title: Cholangiocarcinoma tumorigenesis in Abcb4(Mdr2)-/- and wild-type mice by transposon-based oncogenes integration

first: Pinzhu

last: Huang

state: MA

address: 330 Brookline Ave

department: Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition

institute: Beth Israel Deaconess Medical Center

summary: Cholangiocarcinoma (CCA) is a dreaded complication of primary sclerosing cholangitis (PSC), difficult to diagnose and associated with high mortality. Lack of animal models of CCA recapitulating the hepatic microenvironment of sclerosing cholangitis hinders development of novel treatments. We have developed and characterized a new mouse model of PSC-CCA through hydrodynamic tail vein injection of oncogenes pT3-EF1a-HA-myrAKT (AKT) and pT3-EF1a-YapS127A (YAP1), termed SB CCA.Mdr2-/-, which features reliable tumor induction in PSC-like background of biliary injury and fibrosis. To unravel the potential molecular profile of CCA developed in different liver context, we profiled the transcriptomes of tumor-bearing fibrotic (Mdr2-/-) (n = 6) and tumor-bearing wild-type liver tissues (n = 6) and compared to corresponding control nontumor-bearing fibrotic (Mdr2-/-) (n = 6) and healthy liver tissues (n = 6). RNA-seq analysis revealed profound transcriptional differences in CCA evolving in PSC-like context, compared to CCA in healthy liver.

overall_design: Profiling of individual tumor-bearing liver tissues with transduction of AKT/YAP1 (CCA.Mdr2-/- and CCA.Wild-type, n = 6 for each) and context matched non-tumor liver tissues with empty transduction (Ctrl.Mdr2-/- and Ctrl.Wild-type. Differentially expressed genes were determined from 2 comparisons:  CCA.Mdr2-/- vs. Ctrl.Mdr2-/-, and CCA.Wild-type and Ctrl.WT.

first: Yury

middle: V

last: Popov


title: Epigenetic recording of stimulation history reveals BLIMP1-BACH2 balance in determining memory B cell fate upon recall challenge

status: Public on Jun 04 2024

city: Beijing

first: Hai

last: Qi

address: Haidian District

country: China

institute: Tsinghua University

type: Expression profiling by high throughput sequencing

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Adrenal gland macrophages regulate glucocorticoid production through Trem2 and TGFβ [scRNA-seq]

status: Public on Jun 03 2024

city: Minneapolis

first: Jesse

middle: W

last: Williams

state: MN

address: 2101 6th St. SE

department: Dept. Integrative Biology & Physiology

country: USA

institute: University of Minnesota

summary: To elucidate how adrenal gland immune cells response to acute or chronic stressors

overall_design: CD45+ cells were FACS sorted from single cell suspensions from mouse adrenal glands from C57bl/6 mice challenged for 12-hours cold (4-degree Celcius) exposure or Ldlr-/- mice fed a high fat diet for 8-weeks to induce chronic atherosclerosis.

first: Yingzheng

last: Xu


title: Adaptation to an acid microenvironment promotes pancreatic cancer organoid growth and drug resistance [acid]

city: Copenhagen

first: Yifan

last: Dai

address: Ole Maaløes Vej 5

department: Department of Biology

country: Denmark

institute: Copenhagen University

summary: We investigated transcriptomic changes in pancreatic ductal organoids under adaptation to an acidic extracellular environment.

overall_design: Wild type ("mN10"), KrasG12D ("mP4"), p53 knockout ("mP4 p53KO"), and Trp53R273H ("mP4 p53R273H") organoids were gradually acid adapted from pH 7.4 to pH 6.7 ("AA") by decreasing pH of their culture medium by approximately Δ=0.125 pH every week for nine weeks. After reaching the desired pH of 6.7, the organoids were cultured for 2 weeks (week 8-9) at pH 6.7. Then, acid adapted organoids were split into two fractions: i) cultured at pH 6.7 until week 11, and ii) cultured at their physiological pH of 7.4 until week 11 ("AA->pH7.4").

first: Arnaud

last: Stigliani

first: Renata

last: Ialchina

first: Dominika

last: Czaplinska

first: Jiayi

last: Yao

first: Henriette

last: Berg Andersen

first: Stine

last: Falsig Pedersen

first: Albin

last: Sandelin


title: Host-Pathogen Interactions in the Plasmodium-Infected Mouse Liver at Spatial and Single-Cell Resolution (VISIUM)

city: Stockholm

first: Franziska

last: Hildebrandt

address: Svante Arrhenius Väg 20C, Stockholm

country: Sweden

institute: Stockholm University

summary: During liver infection, the malaria parasite undergoes massive replication whilst remaining clinically silent. Spatial coordination of immune response regulation and metabolic zonation during malaria infection in the true tissue context remains unexplored. We perform spatial transcriptomics combined with snRNA-seq over multiple time points to delineate transcriptional programs of host-pathogen interactions across P. berghei-infected liver tissues. Our data suggest changes in gene expression related to lipid metabolism adjacent to infected hepatocytes, particularly modulation of the expression of genes involved in peroxisome proliferator-activated receptor pathway signaling. The data further indicates the presence of inflammatory hotspots with differential inflammation programs along the lobular axis in infected tissues. Additionally, upregulation of genes involved in inflammation is observed, but considerably delayed, in livers of control mice injected with mosquito salivary gland components. Our study establishes a benchmark for investigating host-parasite interactions, and can easily be implemented to validate de novo malaria drug and vaccine efforts.

sample_organism:
Plasmodium berghei ANKA
Mus musculus

platform_organism:
Plasmodium berghei ANKA
Mus musculus

overall_design: For this study mice were infected with 400.000 sporozoites of P.berghei ANKA and collected after 12, 24 or 38h post-infection. Control mice were challenged with salivary gland lysate of the same number of mosquitos collected for P. berghei ANKA challenges. The Visium (10X Genomics Inc.) protocol, including sequencing and computational analysis was performed for a total of 8 sections of which 4 were infected with Plasmodium berghei parasites and 4 challenged with mosquito salivary gland lysate.  samples collected after 12h and 24h and 38h. Samples were selected based on sectioning and RNA quality.


title: Host-Pathogen Interactions in the Plasmodium-Infected Mouse Liver at Spatial and Single-Cell Resolution (Spatial Transcriptomics 2k)

overall_design: For this study mice were infected with 400.000 sporozoites of P.berghei ANKA and collected after 12, 24 or 38h post-infection. Control mice were challenged with salivary gland lysate of the same number of mosquitos collected for P. berghei ANKA challenges. The full Spatial Transcriptomics 2k array protocol, including sequencing and computational analysis was performed for a total of 38 sections of which 23 were infected with Plasmodium berghei parasites and 15 challenged with mosquito salivary gland lysate. We analyzed 4 biological replicates for infected samples collected after 12h and 24h and 2 biological replicates for infected samples after 38h. For controls, we analyzed livers for 3, 3 and 2 biological replicates, respectively. Samples were selected based on sectioning and RNA quality.


