 31 Ames St

department: Biology

institute: Massachusetts Institute of Technology

summary: Pathogenic Rickettsia species are obligate intracellular bacteria that must reside in a mammalian host or arthropod vector cell to survive. Although these bacteria transition between different intracellular environments during infection, they encode few putative transcription factors, and their gene regulatory networks are largely unknown. Because of their inextricable relationship with eukaryotic cells, transcriptional profiling of the pathogen is complicated by the abundance of contaminating host RNA, especially in infection conditions or stages where the bacterial burden is inherently low. Here, we employ a hybrid capture technique (PatH-Cap) to improve library preparation by enriching bacterial transcripts while depleting host and rRNA molecules. Using PatH-Cap, we explored transcriptional changes throughout the first 24 hours of infection, including infection initiation – an infection stage which has been difficult to profile with standard library preparation methods. We then clustered genes based on their temporal trends, revealing cohorts of genes whose expression is up- or downregulated at different stages of infection. We also highlighted the diverse temporal expression trends of genes with known roles in growth and pathogenesis, including translation and cell division genes, secreted effectors, and secretion system components. Lastly, we identified 310 antisense RNA molecules, many of which also showed strong temporal trends. This work demonstrates that sensitive transcriptional profiling approaches like PatH-Cap hold great promise for dissecting gene expression networks driving infection in intracellular pathogens that have historically posed significant technical challenges.

overall_design: Hybrid capture (PatH-Cap) proof of concept with the intracellular bacterium, R. parkeri, at 0, 24, and 48 hours post infection (Samples 1-32). Time course PatH-Cap RNA-seq of R. parkeri during the first 24 hours of infection (Samples 33-86)

first: Vincent

last: Butty

first: Stuart

last: Levine

first: Rebecca

last: Lamason


title: Epigenetic Signatures of Brain Aging by Single Nucleus Methylation Sequencing [FC-8wk-m-018]

first: Joseph

last: Ecker

summary: Illustrating aging effects on the epigenomic features of brain is critical to understand brain functional decline and neurodegeneration. Single nucleus methylation sequencing was applied to brain regions of mouse at the age of 2, 9 and 18 months. We use these data to identify the epigenomic changes with aging in mouse brains.

first: Wei

last: Tian

first: Anna

last: Bartlett

first: Jordan

last: Altshul

first: Rosa

last: Gomez Castanon

last: Nery

first: Jacinta

last: Lucero

first: Julia

last: Osteen

first: Huaming

first: M

last: Behrens


title: Mutant SRSF2-associated impaired erythropoiesis is defined by increased mTORC1 signaling due to FYN missplicing

first: Edie

middle: I

last: Crosse

address: 1100 Fairview Ave N

institute: Fred Hutchinson Cancer Center

summary: Using mouse models and primary human samples, we investigated the impact of SRSF2 mutations on erythropoiesis. We found reduced erythropoiesis in Srsf2P95H versus wild-type mice upon stress-induced erythropoiesis and identified that SRSF2 mutations correlate with reduced hemoglobin in JAK2-mutant patients with myeloproliferative neoplasms (MPN). Consistent with this, Jak2V617F-Srsf2P95H versus Jak2V617F mice displayed reduced red blood cell counts and erythroid precursor frequencies. RNA-sequencing on erythroid precursors showed reduced expression of heme metabolism and mitotic spindle-related genes, and increased expression of mTORC1 signaling in Srsf2P95H versus wild-type cells. RNA splicing analyses on the same cells and on human patient samples identified aberrant FYN splicing in SRSF2mut cells, with increased aberrant FYNB over normal FYNT transcripts. FYNB, but not FYNT, expression resulted in reduced erythroid differentiation and increased phosphorylation of mTORC1 downstream target S6 kinase. Additionally, increased S6 phosphorylation was confirmed in primary Srsf2P95H erythroid cells. mTORC1 pathway inhibition using rapamycin normalized FYNB- and Srsf2P95H-induced impaired erythropoiesis and significantly increased erythroid colony formation of SRSF2-mutant myelodysplastic syndrome (MDS) bone marrow cells. Our data reveal targetable molecular mechanisms of impaired erythropoiesis in SRSF2-mutant cells.

overall_design: Mx1-Cre and Mx1-Cre Srsf2P95H/+ mice were subcutaneously injected with phosphate-buffered saline (PBS) or phenylhydrazine (PHZ, 30 mg/kg) on days 0 and 1, followed by sorting and RNA-sequencing of bone marrow-derived CFU-E cells on day 4.

last: Marneth

first: Jonas

last: Jutzi

first: Ann

last: Mullally

first: Robert

last: Bradley

first: Chulwoo

last: Kim


title: Bulk RNA-sequencing of pigs with UBE3A maternal deletion [Pig_CSC_bulkRNAseq]

city: College Station

first: Sarah

last: Christian

state: TX

address: 4467 TAMU

institute: Texas A&M University

summary: Angelman syndrome is a neurodevelopmental disorder caused by the loss of the maternal allele of the ubiquitin-protein ligase E3A (UBE3A) gene.  UBE3A is imprinted with maternal-allelic expression in neurons of the central nervous syndrome (CNS) and biallelically expressed in other cell types.  Consequently, in Angelman syndrome, UBE3A is absent in CNS neurons and reduced by half in other cells.  Using bulk RNA-sequencing, we show that gene expression is dysregulated in the cervical spinal cord of pigs with a UBE3A maternal deletion.

overall_design: Three wildtype pigs were compared to three pigs with a maternally-inherited deletion of the Ubiquitin-protein ligase E3A (UBE3A) gene. Cervical spinal cord tissue was isolated from male pigs approaching sexual maturity (140 days). Differential expression analysis was used to compare differences in gene expression that occur with maternal UBE3A loss.

first: Ashley

last: Coffell

first: Livia

last: Schuller

first: Scott

middle: V

last: Dindot


title: Bulk RNA-sequencing of pigs with UBE3A maternal deletion [Pig_Cortex_bulkRNAseq]

summary: Angelman syndrome is a neurodevelopmental disorder caused by the loss of the maternal allele of the ubiquitin-protein ligase E3A (UBE3A) gene.  UBE3A is imprinted with maternal-allelic expression in neurons of the central nervous syndrome (CNS) and biallelically expressed in other cell types.  Consequently, in Angelman syndrome, UBE3A is absent in CNS neurons and reduced by half in other cells.  Comparing both bulk and single nuclei RNA-sequencing, we show that gene expression is dysregulated in neuronal subtypes in the frontal cortex of pigs with a UBE3A maternal deletion.

overall_design: Three wildtype pigs were compared to three pigs with a maternally-inherited deletion of the Ubiquitin-protein ligase E3A (UBE3A) gene. Cortical tissue was isolated from the brains of male pigs approaching sexual maturity (140 days). Differential expression analysis was used to compare differences in gene expression that occur with maternal UBE3A loss.


title: Rotational settings quantize nucleosome movement by chromatin regulators

city: Aurora

first: Srinivas

last: Ramachandran

state: CO

department: Biochemistry and Molecular Genetics

institute: University of Colorado School of Medicine

summary: Proper nucleosome positioning is essential for gene regulation and genomic integrity. The need for nucleosomes to be assembled throughout the genome has to be balanced with known intrinsic sequence preferences of nucleosomes. Current models posit that chromatin regulators override the intrinsic preferences to establish the nucleosome landscapes observed in vivo. Here, in contrast, we find that chromatin regulators take advantage of both the preference of the nucleosomes for A/T dinucleotides to face inward and G/C outward every 10 bp relative to the histone surface and the existing dinucleotide frequencies of the genome to move nucleosomes in discrete 10 bp steps. We find that the rotational settings of nucleosomes are identical in vivo and in vitro in yeast. Remodelers and transcription elongation change the preference among the alternative translational positions 10 bp apart that a given rotational setting allows. Our results support a model where strong rotational settings introduce a 10 bp ratchet for nucleosome mobilization, leading to quantized shifts in nucleosome positions in yeast and mouse cells.