title: CRISPRi Screen of Long Non-coding RNAs Identifies LINC03045 Regulating Glioblastoma Invasion

city: Los Angeles

first: Frank

last: Attenello

address: 1501 San Pablo St

institute: University of Southern California

summary: Introduction: Glioblastoma (GBM) invasion studies have focused on coding genes, while few studies evaluate long non-coding RNAs (lncRNAs), transcripts without protein-coding potential, for role in GBM invasion. We leveraged CRISPR-interference (CRISPRi) to evaluate invasive function of GBM-associated lncRNAs in an unbiased functional screen, characterizing and exploring the mechanism of identified candidates. Methods: We implemented a CRISPRi lncRNA loss-of-function screen evaluating association of lncRNA knockdown (KD) with invasion capacity in Matrigel. Top screen candidates were validated using CRISPRi and oligonucleotide(ASO)-mediated knockdown in three tumor lines. Clinical relevance of candidates was assessed via The Cancer Genome Atlas(TCGA) and Genotype-Tissue Expression(GTEx) survival analysis. Mediators of lncRNA effect were identified via differential expression analysis following lncRNA KD and assessed for tumor invasion using knockdown and rescue experiments. Results: Forty-eight lncRNAs were significantly associated with 33-83% decrease in invasion (p<0.01) upon knockdown. The top candidate, LINC03045, identified from effect size and p-value, demonstrated 82.7% decrease in tumor cell invasion upon knockdown, while LINC03045 expression was significantly associated with patient survival and tumor grade(p<0.0001). RNAseq analysis of LINC03045 knockdown revealed that WASF3, previously implicated in tumor invasion studies, was highly correlated with lncRNA expression, while WASF3 KD was associated with significant decrease in invasion. Finally, WASF3 overexpression demonstrated rescue of invasive function lost with LINC03045 KD. Conclusion:  CRISPRi screening identified LINC03045, a previously unannotated lncRNA, as critical to GBM invasion. Gene expression is significantly associated with tumor grade and survival. RNA-seq and mechanistic studies suggest that this novel lncRNA may regulate invasion via WASF3.

overall_design: Cell line: U87 cells expressing dCas9-KRAB-BFP, sorted 48 hours post-transudction with Fluorescence associated cell sorting (FACS)

first: Kathleen

last: Tsung

first: Eddie

last: Loh


title: RNA-seq analysis on eosinophils

city: Tokyo

first: Takeshi

last: Nitta

address: 7-3-1 Hongo, Bunkyo-ku

department: Department of Immunology,

country: Japan

institute: Graduate School of Medicine and Faculty of Medicine, The University of Tokyo

summary: We performed RNA sequencing (RNA-seq) on eosinophils from thymus, spleen, and lung.

overall_design: RNA-seq analysis on eosinophils isolated from WT or Rag-deficient mice.

first: Ayami

last: Ota


title: A Spatial Transcriptomics Atlas of the Malaria-infected Liver Indicates a Crucial Role for Lipid Metabolism and Hotspots of Inflammatory Cell Infiltration

first: Miren

last: Urrutia Iturritza

address: Svante Arrhenius väg 20C

department: The Department of Molecular Biosciences, The Wenner-Gren Institute (MBW)

overall_design: For this study mice were infected with 400.000 sporozoites of P.berghei ANKA and their livers were collected after 12, 24 or 38h post-infection. Control mice were challenged with salivary gland lysate of the same number of mosquitos collected for P. berghei ANKA challenges and their livers were also collected after 12, 24 or 38h post-injection. Nuclei were isolated from the snap frozen liver tissues, and two biological replicates of each condition were pooled. Thus, the full single-nuclei RNA sequencing protocol, including sequencing and computational analysis, was performed for a total of 6 liver samples, of which 3 were infected with Plasmodium berghei parasites and 3 challenged with mosquito salivary gland lysate.

first: Christian

last: Zwicker

first: Bavo

last: Vanneste

first: Noémi

last: Van Hul

first: Elisa

last: Semle

first: Jaclyn

last: Quin

first: Tales

last: Pascini

first: Sami

last: Saarenpää

first: Mengxiao

last: He

first: Emma

middle: R

last: Andersson

first: Charlotte

middle: L

last: Scott

first: Joel

last: Vega-Rodriguez

first: Joakim

last: Lundeberg

first: Johan

last: Ankarklev


title: SEX-DEPENDENT APOE4 NEUTROPHIL-MICROGLIA INTERACTIONS DRIVE COGNITIVE IMPAIRMENT IN ALZHEIMER'S DISEASE [Neutrophils_Spleen_Brain_E3_E4]

city: Boston

first: Neta

last: Rosenzweig

state: Massachusetts

address: 60 Fenwood Road, 10002K, Brigham and Women’s Hospital

institute: Brigham and Women's Hospital, Harvard Medical School

summary: APOE4 is the strongest genetic risk factor for Alzheimer's disease (AD) with increased odds ratios in females. Targeting amyloid plaques show modest improvement in male non-APOE4 carriers. Leveraging transcriptomics across APOE variants in both sexes, multiplex flow cytometry and validation in two independent cohorts of APOE4 female AD patients, we identify a new subset of neutrophils associated with cognitive impairment. This phenotype is defined by increased IL-17 and IL-1 co-expressed gene modules in blood neutrophils of cognitively impaired female APOE e4 carriers, showing increased infiltration to the AD brain. APOE4 female IL-17+ neutrophils upregulated the immunosuppressive cytokines IL-10 and TGFb, and immune checkpoints including LAG-3 and PD-1, associated with accelerated immune aging. Deletion of APOE4 in neutrophils reduced this immunosuppressive phenotype and restored microglial response to neurodegeneration (MGnD), limiting plaque pathology in AD mice. Mechanistically, IL-17Fupregulated in APOE4 neutrophils interacts with microglial IL-17RA to suppress the induction of MGnD phenotype, and blocking this axis supported cognitive improvement in AD mice. These findings provide a translational basis to target IL-17F in APOE e4 female carriers with cognitive impairment.

overall_design: Transcriptomic investigation of splenic and brain neutrophils from transgenic female APP/PS1 mice carrying the human APOE3 or APOE4 allele.

first: Kilian

last: Kleemann

first: Thomas

last: Rust

first: Madeline

last: Grucci

first: Michael

last: Aronchik

first: Nieske

last: Brouwer

first: Madison

last: Carpenter

first: Isabel

last: Valenbreder

first: Joya

last: Cooper-Hohn

first: Malvika

last: Iyer

first: Rajesh

middle: K

last: Krishnan

first: Kisha

last: Sivanathan

first: Wesley

last: Brandão

first: Taha

last: Yahya

first: Ana

last: Durao

first: Jean

middle: P

last: Chadarevian

first: Zhuoran

last: Yin

last: Properzi

first: Roni

last: Nowarski

first: Hayk

last: Davtyan

first: Mathew

last: Blurton-Jones

first: Hyun

middle: S

last: Yang

first: Reisa

last: Sperling

first: Bart

middle: J

last: Eggen

first: Oleg

last: Butovsky


title: The impact of HPV8 on Lrig1+ and CD34+ hair follicle keratinocyte stem cell populations

city: Cardiff

first: Girish

last: Patel

address: Maindy Road

country: United Kingdom

institute: Cardiff University

summary: Despite epidermal turnover, the skin is host to a complex array of microbes including viruses, such as the human papillomavirus (HPV), which must infect and manipulate skin keratinocyte stem cells (KSC) to survive. This crosstalk between the virome and KSC populations remains largely unknown. Here, we investigated the effect of HPV8 on KSCs using various mouse models. We observed that the HPV8 early region gene E6 specifically caused Lrig1+hair follicle junctional zone KSC proliferation and expansion, which would facilitate viral transmission. Within Lrig1+KSCs specifically, HPV8 E6 bound intracellular p300 to phosphorylate the STAT3 transcriptional regulatory node. This induces a transcriptional switch from TAp63 to ΔNp63 expression, resulting in expansion of KSC into the overlying epidermis. HPV8 was associated with 70% of human actinic keratoses (AK). Together these results redefine human AK as an expansion of KSC, which lack protecting melanosomes and are thus susceptible to sun-light-induced malignant transformation.