overall_design: MNase-seq of wild-type mouse embryonic stem cells with multiple time-points

first: Van

last: La

first: Abby

last: Trouth

first: Vijay

last: Ramani


title: Bronchial epithelial cell-derived extracellular vesicles drive inflammasome activation and NTHi infection in COPD

city: Belfast

first: Clifford

last: Taggart

address: 97 Lisburn Road

country: United Kingdom

institute: Queen's University Belfast

summary: Extracellular vesicles (EVs) are lipid-membrane bound vesicles that can be beneficial or detrimental depending on the content they carry. As epithelial cells are the first line of defense against harmful particles, this work explored the role of COPD bronchial epithelial cell-derived EVs (CepEVs) in the pathogenesis and progression of chronic obstructive pulmonary disease (COPD). RNA sequencing of macrophages stimulated with CepEVs revealed the upregulation of various inflammasome-related genes, alongside significant IL-1b and IL-18 release, which could be attenuated with caspase-1 or NLRP3 inhibition. The proteome of CepEVs was also assessed, which highlighted a significant reduction in antibacterial proteins compared to healthy EVs (HepEVs). When functionally assessed in NTHi infection of THP-1 cells, pre-incubation with HepEVs stimulated NTHi clearance and reduced pro-inflammatory cytokine release by macrophages, which was reduced in CepEV-stimulated cells. This study shows for the first time that CepEVs are able to both prime and activate the inflammasome in healthy macrophages, and highlights EV-induced inflammasome inhibition as a potential therapeutic target for the dysregulated inflammation seen in COPD. Alongside the inflammasome, we were also able to show that CepEVs are deficient for multiple antibacterial proteins, and that one or more of these proteins are essential in mounting an immune response against NTHi in macrophages. This finding contributes to a potential therapeutic pipeline through the supplementation of the depleted antibacterial proteins in CepEVs, allowing for efficient bacterial clearance and reduced consequential inflammatory burden. CepEV co-incubation resulted in a persistent state of inflammation and infection. Both sets of findings contribute to the overall knowledge of COPD pathogenesis, and highlight epithelial EVs as key players in the propagation of inflammation and susceptibility to infection.

overall_design: RNA-seq analysis of THP-1 cells stimulated with COPD bronchial epithelial cell-derived EVs or healthy bronchial epithelial cell-derived EVs for 48hrs


title: TrAEL-seq captures DNA replication

first: Laura

last: Biggins

address: Babraham Research Campus

department: Bioinformatics

institute: The Babraham Institute

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Impact of maternal compensation on developmental phenotypes in a zebrafish model of severe congenital muscular dystrophy

city: New Brunswick

first: M. Chiara

last: Manzini

state: NJ

address: 89 French Street, Child Health Institute of New Jersey

department: Neuroscience and Cell Biology

institute: Rutgers Robert Wood Johnson Me

summary: Genetic compensation is a common phenomenon in zebrafish in response to genetic alterations. Differences between genetic and morpholino-mediated zebrafish models of human diseases have led to significant difficulties in phenotypic interpretation and translatability. One form of compensation is the maternal deposit of mRNAs and proteins to the oocyte that supports developmental processes before zygotic genome activation. In this study, we generated a zebrafish model of severe congenital muscular dystrophy (CMD) by targeting protein O-mannose N-Acetylglucosaminyltransferase 2 (pomgnt2), a maternally provided gene that maintains cell-extracellular matrix interactions through glycosylation and leads to congenital muscular dystrophy when mutated. Zygotic knockouts (ZKOs) retain protein function in the first week post fertilization and survive to adulthood, only developing muscle disease later in life. In contrast, maternal-zygotic KOs (MZKOs) generated from ZKO females develop early-onset muscle disease, reduced motor function, neuronal axon guidance deficits, and retinal synapse disruptions recapitulating features of the human presentation. While assessing transcriptional changes linked to disease progression, the availability of embryos obtained from different breeding strategies also allowed for a direct comparison of ZKOs and MZKOs to define the impact of having a KO mother. We found that offspring from a ZKO mother, independently of genotype, show distinct expression patterns from animals obtained from heterozygous breedings. Some of these changes reflect changes in metabolic function, possibly stemming from maternal metabolic disruption. These findings will not only be applicable for other CMD models targeting maternally provided genes, but also provide new insight into modeling disease using maternal-zygotic mutants.

overall_design: RNA seq profiling of 5 days and 10 days post-fertilization zebrafish larvae with frameshift mutations in pomgnt2 generated from pomgnt2+/- and pomgnt2-/- female breeders

first: Kyle

middle: P

last: Flannery

first: Shorbon

last: Mowla

first: Namarata

last: Battula

first: L. Rose

last: Clark

first: Callista

last: Oliveira

first: Lillian

last: Simhon

first: Deze

last: Liu

first: Cynthia

last: Venkatesan

first: Brittany

middle: F

last: Karas

first: Kristin

last: Terez

first: Daniel

last: Burbano


title: RUVBL1 and RUVBL2 are druggable MYCN regulators in neuroblastoma

city: Ghent

first: Sarah-Lee

last: Bekaert

state: No state

address: Corneel Heymanslaan 10

country: Belgium

institute: UGent

summary: High-risk neuroblastoma (NB), accounting for approximately 50% of all cases, exhibits high relapse rates and life-threatening toxicities despite intensive therapies, highlighting the need for safer and more effective treatments. Analysis of publicly available NB transcriptomic datasets identified RUVBL2 as a key node in the MYC(N) regulatory network. High MYC signature scores were observed in high-risk NB cohorts, and multivariate Cox regression analysis showed that RUVBL1/2 expression is an independent predictor of both overall and event-free survival. Experimental validation through cell culture, western blotting, qPCR, and RNA-seq demonstrated that knockdown or pharmacological inhibition of RUVBL1/2 using CB-6644 induced replication stress, leading to cell cycle arrest, DNA damage, and apoptosis. Mechanistically, RUVBL1/2 regulated ATR and ATM protein stability and transcriptionally controlled MYC(N) expression. These findings indicate that RUVBL1 and RUVBL2 are novel regulators of the DNA damage response with both therapeutic and prognostic potential in high-risk NB.

overall_design: Identification of MYCN, RUVBL1 and RUVBL2  and various histone mark bindingsites through CUT&RUN sequencing in the neuroblastoma cell lines CLB-Bar

first: Joachim

last: Siaw

first: Kaat

last: Durinck

first: Jimmy

last: Van den Eynden


title: Different modes of engagement with the nucleosome acidic patch yield distinct functional outcomes

city: Durham

first: Michael-Christopher

last: Keogh

state: NC

address: 6 Davis Dr

institute: EpiCypher, Inc

summary: The nucleosome acidic patch is a hub of coordinated engagement by proteins that regulate genomic function. Here we report that S. cerevisiae Dot5 contains an arginine-rich HMGN-like motif that mediates nucleosome acidic patch binding and is required for the cell growth, DNA repair and heterochromatin defects that are exhibited when the protein is overexpressed. The heterologous expression of single chain antibodies to the nucleosome acidic patch confers a similar range of phenotypes, with the most severe observed when an ‘arginine-anchor’ mode of binding analogous to many endogenous factors is employed. This highlights a delicate balance between nucleosome acidic patch interactors that are critical for normal cellular functions and dysregulated in disease.

overall_design:
Genome binding/occupancy profiling by high throughput sequencing
CUT&RUN reactions performed in yeast (S. cerivisiae) cells

first: Ujani

last: Chakraborty

first: Emma

last: Saccone

first: Grisel

last: Cruz-Becerra

first: Laiba

last: Khan

first: Nina

last: Arslanovic

first: Rhiannon

last: Aguilar

first: Susan

last: Gloor

first: Sabrina

last: Hunt

first: Heather

last: Folkwein

first: Natalia

last: Ledo Husby

first: Keith

last: Maier

first: Matthew

last: Marunde

first: Noah

last: Schomburg

first: Anup

last: Vaidya

first: Martis

last: Cowles

first: Bryan

last: Venters

first: George

last: Kassavetis

first: Zu-Wen

last: Sun

first: James

last: Kadonaga

first: Jean-Paul

last: Armache

first: Jessica

last: Tyler


title: Maintenance of Embryonic Positional Identity Programs in the Adult Sheep Tail: Evidence from HOXB13 Spatial RNA Expression Gradients

city: München

first: Caroline

last: Friedel

address: Amalienstr. 17

department: Institut für Informatik

country: Germany

institute: Ludwig-Maximilians-Universität München

summary: Body axis patterning in vertebrates is controlled by HOX genes during embryogenesis, with their expression gradients defining spatial identity along the anterior–posterior axis. While their developmental roles are well known, less is understood about their persistence and function in adult tissues. Tail length in sheep provides a model for studying the phenotypic effects of HOX gene variants. We used Improved Jezersko–Solčava sheep, which segregate for both tail length and HOXB13 promoter variants, to examine genotype–phenotype associations. The HOXB13 genotype was the main determinant of adult tail length, acting primarily by modulating caudal vertebral number. RNA‑seq and qPCR analyses revealed a clear anterior–posterior HOXB13 expression gradient in adult tail skin and bones, with stronger distal expression in short‑tailed animals. These results provide in vivo evidence that spatially patterned HOX gene expression persists in adulthood, suggesting that residual developmental programs may maintain regional identity in adult tissues.

overall_design: RNA-seq profiling was performed on tail skin samples collected post-mortem from adult Improved Jezersko–Solčava rams with homozygous short-tail (A/A) or long-tail (D/D) HOXB13 genotypes. For each genotype (A/A: N=4; D/D: N=3), tissue was sampled at three defined anatomical positions along the anterior–posterior tail axis (base, mid-tail, tip) to investigate spatial expression gradients and genotype-specific transcriptional differences.

first: Simon

last: Horvat

first: Stefan

last: Kreb

first: Ivica

last: Medugorac


title: Peptidyl-tRNA hydrolase 2 is an essential negative regulator of peripartum cardiomyopathy with maternal heart failure (human and mouse RNA-seq)

city: Torrance

first: Michelle

last: Matter

address: 1124 West Carson Street

department: Genomics

institute: The lundquist Institute

summary: The peptidyl-tRNA hydrolase 2 (PTRH2, Bit-1; BIT1) gene plays a pro-survival role during development with loss of function gene mutations causing congenital infantile multisystem disease (IMNEPD). In wild-type female mice hearts, ptrh2 protein levels significantly increase during pregnancy and decrease postpartum, demonstrating a protective role in response to pregnancy-initiated cardiac stresses. Peripartum cardiomyopathy (PPCM) is due to dysregulated protective signaling in the pregnant heart. The genetic and molecular mechanisms underlying PPCM remain poorly defined with no specific therapies. Here, we engineered a cardiac-specific Ptrh2 knockout (Ptrh2-CKO) mouse and show these maternal mice develop left ventricular systolic dysfunction, exhibit high rates of postpartum heart failure, and model key features of human PPCM. Infusion of a caspase 3-specific inhibitor attenuated the PPCM phenotype. Collectively, our findings demonstrate Ptrh2 is a negative regulator of pregnancy-induced cardiac stresses by activating pro-survival signals and blocking apoptotic signals, suggesting Ptrh2 may be a therapeutic target for the treatment of PPCM

overall_design: RNA-seq of LV tissue from donor and PPCM patients. The hypothesis tested in the present study was that pregnancy induced stress alters gene expression that may lead to maternal heart failure. Results identify up-and down-regulated genes that are altered in PPCM/DCM (humans and mice).

first: Vanessa

last: Montoya-Uribe

first: Pooja

last: Choubey


title: Influenza hijacks circulating myeloid cells to inflict Type-I Interferon-fueled damage in the heart

first: Filip

last: Swirski

address: 1470 Madison Avenue, 7th Floor

department: Cardiovascular Research Institute

institute: Icahn School of Medicine at Mount Sinai

summary: Infections by influenza A viruses remain a threat to human health. Although much of the virus’s pathogenesis is confined to the lung, there is evidence that IAV inflames and damages other organs, including the heart. Our understanding of how this occurs, however, is lacking. Here, we identify a hijacked heart-lung axis, whereby influenza infects a relatively minor and ontologically unusual monocyte which preferentially migrates to the heart, differentiates into a cardiac macrophage, and disseminate the virus exclusively to cardiomyocytes, which then support active viral replication. The arrival of influenza in the heart initiates TLR3-dependent IFN-I production by myeloid cells, which signal through the IFN-I receptor (IFNAR1) on cardiomyocytes to cause damage and compromise heart function. Therapeutically dampening IFNAR1 on cardiomyocytes or limiting monocyte accumulation protects the heart while allowing for anti-viral immunity in the lung. Our results uncover an axis for intervention to mitigate cardiovascular risk following influenza infection.

sample_organism: Influenza A virus (A/Puerto Rico/8/34/Mount Sinai(H1N1))

overall_design: All experiments were performed on sex- and age-matched mice between the ages of 5 and 12 weeks. In certain experiments, where indicated, aged mice were used. A group of mice were infected with Influenza A virus. Heart tissues from a group of infected and uninfected mice were extracted and single-nuclear RNA-seq (10X Genomics) was performed.

first: Jeffrey

last: Downey

first: Ana

last: Oliviera-Coelho

first: Mate

middle: G

last: Kiss

first: Emir

last: Radkevich

first: Darwin

last: D'Souza

first: Zhihong

last: Chen

first: Seunghee

last: Kim-Schulze


title: Functional characterization of GWAS findings highlights a role for REST in endothelial plasticity and atherosclerosis [RNA-seq]

city: Columbus

first: Marios

last: Arvanitis

state: OH

address: 473 W 12th Ave

institute: The Ohio State University

summary:
Background: Genome-wide association studies (GWAS) have identified multiple novel loci that contribute to coronary artery disease (CAD) pathogenesis, but the mechanisms of these associations remain largely unknown.
Methods: In this study we used a multi-trait colocalization approach to prioritize novel endothelial specific loci for atherosclerosis. We combined computational methods with in vitro assays and mouse models to study one of those new loci targeting gene REST.
Results: A multi-trait colocalization approach across expression quantitative trait loci (eQTL) in atherosclerosis-relevant cell types followed by in vitro CRISPR interference revealed that a conserved regulatory element in a chromosome 4 genetic locus increases risk of CAD and decreases the
expression of REST, a transcriptional repressor, in endothelial cells. Pcsk9-overexpressing mice with an endothelial-specific knockout of Rest exhibited increased atherosclerotic plaque formation in their aortas, with increased macrophage and lipid deposition within the plaque after 16 weeks of high-fat diet exposure compared to littermate controls. RNA-seq in human aortic endothelial cells (HAEC) after REST silencing followed by assessment of protein expression revealed that REST silencing triggers endothelial-to-mesenchymal transition (endMT). Consistently, REST silencing increased endothelial permeability and migration in vitro. Single nucleus RNA sequencing in endothelial lineage traced atherosclerotic mice with Rest knock-out revealed evidence of endothelial TGFb signalling activation and of transition smooth muscle-like cells in atherosclerotic aortas upon genetic knockout of Rest. CUT&Tag sequencing did not identify any known TGFb effector genes as direct REST transcriptional targets. Instead, joint analysis of CUT&Tag with RNA-seq, highlighted L1CAM, a known endMT activator, and its interactors as the most significant gene-set directly affected by REST in the endothelium. Simultaneous silencing of L1CAM and REST in HAEC inhibited the upregulation of mesenchymal genes and the enhanced migration induced by REST silencing and diminished the upregulation of several TGFb effectors overexpressed upon REST silencing.
Conclusion: In summary, our data reveal the novel role of REST as a repressor in endothelial cells that functions to constitutively inhibit endMT and protect against atherosclerosis.
Key words: GWAS, NRSF/REST, endothelial to mesenchymal transition, CRISPRi, CUT&TAG, endothelial cells, L1CAM, atherosclerosis

overall_design: To evaluate cellular mechanisms affected by REST in the endothelium we performed silencing of REST in HAEC followed by RNA-seq

first: Charles

last: Lowenstein

first: Pablo

last: Toledano-Sanz


title: Functional characterization of GWAS findings highlights a role for REST in endothelial plasticity and atherosclerosis [snRNA-seq]

overall_design: We assess whether endMT is observed in vivo during atherosclerosis upon endothelial Rest knock-out. To do that we crossed our endothelial Rest knock-out mice and corresponding endothelial Cre mice controls with a reporter mT/mG mouse. By treating these mice with tamoxifen after 8 weeks of age, we are able to simultaneously knock-out endothelial Rest and label endothelial cells with green fluorescence permanently, thereby permitting lineage tracing of those cells during atherosclerosis progression. We injected those mice with AAV8-m-Pcsk9 and exposed them to 16 weeks HFD as above to induce atherosclerosis. We subsequently isolated their aortas and performed single nucleus RNA-sequencing


title: Functional characterization of GWAS findings highlights a role for REST in endothelial plasticity and atherosclerosis [CUT&TAG]