overall_design: To determine why HPV8 induced selective proliferation of the Lrig1+hair follicle junctional zone keratinocyte stem cells, but not other keratinocyte stem cells, we undertook a transcriptomic analysis of adjacent stem cell populations defined by Lrig1 and CD34 expression in adult mice. Both Lrig1+and CD34+flow-sorted keratinocytes were isolated from dorsal back skin of individual HPV8-CERtg and WT mice. We then performed gene expression profiling analysis on this RNA-seq data.

first: Alex

last: Gibbs

first: Huw

last: Morgan


title: SEX-DEPENDENT APOE4 NEUTROPHIL-MICROGLIA INTERACTIONS DRIVE COGNITIVE IMPAIRMENT IN ALZHEIMER'S DISEASE [Human_IL18R1]

first: Neta

last: Rosenzweig

overall_design: Transcriptomic investigation of Il18R1 high and low neutrophils from female patients.

first: Kilian

last: Kleemann

first: Thomas

last: Rust

first: Madeline

last: Grucci

first: Michael

last: Aronchik

first: Nieske

last: Brouwer

first: Madison

last: Carpenter

first: Isabel

last: Valenbreder

first: Joya

last: Cooper-Hohn

first: Malvika

last: Iyer

first: Rajesh

last: Krishnan

last: Sivanathan


title: Histone serotonylation regulates ependymoma tumorigenesis through neurodevelopmental pathways [RNA-seq]

city: Houston

first: Hsiao-Chi

last: Chen

state: TX

address: 1 Baylor Plaza

institute: Baylor College of Medicine

summary:
To profile gene expression differences between mouse non-tumor cortex versus ZFTA-RELA ependymoma versus ETV5 overexpressing tumors
Furthermore, we also sought to profile gene expression differences between ZFTA-RELA ependymoma versus NPY overexpressing tumor

overall_design: Comparative gene expression profiling analysis of RNA-seq data for bulk tissues

first: Benjamin

last: Deneen


title: Endothelial LATS2 is a suppressor of bone marrow fibrosis

city: Münster

first: Paul-Georg

last: Majev

address: Röntgenstraße 20

department: Tissue Morphogenesis

country: Germany

institute: Max Planck Institute for Molecular Biomedicine

summary: The skeletal system vasculature and specialized capillary subtypes regulate crucial elements of bone growth and regeneration. However, little is known about endothelial cell (EC) activation and plasticity during bone remodeling. Here, we demonstrate that the inactivation of the gene encoding Hippo kinase large tumor suppressor kinase 2 (Lats2) in ECs results in the development of a vascular defect in the metaphysis, which favours accumulation of hypertrophic chondrocytes and induces bone marrow fibrosis in the bone.

overall_design: Examination of endothelial and bone stromal cells in Lats2iECKO and control mice. Two samples per condition.

first: Kishor

last: Sivaraj

first: Ralf

middle: H

last: Adams


title: Histone serotonylation regulates ependymoma tumorigenesis through neurodevelopmental pathways [ChIP-Seq]

summary:
To profile H3K4me3Q5Ser (5HT-H3) distribution in mouse ZFTA-RELA ependymoma tumors
Furthermore, we also sought to examine H3K27ac and H3K27me3 distribution in mouse ZFTA-RELA ependymoma versus ETV5 overexpressing tumors

overall_design: Chromatin immunoprecipitation (ChIP-Seq) for 5HT-H3, H3K27ac, H3K27me3 and ETV5 in mouse ZFTA-RELA ependymoma tumors


title: Histone serotonylation regulates ependymoma tumorigenesis through neurodevelopmental pathways


title: Effects of Klf4 knockdown in GT1-7 cells

city: Milwaukee

first: Justin

last: Grobe

address: 8701 W Watertown Plank Road

institute: Medical College of Wisconsin

summary: The goal is to examine the relative abundance of Agrp transcripts and changes to gene expression within GT1-7 cells (immortalized mouse hypothalamic cell line, used as a model of AgRP neurons) treated with Klf4 siRNA, scramble siRNA, or Ca2+ DMEM (vehicle).

overall_design: Exon count and differential gene expression analyses from RNA-seq data from GT1-7 cells comparing a Klf4 siRNA, scramble siRNA and a Ca2+ free vehicle group

first: McKenzie

last: Ritter


title: Effects of Klf4 deletion from Agrp expressing cells under chow or HFD conditions

summary: The goal is to examine the relative abundance of Agrp transcripts and changes to gene expression within the ARC of Agrp-Cre x Klf4-flox KO and control littermates after either regular chow or 10 weeks of HFD

overall_design: Exon count and differential gene expression analyses from RNA-seq data from ARC samples of Agrp-Cre x Klf4-flox KO and control littermates after either regular chow or 10 weeks of HFD


title: Screening by deep sequencing reveals mediators of microRNA tailing in C. elegans

status: Public on Jun 01 2024

city: Bethesda

first: Katherine

last: McJunkin

state: MD

address: 50 South Drive, Building 50 Room 3148

institute: National Institutes of Health

type: Non-coding RNA profiling by high throughput sequencing

summary: microRNAs are frequently modified by addition of untemplated nucleotides to the 3' end, but the role of this tailing is often unclear. Here we characterize the prevalence and functional consequences of microRNA tailing in vivo, using Caenorhabditis elegans. MicroRNA tailing in C. elegans consists mostly of mono-uridylation of mature microRNA species, with rarer mono-adenylation which is likely added to microRNA precursors. Through a targeted RNAi screen, we discover that the TUT4/TUT7 gene family member CID-1/CDE-1/PUP-1 is required for uridylation, whereas the GLD2 gene family member F31C3.2-here named GLD-2-related 2 (GLDR-2)-is required for adenylation. Thus, the TUT4/TUT7 and GLD2 gene families have broadly conserved roles in miRNA modification. We specifically examine the role of tailing in microRNA turnover. We determine half-lives of microRNAs after acute inactivation of microRNA biogenesis, revealing that half-lives are generally long (median = 20.7 h), as observed in other systems. Although we observe that the proportion of tailed species increases over time after biogenesis, disrupting tailing does not alter microRNA decay. Thus, tailing is not a global regulator of decay in C. elegans. Nonetheless, by identifying the responsible enzymes, this study lays the groundwork to explore whether tailing plays more specialized context- or miRNA-specific regulatory roles.