summary: Background: Genome-wide association studies (GWAS) have identified multiple novel loci that contribute to coronary artery disease (CAD) pathogenesis, but the mechanisms of these associations remain largely unknown. Methods: In this study we used a multi-trait colocalization approach to prioritize novel endothelial specific loci for atherosclerosis. We combined computational methods with in vitro assays and mouse models to study one of those new loci targeting gene REST. Results: A multi-trait colocalization approach across expression quantitative trait loci (eQTL) in atherosclerosis-relevant cell types followed by in vitro CRISPR interference revealed that a conserved regulatory element in a chromosome 4 genetic locus increases risk of CAD and decreases the expression of REST, a transcriptional repressor, in endothelial cells. Pcsk9-overexpressing mice with an endothelial-specific knockout of Rest exhibited increased atherosclerotic plaque formation in their aortas, with increased macrophage and lipid deposition within the plaque after 16 weeks of high-fat diet exposure compared to littermate controls. RNA-seq in human aortic endothelial cells (HAEC) after REST silencing followed by assessment of protein expression revealed that REST silencing triggers endothelial-to-mesenchymal transition (endMT). Consistently, REST silencing increased endothelial permeability and migration in vitro. Single nucleus RNA sequencing in endothelial lineage traced atherosclerotic mice with Rest knock-out revealed evidence of endothelial TGFb signalling activation and of transition smooth muscle-like cells in atherosclerotic aortas upon genetic knockout of Rest. CUT&Tag sequencing did not identify any known TGFb effector genes as direct REST transcriptional targets. Instead, joint analysis of CUT&Tag with RNA-seq, highlighted L1CAM, a known endMT activator, and its interactors as the most significant gene-set directly affected by REST in the endothelium. Simultaneous silencing of L1CAM and REST in HAEC inhibited the upregulation of mesenchymal genes and the enhanced migration induced by REST silencing and diminished the upregulation of several TGFb effectors overexpressed upon REST silencing. Conclusion: In summary, our data reveal the novel role of REST as a repressor in endothelial cells that functions to constitutively inhibit endMT and protect against atherosclerosis. Key words: GWAS, NRSF/REST, endothelial to mesenchymal transition, CRISPRi, CUT&TAG, endothelial cells, L1CAM, atherosclerosis

overall_design: Genes directly repressed by REST should have their promoter or nearby enhancer sites bound by REST in endothelial cells. To discover those bound regulatory elements, we performed CUT&Tag for REST in HAEC.


title: Transcriptional Profiling of Zebra Finch Hypothalamus After Chronic Heat Call Exposure

city: Clemson

first: Prakrit

last: Subba

state: SC

address: 132 Long Hall, 230 Parkway Drive

department: Biological Sciences

institute: Clemson University

summary: Late-stage zebra finch embryos were exposed to chronic playback of parental heat calls or control sounds to test how acoustic cues of extreme heat reshape hypothalamic gene expression. RNA-seq of medial hypothalamic punches revealed modest changes in neuroendocrine genes but strong downregulation of muscle and cytoskeletal programs, with evidence of altered isoform usage and enrichment in neurovascular cell populations. These findings indicate that parental heat calls drive a non-neuroendocrine transcriptional program underlying early-life developmental plasticity and acoustic-mediated adaptation to environmental extremes in birds.

overall_design: Embryonic zebra finch (Taeniopygia guttata) eggs from breeding pairs were incubated under controlled temperature and randomly assigned to either parental heat-call playback or control-call playback from embryonic day 9 (E9) to E12 (9 h/day). On E13, embryos from both groups were collected within a fixed morning window, and 1 mm medial hypothalamic punches were taken from 100 μm coronal sections at the level of the third ventricle; both males and females were included and sex was recorded for all individuals. RNA isolated from each punch was sequenced (75 bp paired-end, Illumina NextSeq 500), quantified against the zebra finch reference transcriptome, and analyzed at the gene and isoform level using models that tested effects of playback condition and sex, with additional weighted gene co-expression network, pathway enrichment, and cell-type deconvolution analyses to link transcriptional changes to hypothalamic cell populations.

first: Katerina

last: Palios

first: Michael G.

last: Emmerson

first: Elisabetta

last: Versace

first: Katherine L.

last: Buchanan

first: Mylene M.

last: Mariette

first: David F.

last: Clayton

first: Julia M.

last: George


title: Activation of the aryl hydrocarbon receptor in mature adipocytes does not impact diet-induced obesity in mice [scRNA-seq]

city: London

first: LMS

last: Bioinformatics Core

address: Hammersmith Hospital Campus, Du Cane Road

department: Bioinformatics Core

institute: MRC London Institute of Medical Sciences

summary: Background: Despite rising awareness and advances in the field, the prevalence of obesity continues to increase. Current weight-loss treatments are often ineffective long-term, and some are associated with serious side effects. For this reason, alternative pathways need to be explored in order to identify novel pharmacological targets for the treatment of obesity.  In this study, we aim to investigate the role of the ligand-activated transcription factor, aryl hydrocarbon receptor (AHR), in the development of obesity and metabolic syndrome. Methods & Results: Using global and adipocyte-specific Ahr knock-out mouse models, we show that even though global Ahr deletion has some protective effects from diet-induced obesity and metabolic syndrome, adipocyte-specific deletion of Ahr has no effect. Furthermore, using a variety of techniques to isolate mature adipocytes, such as flotation, in-vitro differentiation, and translating ribosome affinity purification, we show no evidence that Ahr is expressed in mature white adipocytes. Finally, using single-cell RNA sequencing on cells from a Cyp1a1 fate reporter mouse line, in which eYFP can be used as a readout of AHR activation, we show that the AHR pathway is active in adipose stem and precursor cells. Using bulk RNA sequencing on adipocyte precursors stimulated with an AHR ligand in-vitro, we show evidence suggesting that Ahr may be involved in the control of adipocyte differentiation. Conclusions: Our study indicates that Ahr may play an important role in adipocyte precursors, particularly in the initiation of adipocyte differentiation. We suggest that these results warrant further research in this mechanism in the context of obesity.

overall_design: Cyp1a1-expressing (eYFP positive) cells from the stromovascular fraction of the perigonadal white adipose tissue of Cyp1a1 reporter mice fed a low or high fat diet for 12 weeks.

first: Julia

last: Vlachaki Walker

first: Marian

last: Dore

first: Chris

last: Schiering


title: Activation of the aryl hydrocarbon receptor in mature adipocytes does not impact diet-induced obesity in mice


title: Gene expression changes were assessed in tumor cells treated with epigenetic inhibitors and after drug withdrawal

city: PITTSBURGH

first: runzi

last: sun

state: PA

address: 203 Lothrop st

institute: university of pittsburgh

summary: Sequential therapy is a promising strategy to enhance antitumor efficacy; however, the rational design of sequential drug treatments remains challenging. Here, we introduce an AI framework, AlphaTherapy, which can design sequential drug combinations for cancer treatment. Analysis of AlphaTherapy-predicted combinations and large-scale in vitro validation revealed that the first drug in effective anti-tumor combinations often targets transcription, particularly epigenetic inhibitors. To investigate how epigenetic drugs enhance the efficacy of subsequent treatments, RNA-seq was performed on tumor cells treated with epigenetic inhibitors followed by drug withdrawal to assess gene expression changes.

overall_design: RNA-seq was performed on the following cell lines under specified treatment conditions: A375 cells: untreated control (DMSO), treatment (2 µM I-BET151 for 72 h), and withdrawal treatment (2 µM I-BET151 for 72 h followed by 72 h without drug). MDA-MB-231 cells: untreated control (DMSO), treatment (2 µM OTX-015, 5 µM GSK126, 5 µM UNC1999, 10 µM CCS1477, and 5 µM A485 for 72 h), and withdrawal treatment (drug treatment for 72 h followed by 72 h without drug). MCF7 cells: untreated control (DMSO), treatment (10 µM CCS1477 for 72 h), and withdrawal treatment (drug treatment for 72 h followed by 72 h without drug).