overall_design: To determine the role of microRNA tailing (i.e. untemplated addition of nucleotides to the 3' end), an RNAi screen was performed to identify enzymes required for microRNA tailing in C. elegans (screen group samples). To determine the role of tailing in rates of microRNA decay, decay rates were profiled in the presence of tailing (sample group 1), or in the context of disrupted tailing by knockdown of tailing enzymes CID-1 or F31C3.2/GLDR-2 (sample group 2). To further determine redundancy of CID-1 or F31C3.2/GLDR-2 with their paralogs, tailing was profiled in double mutant conditions for CID-1/PUP-1 and PUP-2 (sample group 3) or in F31C3.2/GLDR-2 mutant with GLD-2 RNAi (sample group 4). Additional experiments were performed, which were ultimately inconclusive due to technical limitations, to determine requirements for TDMD in C. elegans (sample group 5).

first: Karl-Frédéric

last: Vieux

last: Prothro

first: Leanne

last: Kelley

first: Cameron

last: Palmer

first: Eleanor

middle: M

last: Maine

first: Isana

last: Veksler-Lublinsky


title: Benchmarking pathway analysis methods offer novel prognostic cancer biomarkers and therapeutics: application in bladder cancer

city: WEST LAFAYETTE

first: Majid

last: Kazemian

state: IN

address: 201 S. University Street

department: Biochemistry and Computer Science

institute: Purdue University

summary: The pathogenesis of cancer is typically driven by alterations in multiple cellular pathways that are challenging to identify and target. However, it is unclear how suitable the existing pathway analysis tools are for unbiased discovery and ranking of dysregulated and/or cancer-specific pathways. Here, we created a new platform called Benchmark to evaluate the potential of pathway analysis tools for discovery under experimental conditions. Unexpectedly, we found that despite wide-spread success in confirming hypothesized dysregulated pathways, common pathway analysis tools are less than ideal for unbiased discovery. Nevertheless, our pathway ensemble tool (PET) that combines the rank statistics from the exisiting methods significantly enhanced discovery. We applied PET to transcriptomics data from 12 independent tumor types to identify prognostic pathways. We showed that the genes from prognostic pathways are excellent biomarkers and can define cancer molecular subtypes. Moreover, drug prediction to normalize genes from prognostic pathways identified effective known and novel drugs. Finally, in vitro and/or a xenograft models of bladder cancer treated with the top predicted drug significantly restricted tumor cells. We anticipate that our unbiased approach for pathway discovery will have tangible impacts on cancer management.

overall_design: Comparision of transcriptome of T-24 cells treated with DMSO and cells treated with CCT068127. T-24 (1.5 million) cells were seeded in a 6 well plate. After 12 hrs, cells were treated with either 1 micro molar concentrations of CCT068127 or DMSO for 48hrs. Trypsinized the cells and extracted RNA using Trizol reagent. Experiments were performed in three independent biological replicates.

first: Luopin

last: Wang

first: Aryamav

last: Pattnaik

first: Md

last: Tajmul

first: Behdad

last: Afzali


title: Adaptation to an acid microenvironment promotes pancreatic cancer organoid growth and drug resistance

first: Yifan

last: Dai

overall_design: Refer to individual Series


title: miRNA as biomarkers for fracture healing

status: Public on Jun 02 2024

city: Davos Platz

first: Martin

last: Stoddart

address: Clavadelerstrasse 8

country: Switzerland

institute: AO Research Institute Davos

summary: Fracture non-union is a complex clinical condition affecting many patients worldwide; however, known risk factors alone cannot help in predicting individual risk of progressing towards healing complications. The identification of novel biomarkers in the serum of fracture patients is crucial for early diagnosis and timely patient treatment.   This study focused on the identification of microRNA (miRNA) that could correlate to the process of fracture healing. Toward this aim, serum of fracture patients and healthy volunteers was screened by RNA sequencing to identify differentially expressed miRNA at different times after injury. The results were correlated to miRNA that were found to be differentially secreted in the conditioned medium of human bone marrow mesenchymal stromal cells (BMSCs) during in vitro osteogenesis.

overall_design: RNAseq of serum samples from fracture patients at different timepoints after injury + healthy volunteers as controls; RNAseq of conditioned medium from human bone marrow mesenchymal stromal cells undergoing osteogenic differentiation in vitro.

first: Elena

last: Della Bella

first: Ursula

last: Menzel

first: Andreas

last: Naros

first: Eva

last: Kubosch

first: Mauro

last: Alini


title: Nasal Brushing Molecular Endotyping Distinguishes CRSwNP Patients with Better Response to Dupilumab

city: Tarrytown

first: Kaitlyn

last: Gayvert

state: NY

address: 777 Old Saw Mill River Rd

department: Molecular Profiling & Data Science

institute: Regeneron Pharmaceuticals

summary:
There is evidence of pathophysiologic diversity in chronic rhinosinusitis with nasal polyps (CRSwNP), but data characterizing the molecular endotypes of CRSwNP and their association with treatment is lacking. The objective of this study was to identify gene signatures associated with CRSwNP endotypes, clinical features, and dupilumab treatment response.
Two distinct transcriptional clusters (C1 and C2) were identified, both with elevated type 2 biomarkers. At baseline, C2 patients had higher mean Nasal Polyp Score and higher type 2 biomarker levels than C1 patients. At Week 24, significant improvements in clinical outcomes (dupilumab vs placebo) were observed in both clusters, although the magnitude of improvements was significantly greater in C2 than C1, and more C2 patients demonstrated clinically meaningful responses. Gene sets enrichment analyses supported the existence of two molecular endotypes: C2 was enriched in genes associated with type 2 inflammation (including periostin, cadherin-26, and type 2 cysteine protease inhibitors), while C1 was enriched in genes associated with T cell activation, interleukin-12, and interferon-γ production.

overall_design: Nasal brushing samples were collected from 89 patients randomized to dupilumab 300 mg every 2 weeks or placebo in the SINUS-52 trial (NCT02898454). Microarrays were used to identify transcriptional clusters and assess the relationship between gene expression and baseline clinical features and clinical response to dupilumab. Endotype signatures were determined using differential expression analysis.

last: Desrosiers

first: Tanya

last: Laidlaw

first: Leda

last: Mannent

first: Kiran

last: Patel

first: Julie

last: Horowitz

first: Nikhil

last: Amin

first: Alexandre

last: Jagerschmidt

first: Jennifer

middle: D

last: Hamilton

first: Wei

last: Lim

first: Sivan

last: Harel


title: Adaptation to an acid microenvironment promotes pancreatic cancer organoid growth and drug resistance [drug]

first: Yifan

last: Dai

summary: We investigated transcriptomic changes in PanIN ("mP4") mouse pancreatic ductal organoids under acute or long-term treatment of Gemcitabine and Erlotinib.

overall_design: The mP4 organoids were gradually adapted to growth in medium containing 10 nM of drugs (Erlotinib, Gemcitabine or combination), by increasing drug concentrations by 1 nM / week over a period of 12 weeks. As controls, we kept organoids at 0 nM drug concentrations for the same time. A subset of control organoids were acutely treated with 10 nM erlotinib, gemcitabine or the combination of the two at 12 weeks. Expression prolifing analysis was conducted based on RNAseq of 33 samples from 2 experiments: drug adaptation experiment (24 samples from week 1 and 12), and acute drug treatment experiment (9 samples).

first: Arnaud

last: Stigliani

first: Renata

last: Ialchina

first: Dominika

last: Czaplinska

first: Jiayi

last: Yao

first: Henriette

last: Berg Andersen

first: Stine

last: Falsig Pedersen

first: Albin

last: Sandelin


title: Gene expression profiling of HIV-associated diffuse large B-cell lymphomas from Malawi

city: Chapel Hill

first: Yuri

last: Fedoriw

state: NC

address: 160 Medical Drive

institute: University of North Carolina

summary: To investigate the biological differences between HIV-, HIV+/ART-experienced and HIV+/ART-naive diffuse large B-cell lymphoma, we performed RNA sequencing of 70 pre-treatment formalin-fixed paraffin-embedded (FFPE) whole lymph node biopsies of diffuse large B-cell lymphoma.

overall_design:
We performed RNA sequencing of 70 pre-treatment formalin-fixed paraffin-embedded (FFPE) whole lymph node biopsies of HIV- and HIV+ (with and without prior antiretroviral therapy exposure) diffuse large-B cell lymphoma.