first: Yue

last: Deng

first: Shuxian

last: Li

first: Xun

last: Huang


title: NSD2 targeting reverses lineage plasticity and drug resistance in advanced prostate cancer [bulk RNA-seq]

first: Jia

middle: Jessie

department: Medicine

institute: Columbia University Irving Medical Center

summary: Treatment with highly potent androgen receptor signaling inhibitors (ARSIs), such as enzalutamide and abiraterone promotes lineage plasticity in metastatic castration-resistant prostate cancer (mCRPC), which results in intra-tumor heterogeneity and emergence of mCRPC subtypes. The histological transformation from adenocarcinoma to aggressive neuroendocrine prostate cancer (CRPC-NE) has been associated with a loss of dependency on lineage-survival signals, leading to targeted drug resistance. Epigenomic reprogramming might be a fundamental driver of lineage plasticity. To determine CRPC-NE vulnerabilities, we have performed image-based screening using a small library of antibodies targeting different histone marks on CRPC-NE organoids derived from genetically engineered mice. We find that the histone mark H3K36me2 and the histone methyltransferase NSD2 (also known as MMSET/WHSC1) play important roles in maintaining the state of CRPC-NE. Knockout of NSD2 or ablation of H3K36me2 using H3.3K36M oncohistone reverted CRPC-NE to CRPC-AR-like phenotype. Simultaneous profiling of the transcriptome and epigenome from single cells collected from control (sgCtrl) and NSD2 knockout (sgNSD2) organoids, or from empty vector (EV) and H3.3K36M transduced organoids confirms the lineage reversal of treatment-resistant cells and maps the re-establishment of canonical AR signaling dependency. Moreover, H3K36me2 or NSD2 depleted mouse and human CRPC-NE organoids responded to enzalutamide treatment in vitro and in vivo, suggesting a restoration of ARSI sensitivity. Most importantly, a small molecule inhibitor of NSD2, which is similar to KTX-1001 (NCT05651932), in combination with enzalutamide leads to growth suppression or apoptosis of mouse and human CRPC-NE organoids, organoids of other CRPC subtypes and xenografts in vitro and in vivo. In conclusion, inhibition of NSD2 reverses lineage plasticity and reverts the state of treatment-resistant cells back into an ARSI-sensitive state. Thus, we suggest that the combination of NSD2 inhibition with AR inhibition may represent a novel therapeutic approach for patients with CRPC-NE and other CRPC subtypes.

overall_design: The mouse NE tumors derived from NPp53 (Nkx3.1CreERT2/+; Ptenflox/flox; TrpP53flox/flox; Rosa26-EYFP) mice were cultured as organoids. To detect intercellular heterogeneity within organoids, I used single-cell RNA sequencing (scRNA-seq) to analyze lineage diversity of five mouse NE tumor organoids on passage 2. I also used single-nucleus ATAC sequencing (snATAC-seq) to profile the chromatin accessibilities of NPPO-1 organoids on passage 8, a multilineage NE tumor organoid line. The non-NE tumor cells from NPPO-1 organoids on passage 3 were isolated by fluorescence-activated cell sorting (FACS) according to a higher side scatter (SSC) value in non-NE compared to NE tumor cells. Some isolated non-NE tumor cells were analyzed by scRNA-seq to detect any contamination by NE tumor cells. Other isolated non-NE tumor cells were transduced with H2B-RFP lentivirus without antibiotic selection. H2B-RFP labeled tumor cells were either cultured alone or co-cultured with NE tumor cells to induce non-NE to NE conversion. To trace cell lineage of H2B-RFP-labeled cells, organoids from either monoculture or co-culture on passage 4 were analyzed by scRNA-seq (gene name: addgene26001). To quantitively compare the histone mark expression between NE and non-NE cells, epigenomic profiling using Cut&Tag was performed on 4 NE and 4 non-NE organoids derived from the same NPp53 background. To accurately quantify gene regulation, bulk RNA-seq was performed alongside epigenomic profiling on the organoids. To study the effects of NSD2 on NE lineage maintenance, we knockout of NSD2 on two mouse NE tumor organoids using CRISPR/Cas9. After antibiotic selection for 14 days, positive-transfected cells were isolated by FACS sorting according to the presence of RFP reporter signal. We used multiome single-nucleus ATAC and RNA sequencing (multiome snATAC + snRNA-seq) to analyze changes of cell lineage and state in sgNSD2 transfected NE tumor organoids in compared with sgControl (sgCtrl) organoids. To provide direct evidence of H3K36me2 histone modification in modulating NE lineage, mouse NE tumor organoids were transduced with lentivirus carrying the gene encoding the H3.3K36M oncohistone that can cause significant reduction in global levels of H3K36me2. After antibiotic selection for 14 days, cells positively transfected with H3.3K36M or empty vector (EV) control were analyzed using multiome snATAC + snRNA-seq to detect changes in cell lineage and state. In parallel, quantitative epigenomic profiling using Cut&Tag was performed on sgCtl and sgNSD2, or EV and H3.3K36M organoids to quantify global epigenomic changes. We have synthesized a small molecular inhibitor of NSD2 (NSD2i), which is similar to KTX-1001 (NCT05651932) in phase 1 clinical trial in patients with relapsed and refractory multiple myeloma. To evaluate if NSD2i recapitulates the effects of genetic interventions in reversal of lineage plasticity and restoring androgen receptor signaling inhibitor (ARSI) sensitivity, 1 µM NSD2i or DMSO was applied every other day on NPPO-1NE organoids cultured in a medium without DHT for 12 days. Organoids treated with either DMSO or 1 µM NSD2i were then collected for multiome snATAC + snRNA-seq analysis or Cut&Tag epigenomic profiling.

first: Kristjan

middle: H

last: Gretarsson

first: Michael

last: Shen


title: RNA-seq of A375 cells treated with I-BET726 alone and sequential combination of I-BET726 and Oxaliplatin

city: Shanghai

first: Xiaohan

last: Chen

address: Tongji University

institute: Tongji University/ major in Bioinformatics

summary: Sequential therapy is a promising approach to enhance antitumor efficacy; however, rational design of sequential drug treatments remains challenging. Here, we applied an AI framework, AlphaTherapy, to identify a specific sequential drug combination of I-BET726 and Oxaliplatin that exhibits antitumor effects in melanoma (A375 cells). To investigate the molecular mechanisms underlying the antitumor activity of this combination, gene expression was profiled by RNA sequencing (RNA-seq) after treatment.

overall_design: RNA-seq was performed on A375 cells under the following conditions: untreated control (DMSO), 2 µM I-BET726 for 72 h, withdrawal treatment (2 µM I-BET726 for 72 h followed by 72 h without drug), and sequential treatment (2 µM I-BET726 for 72 h followed by 10 µM Oxaliplatin for 72 h).


title: NSD2 targeting reverses lineage plasticity and drug resistance in advanced prostate cancer [Cut&Tag]

overall_design: The mouse NE tumors derived from NPp53 (Nkx3.1CreERT2/+; Ptenflox/flox; TrpP53flox/flox; Rosa26-EYFP) mice were cultured as organoids. To detect intercellular heterogeneity within organoids, I used single-cell RNA sequencing (scRNA-seq) to analyze lineage diversity of five mouse NE tumor organoids on passage 2. I also used single-nucleus ATAC sequencing (snATAC-seq) to profile the chromatin accessibilities of NPPO-1 organoids on passage 8, a multilineage NE tumor organoid line. The non-NE tumor cells from NPPO-1 organoids on passage 3 were isolated by fluorescence-activated cell sorting (FACS) according to a higher side scatter (SSC) value in non-NE compared to NE tumor cells. Some isolated non-NE tumor cells were analyzed by scRNA-seq to detect any contamination by NE tumor cells. Other isolated non-NE tumor cells were transduced with H2B-RFP lentivirus without antibiotic selection. H2B-RFP labeled tumor cells were either cultured alone or co-cultured with NE tumor cells to induce non-NE to NE conversion. To trace cell lineage of H2B-RFP-labeled cells, organoids from either monoculture or co-culture on passage 4 were analyzed by scRNA-seq (gene name: addgene26001). To quantitively compare the histone mark expression between NE and non-NE cells, epigenomic profiling using Cut&Tag was performed on 4 NE and 4 non-NE organoids derived from the same NPp53 background. To accurately quantify gene regulation, bulk RNA-seq was performed alongside epigenomic profiling on the organoids. To study the effects of NSD2 on NE lineage maintenance, we knockout of NSD2 on two mouse NE tumor organoids using CRISPR/Cas9. After antibiotic selection for 14 days, positive-transduced cells were isolated by FACS sorting according to the presence of RFP reporter signal. We used multiome single-nucleus ATAC and RNA sequencing (multiome snATAC + snRNA-seq) to analyze changes of cell lineage and state in sgNSD2 transfected NE tumor organoids in compared with sgControl (sgCtrl) organoids. To provide direct evidence of H3K36me2 histone modification in modulating NE lineage, mouse NE tumor organoids were transduced with lentivirus carrying the gene encoding the H3.3K36M oncohistone that can cause significant reduction in global levels of H3K36me2. After antibiotic selection for 14 days, cells positively transduced with H3.3K36M or empty vector (EV) control were analyzed using multiome snATAC + snRNA-seq to detect changes in cell lineage and state. In parallel, quantitative epigenomic profiling using Cut&Tag was performed on sgCtl and sgNSD2, or EV and H3.3K36M organoids to quantify global epigenomic changes. We have synthesized a small molecular inhibitor of NSD2 (NSD2i), which is similar to KTX-1001 (NCT05651932) in phase 1 clinical trial in patients with relapsed and refractory multiple myeloma. To evaluate if NSD2i recapitulates the effects of genetic interventions in reversal of lineage plasticity and restoring androgen receptor signaling inhibitor (ARSI) sensitivity, 1 µM NSD2i or DMSO was applied every other day on NPPO-1NE organoids cultured in a medium without DHT for 12 days. Organoids treated with either DMSO or 1 µM NSD2i were then collected for multiome snATAC + snRNA-seq analysis or Cut&Tag epigenomic profiling.