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Submitter states that missing raw files are due to patient privacy concerns.
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first: Roush

last: Sophia

first: Fedoriw

last: Yuri

first: Tomoka

last: Tamiwe

first: Painschab

last: Matthew


title: Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2 (RNA-seq)

city: Kiel

first: Elmar

last: Wolf

state: Schleswig-Holstein

address: Rudolf-Höber-Str. 1

department: Biochemical Institute

institute: Christian-Albrechts-University Kiel

summary:
Objective:
The hallmark oncogene MYC drives the progression of most tumorstumours, but direct inhibition of MYC by a small-molecule drug has not reached clinical testing. MYC is a transcription factor that depends on several binding partners to function. We therefore explored the possibility of targeting MYC via its interactome in pancreatic ductal adenocarcinoma (PDAC).

Design:
To identify the most suitable targets among all MYC binding partners, we constructed a targeted shRNA library and performed screens in cultured PDAC cells and tumorstumours in mice.Results:Unexpectedly, many MYC binding partners were found to be important for cultured PDAC cells but dispensable in vivo. However, some were also essential for tumorstumours in their natural environment and, among these, the ATPases RUVBL1 and RUVBL2 ranked first. Degradation of RUVBL1 by the auxin-degron system led to the arrest of cultured PDAC cells but not untransformed cells and to complete tumortumour regression in mice, which was preceded by immune cell infiltration. Mechanistically, RUVBL1 was required for MYC to establish oncogenic and immuno-evasive gene expression identifying the RUVBL1/2 complex as a druggable vulnerability in MYC-driven cancer.

Conclusion:
One implication of our study is that PDAC cell dependencies are strongly influenced by the environment, so genetic screens should be performed in vitro and in vivo. Moreover, the auxin-degron system can be applied in a PDAC model, allowing target validation in living mice. Finally, by revealing the nuclear functions of the RUVBL1/2 complex, our study presents a pharmaceutical strategy to render pancreatic cancers potentially susceptible to immunotherapy.

overall_design: 8661, 9091, 9172, 24033 and SB1557 murine PDAC cells were treated with 1 µM CB-6644 or DMSO for 24 h.

first: Markus

last: Vogt

first: Nevenka

last: Dudvarski Stankovic

first: Yiliam

last: Cruz Garcia

first: Julia

last: Hofstetter

first: Katharina

last: Schneider

first: Filiz

last: Kuybu

first: Theresa

last: Hauck

first: Bikash

last: Adhikari

first: Anton

last: Hamann

first: Yamila

last: Rocca

first: Lara

last: Grysczyk

first: Benedikt

last: Martin

first: Anneli

last: Gebhardt-Wolf

first: Armin

last: Wiegering

middle: E

last: Diefenbacher

first: Georg

last: Gasteiger

first: Stefan

last: Knapp

first: Dieter

last: Saur

first: Martin

last: Eilers

first: Mathias

last: Rosenfeldt

first: Florian

last: Erhard

first: Seychelle

last: Vos


title: Contribution of CENP-F to FOXM1-mediated discordant centromere and kinetochore transcriptional regulation

city: Chicago

first: Sakshi

last: Khurana

state: IL

address: SQBRC Room 7-400, 303 E Superior St.

department: Biochemistry and Molecular Genetics

institute: Northwestern University

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

first: Dileep

last: Varma

first: Daniel

last: Foltz


title: Contribution of CENP-F to FOXM1-mediated discordant centromere and kinetochore transcriptional regulation [ATAC-seq]

summary: Proper chromosome segregation is required to ensure genomic and chromosomal stability. The centromere is a unique chromatin domain present throughout the cell cycle on each chromosome defined by the CENP-A nucleosome. Centromeres (CEN) are responsible for recruiting the kinetochore (KT) during mitosis, ultimately regulating spindle attachment and mitotic checkpoint function. Upregulation of many genes that encode CEN/KT proteins is commonly observed in cancer. Here, we show although FOXM1 occupies the promoters of many CEN/KT genes with MYBL2, occupancy is insufficient alone to drive the FOXM1 correlated transcriptional program. We show that CENP-F, a component of the outer kinetochore, functions with FOXM1 to coregulate G2/M transcription and proper chromosome segregation. Loss of CENP-F results in alteration of chromatin accessibility at G2/M genes, including CENP-A, and leads to reduced FOXM1-MBB complex formation. The FOXM1-CENP-F transcriptional coordination is a cancer-specific function. We observed that a few CEN/KT genes escape FOXM1 regulation such as CENP-C which when upregulated with CENP-A, leads to increased chromosome misegregation and cell death. Together, we show that the FOXM1 and CENP-F coordinately regulate G2/M gene expression, and this coordination is specific to a subset of genes to allow for proliferation and maintenance of chromosome stability for cancer cell survival.

overall_design: To evaluate the accessibility changes in normal cells, in reponse to loss of CENP-F and FOXM1, we conducted ATAC-seq in 48 hour siRNA treated hTERT-RPE1.


title: Contribution of CENP-F to FOXM1-mediated discordant centromere and kinetochore transcriptional regulation. [CHIP-Seq]

overall_design: To evaluate the occupancy of transcription factors FOXM1 and MYBL2 in normal hTERT-RPE1 cells, we conducted ChIP-seq and then performed peak calling to visualize binding sites.


title: Contribution of CENP-F to FOXM1-mediated discordant centromere and kinetochore transcriptional regulation. [RNAseq_KD]

overall_design: To evaluate the transcriptomic changes in response to FOXM1 and CENP-F knockdown, we treated hTERT-RPE1 cells with siRNAs. We conducted RNA-seq in siCENP-F, siFOXM1 and siDouble conditions and performed comparative gene expression profiling analysis.


title: Contribution of CENP-F to FOXM1-mediated discordant centromere and kinetochore transcriptional regulation [RNA-seq]

overall_design: To evaluate the transcriptomic changes in response to FOXM1 overexpression, we created a stable Tet inducible FLAG-FOXM1c expressing hTERT-RPE1 cell lines. We conducted RNA-seq in dox and no dox conditions and performed comparative gene expression profiling analysis.


title: Immunological assessment of multiple clinical-stage adjuvants to induce durable immune responses to the R21 malaria vaccine

city: Palo Alto

first: Dmitri

middle: A

last: Kazmin

state: CA

address: 240 Pasteur Dr

department: Institute for Immunity, Transplantation and Infection

institute: Stanford University

summary: Authorization of the Matrix-M-adjuvanted R21 vaccine by three countries and its subsequent endorsement by the World Health Organization (WHO) for malaria prevention in children is a milestone in the fight against malaria. Yet, to meet the unprecedented demand for malarial vaccines, there is a pressing need for additional adjuvants that induce robust and durable vaccine-induced immunity. Here, we performed a comparative assessment of three clinically relevant adjuvants (an alum formulation of the TLR7/8 agonist 3M-052 (3M-052+Alum), the TLR4 agonist GLA-LSQ (GLA in liposome QS-21 formulation), and Matrix-M, the currently approved adjuvant for R21) for their capacity to induce durable immune responses to the R21 malaria vaccine in non-human primates. Immunization of macaques with R21 adjuvanted with 3M-052+Alum on a 0, 8, and 24-week schedule elicited anti-circumsporozoite antibody responses comparable in magnitude to the R21/Matrix-M vaccine and persisted up to 72 weeks with a half-life of 337 (264 – 459) days. A booster dose at 72 weeks induced an antigen-specific recall response, similar to the R21/Matrix-M vaccination. In contrast, R21/GLA-LSQ immunization induced a considerably lower and short-lived response. Consistent with the durability of serum antibody responses, Matrix-M and 3M-052+Alum induced long-lived plasma cells in the bone marrow and other tissues, including the spleen, but GLA-LSQ stimulated only short-lived plasmablasts. Finally, we show distinct innate immune signatures early after vaccination with these adjuvants. While 3M-052+Alum stimulated potent and persistent antiviral transcriptional and cytokine signatures after primary and booster immunizations, Matrix-M induced an enhanced expression of interferon- and Th2-related signatures more highly after the booster vaccination. Collectively, these findings provide a comparative database on the immune responses of three clinically relevant adjuvants with R21 and highlight the promise of 3M-052+Alum as an additional adjuvant for the R21 malaria vaccine.

overall_design: The study involved six groups of 6 - 10 male Rhesus macaques per group. All groups received four doses of 5 g R21 antigen with an adjuvant, as shown in Fig. 1A. The first two groups were immunized with R21 plus 30 or 10 g 3M-052 formulated in 0.5 mg Aluminum hydroxide subcutaneously at weeks 0, 4, 8 and 62. The third group was immunized with R21 plus 30 g 3M-052 formulated in 0.5 mg Aluminum hydroxide via the intramuscular route at the same time points (weeks 0, 4, 8, and 62) as a route comparison with group 1. Groups 4 and 5 were administered with R21 plus GLA-LSQ (25 g + 10 g QS-21) and 50 g Matrix-M via intramuscular route, respectively. The R21/MM group was immunized at weeks 0, 4, 8, and 62, comparable with groups 1, 2, and 3. The R21/GLA-LSQ group was immunized at weeks 0, 4, 8 and 72. Group 6 was immunized with 30 g 3M-052 using a longer interval between immunizations (weeks 0, 8, 24, and 72) to be consistent with our previous studies using HIV antigens (16-18). In summary, the study was designed to compare multiple parameters. Groups 1 and 2 served for dose comparison; groups 3, 4, and 5 for adjuvant comparison; and groups 3 and 6 for a comparison of interval between vaccinations. The animals were subjected to longitudinal sampling of blood and bone marrow for the analysis of immunogenicity and durability of immune responses

first: Prabhu

last: Arunachalam

first: Bali

last: Pulendran


title: Active forms of vitamin D as preventive and therapeutic agents in cutaneous melanoma: review and perspective

city: Birmingham

first: Andrzej

middle: T

last: Slominski

state: AL

address: 1720 2nd Avenue South

department: Dermatology

institute: UAB

summary: WM164, a human melanoma line, was a gift from Dr M. Herlyn (Wistar Institute, Philadelphia, PA, USA). The cells were cultured using DMEM media supplemented with 10% serum (fetal bovine serum while growing cells and charcoal-treated serum during incubation with vitamin D compounds), 1% antibiotics and 5  g/mL insulin (Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured in 75 cm2 plastic bottles (TPP–Techno Plastic Products AG, Trasadingen, Switzerland) with a filter in an incubator at 37  C with 5% CO2. CRISPR/Cas Knock out of the VDR. The clustered regularly interspaced short palindromic repeats (CRISPR) genetic engineering method was carried out to knock out VDR expression in the WM164 cell line as described in (Cancers 2021, 13, 3111. https://doi.org/10.3390/cancers13133111). The resulting cells with the VDR gene knocked out are designated as WM164 VDR KO, and the cells transduced with an “empty” lentivirus served as a control (WM164 scramble). VDR expression was checked for scramble and VDR KO lines before experiments were performed and further every 4 months after lentivirus treatment to ensure that VDR was not expressed in the WM164 KO. Sources of Vitamin D3 Compounds: Both 1,25(OH)2D3 and 25(OH)D3 were purchased from Sigma. 1,20(OH)2D3 and 20(OH)D3 were chemically synthesized. RNA SEQ: VDR-KO or scramble WM164 cells were plated onto 100 mm2 Petri dishes and treated with 1,25(OH)2D3, 25(OH)D3, 20(OH)D3, 1,20(OH)2D3 or ethanol (control) in triplicates.  Cells were incubated in 10% charcoal-treated serum for 24 h. Cells were further harvested and RNA isolated using Absolutly RNA Miniprep Kit  (Stratagen) . Approximately 120 ng/ul of each sample was sent for RNAseq analysis. BGI@CHOP genomics company sequenced the samples on Illumina HiSeq TM, analyzed, and sent us the results.

overall_design:
VDR-KO or scramble WM164 cells were plated onto 100 mm2Petri dishes and treated with 1,25(OH)2D3, 25(OH)D3, 20(OH)D3, 1,20(OH)2D3 or ethanol (control) in triplicates.  Cells were incubated in 10% charcoal-treated serum for 24 h. Cells were further harvested and RNA isolated using Absolutly RNA Miniprep Kit  (Stratagen)

Grant ID: W81XWH2210689
Grant title: Noncalcemic Vitamin D-Hydroxy Derivatives as Preventive and Therapeutic Agents in UVB-Induced Melanoma
Grantee name: Andrzej Slominski
Funding Agency: DOD

Grant ID: 2I01BX004293
Grant title: CYP11A1-derived secosteroids as therapeutic agents in UVB induced skin cancer
Grantee name: Andrzej Slominski
Funding Agency: VA merit

first: Tae-Kang

last: Kim

first: Zorica

last: Janjetovic

first: David

last: Crossman


title: Resistance of Ovarian Cancer Cells to Platinum Requires Exogenous Cholesterol, Scavenger Receptor Class B Type 1, and Glutathione Peroxidase 4

first: Daniela

last: Matei

address: 420 E. Superior St.