middle: M


title: Functional correction and genome integrity with duplex base editing of β-thalassemic hematopoietic stem cells

city: Nicosia

first: Nikoletta

middle: Y.

last: Papaioannou

address: Iroon Avenue

department: Molecular Genetics Thalassemia Department

country: Cyprus

institute: The Cyprus Institute of Neurology and Genetics

summary: Background: Beta-thalassemia is among the commonest monogenic disorders, posing a major global health challenge. Editing of genetic modifiers of β-thalassemia, such as BCL11A erythroid enhancer and HBG promoters, enhances fetal hemoglobin (HbF) expression and confers major therapeutic potential. Double-strand-break (DSB)-independent genome editing tools, such as base editors, are potentially safer and better suited for multiplexed application than traditional DSB-dependent CRISPR/Cas technology. However, harmful inadvertent on- and off-target events remain a concern and must be excluded before clinical application, including chromosomal rearrangements, which are invisible to standard detection technologies. Results: Using primary patient-derived CD34+ cells from three donors, we investigated simplex and duplex BE-based disruption of the BCL11A erythroid enhancer and the BCL11A binding site (-115bp) on the HBG promoter for DNA-level events and functional studies at the RNA, protein, and morphological level. Analyses included direct comparison to DSB-based editing, as the current clinically applied standard, and analysis of DNA recombination events by CAST-seq to allow wider inferences for relative safety of DSB-, BE- and duplex BE-based editing. Our study reveals the effectiveness of duplex base editing, with robust γ-globin and HbF induction and significantly improved functional correction over simplex editing. Moreover, duplex editing resulted in low incidence of simple and complex genomic alterations in both therapeutically relevant target loci. Conclusions: Here we display simultaneous duplex base editing by targeting both BCL11A erythroid enhancer and HBG promoter for functional correction and genome integrity. Our study highlights the efficacy, safety, and therapeutic potential of the present duplex BE approach

overall_design: After the collection of BFU-E colonies and the investigation of editing levels, three colonies from each treatment were selected for RNA sequencing by Genewiz (Germany). For RNA sequencing workflow, the ultra-low Input RNA-seq assay was performed. The RNA was enriched for full-length transcripts by poly(A) selection and was then processed for library preparation including cDNA synthesis and adapter ligation. Samples were sequenced on the Illumina HiSeq or NovaSeq platforms with 2× 150 bp read length. Raw RNA-Seq data in FASTQ format were trimmed to remove adapter sequences and low-quality nucleotides using Trimmomatic v0.36. The trimmed reads were mapped to the Homo sapiens GRCh38 reference genome available on ENSEMBL using the STAR aligner v2.5.2b, a splice-aware aligner that detects splice junctions and incorporates them into the alignment to improve mapping accuracy, generating BAM files. Unique gene hit counts were then obtained for each sample, and using DESeq2, a comparison of gene expression between the CONTROL vs. DSB-Cas9, CONTROL vs. BE-BCL11A, CONTROL vs. HBG and CONTROL vs. 2xBE groups of samples was performed. The Wald test was applied to generate p-values and log2 fold change values, producing tables of differential expression for all genes as well as significantly differentially expressed genes.

middle: Y

first: Petros

last: Patsali

last: Klermund

first: Panayiota

last: Papasavva

first: Geoffroy

last: Andrieux

first: Lola

last: Koniali

first: Basma

last: Naiisseh

first: Soteroula

last: Christou

first: Maria

last: Sitarou

first: Marina

last: Kleanthous

first: Toni

last: Cathomen

first: Carsten

last: Lederer


title: Transcriptomic changes upon doxorubicin treatment in the brain of ApoE3 and ApoE4 rats

first: Chadni

last: Patel

address: 195 Little Albany Street

institute: Rutgers Cancer Institute

summary: Many pediatric cancer survivors experience chemotherapy-induced cognitive impairment (CICI), which negatively impacts the quality of life and the interpatient variability in susceptibility to CICI is not well understood. Among pediatric cancer survivors, those with the E4 allele of Apolipoprotein E (ApoE) are more likely to exhibit cognitive dysfunction than those with the more prevalent ApoE3 allele, suggesting the ApoE4 allele increases susceptibility to CICI. To mimic a curative pediatric treatment regimen, in our experimental model, five-week-old rats homozygous for either human ApoE3 or ApoE4 allele were treated with doxorubicin (2mg/kg/week for 4 weeks) or PBS (saline). This study aims to investigate transcriptomic changes in different regions of brain to determine the impact of doxorubicin treatment in ApoE3 and ApoE4 rats.

overall_design: RNA-Sequencing was performed to characterize changes in different regions of the brain (cortex and hippocampus) of ApoE3 and ApoE4 rats after doxorubicin treatment. Rats were treated with doxorubicin (2 mg/kg/week for 4 weeks) starting at the age of 5 weeeks. 5-6 weeks after the last doxorubicin treatment, the hippocampus and cortex regions of the brains were collected for RNA-seq.

first: Peter

middle: D

last: Cole


title: Blocking protein quality degradation leads to structural stabilization of DHFR indel variants

city: Copenhagen

first: Sven

last: Larsen-Ledet

state: N

address: Ole Maaloes Vej 5

country: Denmark

institute: University of Copenhagen

summary: Gene variants leading to insertions or deletions of amino acid residues (indels) often have detrimental consequences for the folding of the encoded protein. Yet at some positions indels are tolerated or only result in partial unfolding. Typically unfolded proteins are targeted for protein quality control (PQC) degradation via ubiquitin-proteasome system, which in yeast is mediated by specific E3 ubiquitin-protein ligases, including Ubr1 and San1. Here we systematically probed the folding of a library of indel variants in the DHFR protein using sensitive yeast-based protein folding reporter. We show that deletion of Ubr1 and San1 leads to a greater fraction of folded DHFR indel variants, primarily positioned towards the N- and C-termini regions in DHFR. Intriguingly, most of the DHFR indels that are structurally stabilized in the E3 knockout strains, are also stabilized at lowered temperatures and upon binding the DHFR inhibitor methotrexate. This suggests that blocking PQC degradation can restore function to partially unfolded hypomorph variants, thus providing a potential therapeutic avenue for protein misfolding diseases.

overall_design: The human DHFR sequence was divided into five tiles. In each tile, we generated indel libraries comprising all possible deletions and insertions of a single glycine residue that was inserted into CPOP folding sensor and cloned into pAG415. The libraries were transformed into the ura5Δura10Δ strain (WT strain), ura5Δura10Δubr1Δ strain (Ubr1 strain), and ura5Δura10Δsan1Δ strain (San1 strain). Cells were plated on medium without uracil at 30 °C and 35 °C to select for folding of DHFR indel variants. Based on the relative change in frequency, we calculated a CPOP folding score of each DHFR indel variant.

first: Rasmus

last: Hartmann-Petersen


title: ADH1C downregulation is a key hypoxia response in colon epithelium

city: Baltimore

first: Maged

last: Zeineldin

state: MD

address: 1812 Ashland Ave

institute: Johns Hopkins University School of Medicine

summary: Defining how normal epithelial cells adapt to microenvironmental perturbations may reveal molecular vulnerabilities obscured later in carcinogenesis. Although the colon is susceptible to hypoxic injury and hypoxia is common in colorectal cancer (CRC), the insight into how non-neoplastic colon epithelium responds to hypoxia is lacking. To discover these mechanisms, human colon organoids (colonoids) were subjected to sustained hypoxia in vitro with characterization of consequent phenotypes and transcriptional changes. Hypoxia tolerance in human colonoids resulted in robust downregulation of alcohol dehydrogenase 1C (ADH1C), previously associated with poor prognosis and metabolic reprogramming in CRC. In normal crypts, ADH1C transcripts localized to transit amplifying (TA) and progenitor epithelial cells, and colon epithelial ADH1C downregulation was validated in archival tissue from patients with acute ischemic colitis, a form of hypoxic injury. Hypoxia induced PHGDH (the first and rate-limiting step in the serine synthesis pathway, SSP) in colonoids in an ADH1C-dependent manner, and the SSP contributed to neutralization of hypoxia-induced reactive oxygen species (ROS) in colonoids. Hypoxia-induced ADH1C downregulation was also associated with reduced TA cell signatures and increased expression of regeneration-associated stem cell marker FGFBP1. Finally, ADH1C-low CRC showed significant enrichment for hypoxia-associated colon epithelial signatures. Taken together, these results establish ADH1C as a mediator of colon epithelial hypoxia responses, metabolic reprogramming, and epithelial identity with relevance to human CRC.

overall_design:
RNA-seq for 4 differed colon epithelial organoid lines grown under either abient oxygen concentration or at 0.2% oxygene for one week. RNA-seq as paired end sequencing.
RNA-seq for 3samples of ischemic colitis. Regions with ischemia compared to normal regions in the same patients.

first: Tianhao

last: Bi

first: Varuni

last: Rastogi

first: Yi

last: Dong

first: Reem

last: Abu-Shamma

first: Tatianna

last: Larman


title: Autocrine TGFβ2 enforces a transcriptionally hybrid cell state in Ewing sarcoma

city: Seattle

first: Emma

last: Wrenn

state: WA

address: 1920 Terry Ave

institute: Seattle Children's Research Institute

summary: Sub-populations of cancer-associated fibroblast (CAF)-like tumor cells deposit extracellular matrix (ECM) proteins that support Ewing sarcoma (EwS) progression and metastasis. We previously showed a hallmark of CAF-like EwS cells is their hybrid transcriptional state wherein the driver fusion oncogene, EWS::FLI1, maintains activation of proliferative programs but loses capacity to repress mesenchymal genes. Here, we studied primary patient tumors and cell line models to identify molecular drivers of this hybrid state. Our data reveal that hybrid EwS cells are induced and maintained by a TGFβ signaling positive feedback loop. Hybrid cells de-repress TGFBR2 and upregulate expression and secretion of TGFβ2 to sustain pathway activation and ECM deposition. While TGFβ ligands can potently induce growth arrest in cells of epithelial origin, we show that TGFβ1 and TGFβ2 have no impact on EwS proliferation but instead promote cell invasion. Thus, stroma and tumor-derived TGFβ ligands induce and maintain hybrid EwS cells to promote metastatic phenotypes.

overall_design: Poly(A)-capture RNA-seq was performed on RNA (purified using Qiagen RNeasy kit) from TC71, A673, CHLA10, and PDX305 cells treated with TGFβ1 (10 ng/mL), TGFβ2 (10 ng/mL), vactosertib (1uM), or vehicle controls for 24hrs. Paired end 150 bp sequencing was performed on a NovaSeq X Plus.

first: Beth

last: Lawlor


title: Spatio-temporal chromosomal arrangements by late-replicating heterochromatin

city: Bangalore

first: Kaustuv

last: Sanyal

state: Karnataka

address: Jakkur

department: Molecular Biology and Genetics Unit

institute: Jawaharlal Nehru Center for Advanced Scientific Research

summary: Centromere clustering orchestrates the three-dimensional genome organization and, in turn, impacts genome stability. In this study, we report the principles governing the chromosome-scale architectural changes associated with centromere clustering and declustering in Cryptococcus neoformans, an opportunistic human fungal pathogen. We demonstrate that centromeres and telomeres in C. neoformans are scattered around the nuclear periphery during interphaseG1, resembling metazoans rather than model budding yeasts. Hi-C data of the synchronised cells reveal centromere and telomere-associated heterochromatic regions are organized as separate compartments at the nuclear periphery, distinct from the euchromatic regions, that eventually shape the global organization of interphaseG1 chromosomes. Polymer modeling of the Hi-C data at interphase and anaphase stages strongly supports a global reorganization of chromosomes from the non-Rabl to the Rabl-like chromosomal arrangement. Most strikingly, while centromeres are clustered and replicate early in most yeasts, our sort-seq studies provide evidence that C. neoformans centromeres replicate late during the S-phase.  In summary, we uncover yet unknown principles governing the chromosome-scale architecture that link centromere DNA replication timing, spatial centromere positioning, functional compartmentalization of chromosomes, and dynamic changes in the global three-dimensional genome architecture in a species that shared a common ancestor with metazoans more than a billion years ago.

overall_design: Hi-C of synchronized Cryptococcus neoformans cells at G1, metaphase and anaphase stages of cell cycle.

first: Satya Dev

last: Polisetty

first: Shuvadip

last: Dutta

first: Rakesh

middle: N

last: Vadnala

first: Dimple

last: Notani

first: Ranjith

last: Padinhateeri


title: Combined epigenetic therapy with FACT and BET inhibitors remodels chromatin and disrupts oncogenic transcription in Diffuse Midline Glioma [Xenium]

city: Kensington

first: Holly

last: Holliday

address: Lowy Cancer Research Centre

country: Australia

institute: Children's Cancer Institute

summary: Aberrant epigenetic regulation is a hallmark of Diffuse Midline Glioma (DMG), an incurable pediatric brain tumor. The H3K27M driver histone mutation leads to transcriptional dysregulation, indicating that targeting the epigenome and transcription may be key therapeutic strategies against this highly aggressive cancer. One such target is the Facilitates Chromatin Transcription (FACT) histone chaperone. We found FACT to be enriched at developmental gene promoters, coinciding with regions of open chromatin and binding motifs of core DMG regulatory transcription factors. Furthermore, FACT interacted and co-localized with the Bromodomain and Extra-Terminal Domain (BET) protein BRD4 at promoters and enhancers, suggesting functional cooperation between FACT and BRD4 in DMG. In vitro, a combinatorial therapeutic approach using the FACT inhibitor CBL0137, coupled with BET inhibition revealed potent and synergistic cytotoxicity across a range of DMG cultures. These results were recapitulated in vivo, significantly extending survival in three independent orthotopic PDX models of DMG. Mechanistically, we show that CBL0137 treatment decreased chromatin accessibility, synergizing with BET inhibition to cause broad transcriptional collapse, silencing several key oncogenes including MYC, PDGFRA, MDM4 and SOX2, as well as causing alterations to the splicing landscape. Notably, this combination also elicited immune-related effects, including activation of the interferon response and antigen presentation mechanisms in DMG cells and induction of an activated state in macrophages and T cells, as demonstrated in an immunocompetent setting with spatial transcriptomics. Altogether, our data highlights the therapeutic promise of simultaneously targeting FACT and BET proteins in DMG, offering a dual tumor-intrinsic and immune-mediated strategy for combating this devastating pediatric brain tumor.

overall_design: 5-7 weeks old female C57BL6 mice were intracranially injected into the brainstem with IUE 24B7 cells that harbor H3.3K27M mutation generated in (doi: 10.1093/neuonc/noz197 and 10.1093/noajnl/vdac079). The timing of treatment initiation was 18 days post intracranial surgery. The drug treatments were as follows: CBL0137 - dissolved in 5% dextrose, CBL0137 was administered intravenously at a dosage of 40 mg/kg/day. The treatment was given once every four days for a total of three weeks. JQ1 - dissolved in DMSO and subsequently in a solution containing 0.9% saline with 5% PEG400 and 5% TWEEN-80, JQ1 was administered at a dosage of 50 mg/kg/day intraperitoneally. The treatment was given five days a week over three weeks. Brain samples were collected on the final day of treatment and fixed in a 10% formalin neutral buffered solution (Sigma-Aldrich). The fixed samples were then embedded in paraffin wax, and 5 µm sections were cut and mounted onto Xenium slides, with two brains per slide. Xenium Prime In Situ (10x Genomics) was performed according to the manufacturer’s instructions using the 5000 pre-designed gene panel and cell segmentation staining. Briefly, priming oligos were hybridized to the target RNA, followed by site-specific cleavage using RNase treatment to release the RNA strand from the hybridized oligo. The 5000-gene panel was then added to the sections to hybridize with the RNA, followed by ligation to generate circular DNA probes, which were subsequently amplified via rolling circle amplification. After a series of ethanol washes, the tissue was blocked and stained with the cell segmentation stain and DAPI. Slides were then loaded into the Xenium Analyzer for imaging and decoding. Spatial Transcriptomics Services for this study were provided by the Garvan Genomics Platform at the Garvan Institute of Medical Research.