institute: Northwestern University Feinberg School of Medicine

summary: Essentially, all patients diagnosed with ovarian cancer (OC) are treated with platinum (Pt); however, Pt resistance (Pt-R) rapidly develops. We show OC cells resistant to Pt increase intracellular cholesterol by increasing expression of the high-density lipoprotein (HDL) receptor, scavenger receptor class B type 1 (SR-B1). Expression of SR-B1 was associated with chemoresistance in OC cells and tumors. SR-B1 blockade using synthetic cholesterol-poor HDL-like nanoparticles (HDL NPs) diminished cholesterol uptake leading to cell death of Pt-R OC cells in vitro and suppressed Pt-R OC tumor growth in vivo. Reduced cholesterol accumulation in Pt-R OC cells induced lipid oxidative stress through reduction of glutathione peroxidase 4 (GPx4) expression leading to ferroptosis. Reduction of GPx4 expression in OC cells re-sensitized Pt-R cells to Pt by reducing cholesterol uptake and enhancing the accumulation of lipid reactive oxygen species (L-ROS). Mechanistically, GPx4 knockdown in OC cells was associated with reduced expression of the histone acetyltransferase EP300, leading to reduced H3K27 acetylation around the SREBP2 promoter suppressing its expression. As SREBP2 regulates SR-B1 transcription, these data demonstrate chemoresistance and cancer cell survival under high ROS burden obligates high GPx4 and SR-B1 expression through SREBP2. Targeting SR-B1 to modulate cholesterol uptake inhibits this axis and causes ferroptosis in vitro and in vivo in Pt-R OC.

overall_design: To study the role of GPX4 in regulating chemoresistance of ovarian cancer, RNA sequencing was conducted on platinum resistant OVCAR5 (pt-R) ovarian cancer cells transfected with shRNA targeting GPX4 and sh control RNA (shctrl). Transcriptome of OVCAR5 Pt-R cells with GPX4 knocking-down was compared with control cells.


title: Identification of Ppy-lineage cells as a novel origin of pancreatic ductal adenocarcinoma

city: Maebashi

first: Yuko

last: Nakagawa

state: Gunma

address: 3-39-15 Showa-machi

department: Institute for Molecular & Cellular Regulation

institute: Gunma University

summary: The Ppy-gene encodes the pancreatic polypeptide (PP) secreted by PP- or - cells, an endocrine cell type located in the islet periphery. For a detailed characterization of PP cells, we aimed to establish PP cell lines. To this end, we generated Ppy-Cre;Rosa26-CAG-LSL-Large T mice, in which the SV40 large T antigen (TAg) is expressed in Ppy-expressing cells upon Cre-loxP-mediated recombination. Ppy-Cre;Rosa26-CAG-LSL-Large T mice, surprisingly, developed pancreatic ductal adenocarcinoma (PDAC) rapidly by 3 to 4 weeks of age, while mice with insulin2-Cre-mediated activation of TAg developed insulinomas. This suggests that PDACs could arise from the islet/endocrine cells, which is rather unexpected as PDACs are generally believed to originate from the pancreatic acinar or ductal cells. RNA-seq analysis of transformed Ppy-lineage cells in 7-day old islets showed downregulation of endocrine genes, upregulation of exocrine, ductal genes, and upregulation of PDAC-related genes and pathways, respectively. These results suggest that expression of an oncogene in Ppy-lineage cells leads to a fate switch in these endocrine precursors that causes them to adopt a PDAC cell fate. Our findings revealed that Ppy-lineage cells may be one of the origins of PDAC and provide novel insights into the heterogeneity in pathogenesis of pancreatic cancer and its personalized therapy.

overall_design: To determine the molecular characteristics and gene expression profile of Ppy-lineage lesion-forming cells, we performed RNA-seq and gene ontology analysis of Ppy-lineage cells with and without antigen activation using7-day oldreporter mice which express both tdTomato and TAg. We profiled total of 100 PpyCre/+; Rosa26tdTomato/+ (control) cells and 100 RTT (transformed) cells pooled from n=3 mice for each group using RNA-seq analysis.

first: Ofejiro

middle: B

last: Pereye

first: Takashi

last: Sato

first: Ayako

last: Fukunaka

first: Shuhei

last: Aoyama

first: Yuya

last: Nishida

first: Hirotaka

last: Watada

first: Reika

last: Kawabata

first: Tetsunari

last: Oyama

first: Hiroki

last: Mizukami

first: Akihisa

last: Fukuda

first: Yoshio

last: Fujitani


title: A systematic dissection of determinants and consequences of snoRNA-guided pseudouridylation of human mRNA

city: Tel Aviv

first: Aldema

last: Sas-Chen

state: Israel

address: Tel Aviv University

department: Shmunis School of Biomedicine and Cancer Research

country: Israel

institute: Tel Aviv University

summary: RNA can be extensively modified post-transcriptionally with >170 covalent modifications, expanding its functional and structural repertoire. Pseudouridine (Ψ), the most abundant modified nucleoside in rRNA and tRNA, has recently been found within mRNA molecules. Due to technical challenges, it remained unclear whether pseudouridylation of mRNA can be snoRNA-guided, which has important implications for understanding the physiological target spectrum of snoRNAs and for their potential therapeutic exploitation in genetic diseases. Here, using a massively parallel reporter based strategy we simultaneously interrogate Ψ levels across hundreds of synthetic constructs with predesigned complementarity against endogenous snoRNAs. Our results demonstrate that snoRNA-mediated pseudouridylation can occur on mRNA targets. However, this is typically achieved at low efficiencies, and is constrained by the localization of mRNA, by the expression levels of snoRNAs and by the length of the snoRNA:mRNA complementarity stretches. We exploited these insights for the design of snoRNAs targeting pseudouridylation at premature termination codons, which was previously suggested to suppress translational termination. However, in contrast to previous studies, in this and follow-up experiments we observe no evidence for readthrough of pseudouridylated stop codons. Our study enhances our understanding of the scope, ‘design rules’ and constraints of snoRNA-mediated pseudouridylation, and challenges a key functional outcome associated with this modification.

overall_design:
Experiment #1: 2 samples of Pol-I- and 2 samples of Pol-II-driven libraries transfected into HEK-293T cells, each treated with CMC or input. Extracted RNA underwent targeted psi-seq to determine pseudouridine level of constructs (used to create Table S2).
Experiment #2: three samples of Pol-II-driven libraries co-transfected with ACA21 and ACA61 overexpressing plasmids, or with a control (empty) overexpression plasmid or with no additional vectors. Each sample was treated with CMC. all 3 samples underwent targeted psi-seq (used in Table S2). overexpression and empty plasmid samples also underwent RNA-seq to determine abundance of snoRNA (used to create Table S5).
Experiment #3: two samples of Pol-II-driven libraries co-transfected with either control or DKC1-targeting siRNAs, each treated with CMC or input. All 4 samples underwent targeted psi-seq to determine pseudouridine levels of construct (used to create Table S2) and RNA-seq to determine abundance of DKC1 and snoRNAs (used to create Table S4). The siControl sample treated with CMC also underwent psi-seq to determine pseudouridine level of endogenous sites in rRNA (used to create Table S6).
Experiment #4: a single sample from either HEK-293T, A549, MCF7 or K562 was transfected with a Pol-II-driven library and treated with CMC or input. Samples underwent both targeted psi-seq and RNA-seq to determine pseudouridine level of constructs (used to create Table S2) and abundance of snoRNAs (used to create Table S7).
Experiment #5: total RNA that was extracted from HEK293T samples are used to generate cDNA libraries using a psi-seq protocol to assess pseudouridine levels in endogenous rRNA targets (used to create Table S6).
Experiment #6: DNA reporters harboring a single thymidine were in vitro transcribed in the presence of uridine or pseudouridine to assess the effect of bases downstream to the modified site on psi-rate measured by targeted psi-seq (used to create Table S3).
Experiment #7: plasmids expressing a bi-cistronic fluorescent reporter separated by a sequence element including a premature stop codon from the DMD gene were transfected into HEK-293T cells alongside a WT or modified sno61 construct targeting the DMD stop codon for pseudouridylation. Purified RNA was pooled, ribo-zero treated and was used to generate a pooled psi-seq library (used to create Figure 5b).