title: Combined epigenetic therapy with FACT and BET inhibitors remodels chromatin and disrupts oncogenic transcription in Diffuse Midline Glioma [Nascent RNA-Seq]

overall_design: H3.3 K27M DMG cells (HSJD-DIPG007 and SU-DIPGVI) were treated with 100µM 4SU to label nascent transcripts, and with CBL0137 (1µM for DIPG7 and 2µM for DIPGVI) or JQ1 (1µM) as single agents and in combination for 2h. Vehicle cotrol cells were treated with DMSO at the same concentration. RNA was then extracted and sequenced.

first: Chelsea

last: Mayoh


title: Activation of the aryl hydrocarbon receptor in mature adipocytes does not impact diet-induced obesity in mice [RNA-seq]

first: LMS

last: Bioinformatics Core

overall_design: Cultured mesenchimal lineage cells from the stromovascular fraction of mouse perigonadal white adipose tissue. Cells were treated with FICZ or vehicle control for 6hr in vitro.

first: Julia

last: Vlachaki Walker

first: Marian

last: Dore

first: Chris

last: Schiering


title: Defective RNA processing and ELOA-mediated transcriptional elongation in reversible cellular senescence suggest aging by transcription

city: Chicago

first: Ali

last: Shilatifard

state: IL

address: 320 E Superior St

department: Department of Biochemistry and Molecular Genetics

institute: Northwestern University Feinberg School of Medicine

summary: Elongation factors control post-initiation steps of transcription by RNA polymerase II (RNAPII). We have established distinct mechanistic roles for the essential elongation factors PAF1, NELF, SPT5, and SPT6 via acute depletion of each individually in auxin-inducible degron lines. Here, we leverage these degron lines to explore the regulatory intersection of elongation control and mRNA processing. Integrating long- and short-read RNA-seq data to quantify transcript isoform usage at single-molecule resolution, we identify elongation factor-specific RNA processing regulons including a cellular senescence-enriched regulon shared by NELF and SPT6. We then show that long-term depletion of NELF or SPT6 results in reversible growth arrest following early upregulation of a small group of genes, which include the senescence-associated genes CDKN1A (p21) and CCN2. We perform genetic suppressor screens that implicate the elongation factor Elongin A (ELOA) in NELF or SPT6 depletion-induced growth arrest. ELOA KO suppresses NELF depletion-induced mRNA processing defects and the 3’ extension of RNAPII occupancy past transcription end sites (TES) at genes induced by NELF depletion. ELOA also occupies TES-proximal regions under normal conditions, and acute ELOA depletion results in a loss of RNAPII processivity at the 3’ end of genes, opposing the effects of NELF or SPT6 depletion. Finally, we demonstrate that genetic ELOA loss confers a growth advantage to aging human primary dermal fibroblasts. These findings establish the existence of novel ELOA-dependent mechanisms regulating transcription termination-coupled processes, and links these to the complex phenomena of cellular senescence and aging.

overall_design: Long-read profiling of transcriptome in control and perturbed DLD1 cells to investigate the impact of the depletion of transcriptional elongation factors PAF1, SPT5, SPT6, NELF-C, and ELOA

first: Saeid

last: Parast

first: Simai

last: Wang

first: Deniz

last: Olgun

first: Marta

last: Iwanaszko

first: Vijay

last: Ramani


title: Iron deficiency induces maturation-dependent loss of pancreatic β-cells.

city: Brussels

first: Yue

last: Tong

state: Brussels

address: Route de Lennik, 808

institute: Université Libre de Bruxelles

summary: Pancreatic β-cells maintain glucose homeostasis by secreting insulin in response to rising blood glucose, a process fueled by mitochondrial ATP production. Iron, a core cofactor in the electron transport chain, is essential for this metabolic coupling. While the cytotoxic effects of iron overload are well known, the role of iron sufficiency during β-cell development remains unclear. Here, we identify a maturation-dependent requirement for iron in mouse and human β-cells. Using chemical chelation and genetic disruption of transferrin receptor (TFRC)-mediated uptake, we show that immature β-cells depend on iron during metabolic transition to functional maturity. Iron restriction at this stage impairs oxidative metabolism and compromises survival. In contrast, mature β-cells remain resilient to iron depletion, revealing a developmental switch in iron dependency. These findings establish iron as a key metabolic cue in β-cell development and suggest strategies to generate fully functional stem cell-derived β-cells for diabetes modeling and cell replacement therapy.

overall_design: We performed bulk-RNA sequencing on magnetic-activated cell sorting (MACS)-purified iPSC-derived stage 7 β-like cells exposed to DFO (100 μM, Vehicle H2O) with a composition of 75% INS single positive cells quantified by immunocytochemistry staining, n=4 independent differentiations.

first: Annelore

last: Van Mulders

first: Xiaoyan

last: Yi

first: Miriam

last: Cnop

first: Willem

last: Staels


title: VHL synthetic lethality screens uncover CBF-β as a negative regulator of STING

city: Cambridge

first: Niek

last: Wit

state: Not US or Canada

address: Puddicombe Way

department: Department of Medicine

institute: University of Cambridge

summary: Clear cell renal cell carcinoma (ccRCC) represents the most common form of kidney cancer and is typified by biallelic inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene. Here, we undertake genome-wide CRISPR/Cas9 screening to reveal synthetic lethal interactors of VHL, and uncover that loss of Core Binding Factor β (CBF-β) causes cell death in VHL-null ccRCC cell lines and impairs tumour establishment and growth in vivo. This synthetic relationship is independent of the elevated activity of hypoxia inducible factors (HIFs) in VHL-null cells, but does involve the RUNX transcription factors that are known binding partners of CBF-β. Mechanistically, CBF-β loss leads to upregulation of type I interferon signalling, and we uncover a direct inhibitory role for CBF-β at the STING locus controlling Interferon Stimulated Gene expression. Targeting CBF-β in kidney cancer both selectively induces tumour cell lethality and promotes activation of type I interferon signalling.

overall_design: ChIP-seq of CBF-β in 7860 with empty vector control or CBF-β overexpression

first: Pauzaite

last: Tekle

first: Wit

last: Niek

first: Bertlin

last: James

first: Nathan


title: Recruitment of BRD4 to the ASXL1 genomic targets depends on the extra-terminal domain of BRD4

first: Lu

state: Illinois

address: 303 E. Superior Street Simpson Querrey 7-414

institute: Northwestern University

summary: Additional sex combs like 1 (ASXL1) is a well-established driver of a wide range of cancers. Here, we identified a high level of genetic correlation between ASXL1 and the major transcriptional activator BRD4 in cancer cells and characterized the molecular mechanism underlying this correlation. Structural and biochemical data show the formation of a tight complex between the extraterminal domain of BRD4 (BRD4ET) and the ET-binding motif of ASXL1 (ASXL1EBM). ChIP-seq analysis of ASXL1 impaired in binding to BRD4ET demonstrates that the recruitment of BRD4 to the ASXL1 genomic targets is ASXL1EBM-dependent. We find that the cancer-related truncated variants comprising residues 1-645 and 1-591 of ASXL1 (ASXL11-645 and ASXL11-591) retain BRD4 binding function, with ASXL11-645 showing an enhanced ability to recruit BRD4 to promoters of ASXL1 target genes. Genomic data from six cancer types reveals a strong positive ASXL1-BRD4 relationship, with BRD4 occupying the ASXL1 promoter and thus further suggesting a possible feed-forward mechanism. Our findings shed light on the mechanistic details by which ASXL1 associates with BRD4 and may provide potential therapeutic avenues for developing cancer treatments through targeting the ASXL1-BRD4 axis.

overall_design: HEK293T cells were transduced with lentivirus expressing either GFP, GFP-tagged ASXL1-1-645-WT, or GFP-ASXL1-1-645-I574D. The ChIP-seq experiments were conducted with GFP monoclonal antibody and BRD4 antibody to determine the impact of ASXL1 cancer-specific mutation on BRD4 occupancy at the genome wide level

first: Zibo

last: Zhao


title: Gene expression analysis of NCI-H889 cells transfected with siRNA against BAF53B

city: Toyoake

first: Kouhei

last: Sakurai

address: 1-98 Dengakugakubo, Kutsukake-cho

department: Department of Joint Research Laboratory of Clinical Medicine, School of Medicine

country: Japan

institute: Fujita Health University

summary:
This study aimed to reveal downstream genes of BAF53B in a small cell lung cancer cell line NCI-H889.
We performed gene expression microarray after BAF53B knockdown in NCI-H889 cells to reveal the potential downstream genes.

overall_design: NCI-H889 cells were transfected with si-negative control (si-NC) or siRNA against BAF53B (si-BAF53B), and gene expression microarray was performed.