first: Ronit

last: Nir

first: Schraga

last: Schwartz


title: Xanthomonas citri pv fuscans transcriptomic changes during the contamination of  Phaseolus vulgaris seeds

city: Beaucouzé

first: Martial

last: Briand

address: 42, rue Georges Morel

country: France

institute: INRAE

summary: To investigate the bacterial functions involved in seed contamination during their formation, we sequenced bacterial mRNA at 24, 35 and 42 days after pollination (DAP). Gene expression was compared between the different stages.

overall_design: Bean plants were inoculated at pre-flowering stage. Three samples (one sample corresponding to the seeds of three plants) were harvested at each sampling date. Two independant experiments were performed. Bacterial mRNA were sequenced using SureSelectXT RNA Direct technology (Agilent), based on the capture of bacterial sequences with oligonucleotidic probes.

first: Armelle

last: Darrasse

first: Matthieu

last: Barret

first: Jérôme

last: Verdier


title: Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2 (Screen)

first: Elmar

last: Wolf

sample_organism:
Mus musculus
synthetic construct

platform_organism:
Mus musculus
synthetic construct

overall_design: Pooled shRNA Screen in cultured KPC cells, treatment with doxycycline or ethanol (3 replicates); pooled shRNA Screen in KPC allograft tumors, treatment with doxycycline or ethanol (5 replicates)

first: Markus

last: Vogt

first: Nevenka

last: Dudvarski Stankovic

first: Yiliam

last: Cruz Garcia

first: Julia

last: Hofstetter

first: Katharina

last: Schneider

first: Filiz

last: Kuybu

first: Theresa

last: Hauck

first: Bikash

last: Adhikari

first: Anton

last: Hamann

first: Yamila

last: Rocca

first: Lara

last: Grysczyk

first: Benedikt

last: Martin

first: Anneli

last: Gebhardt-Wolf

first: Armin

last: Wiegering

last: Diefenbacher

first: Georg

last: Gasteiger

first: Stefan

last: Knapp

first: Dieter

last: Saur

first: Martin

last: Eilers

first: Mathias

last: Rosenfeldt

first: Florian

last: Erhard

first: Seychelle

last: Vos


title: Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2 (SLAM-Seq)

first: Elmar

last: Wolf

summary: The hallmark oncogene MYC drives the progression of most human tumors, but direct inhibition of MYC by small molecules has not yet entered the clinical testing phase. As a transcription factor, MYC is dependent on a variety of binding partners for its molecular oncogenic function. Here, we explored the possibility of targeting MYC via its interactome, through a genetic screen in pancreatic ductal adenoma carcinoma (PDAC) models. While several MYC-binding partners are important for cultured PDAC cells but dispensable for tumors in their natural environment in mice, the nuclear AAA-ATPases RUVBL1 and -2 are most essential among all MYC-binding partners in vivo. Induced degradation of RUVBL by the auxin-degron system leads to rapid arrest of PDAC cells in culture and complete regression of tumors in mice, which is preceded by immune cell infiltration. Mechanistically, RUVBL is required for MYC-mediated elongation of RNA polymerase II (RNAPII) and thus for the establishment of MYC dependent oncogenic gene expression patterns. Overall, three relevant observations emerged from our study. First, our study shows that tumor cell dependencies are strongly influenced by their environment and that genetic screens in cultured cells need to be carefully validated in vivo. Second, we have shown that the auxin-degron system can be applied in a PDAC model, allowing target validation and molecular analysis in living mice. Third, our study identifies RUVBL1 as a druggable vulnerability in MYC driven cancer.

overall_design: KPC-AID-Ruvbl1 cells were treated with 1 µM 5-Ph-IAA or DMSO (depletion of AID-Ruvbl1) for 3 h or 15 h (3 replicates) followed by 2 h labelling with 800 µM 4sU. One additional sample per condition was not labelled with 4sU; KPC-AID-Ruvbl1 cells were treated with 1 µM MZ-1 or DMSO (depletion of BRD4) for 5 h (3 replicates) followed by 2 h labelling with 800 µM 4sU. One additional sample per condition was not labelled with 4sU; KPC-MYC-ER cells were treated with 1 µM CB-6644 (inhibition of RUVBL1/2) or DMSO for 20 h followed by 4-hydroxytamoxifen (activation of MYC-ER) or ethanol by 4 h (3 replicates) followed by 2 h labelling with 400 µM 4sU. One additional sample per condition was not labelled with 4sU.

first: Markus

last: Vogt

first: Nevenka

last: Dudvarski Stankovic

first: Yiliam

last: Cruz Garcia

first: Julia

last: Hofstetter

first: Katharina

last: Schneider

last: Kuybu


title: Fat graft survival requires metabolic reprogramming towards glycolytic pathway

city: Daegu

first: Taehee

last: Jo

address: 1035 Dalgubeol daero

department: Department of Plastic and Reconstructive Surgery

country: South Korea

institute: Keimyung University Dongsan Hospital

summary:
Background: The mechanisms underlying fat graft survival are poorly understood. Here, we performed unbiased transcriptomics analysis of mouse fat graft model to find out the molecular mechanism of free fat graft survival.
Methods: We conducted RNA-sequencing (RNA-seq) analysis of mouse free subcutaneous fat graft model at day 3 and 7 following grafting (n=5). High-throughput sequencing was performed as paired-end using NovaSeq6000 (Illumina, CA, USA). The calculated transcripts per million (TPM) values were processed for principal component analysis (PCA), unsupervised hierarchically clustered heat map generation, and Gene set enrichment analysis.
Results: PCA and heat map data revealed global differences in the transcriptome of grafted fat when compared with non-graft control. The top meaningful up-regulated gene sets in the grafted fat were related to epithelial mesenchymal transition and hypoxia by day 3, and angiogenesis by day 7. Mechanistically, the glycolysis pathway was commonly upregulated in the grafted fat at day 3 (FDR q=0.012) and day 7 (FDR q=0.084). In subsequent experiments, pharmacological inhibition of glycolytic pathway in the grafted fat with 2-Deoxy-D-glucose (2-DG) significantly suppressed fat graft retention rates, both grossly and microscopically (n=5).
Conclusions: Global transcriptome analysis and pharmacologic inhibition study revealed that free adipose tissue grafts undergo metabolic reprogramming towards glycolytic pathway. Whether targeting the pathway wound enhance graft survival rate remains to be investigated.

overall_design: Adipose mRNA profiles of fat grafts: 1) control, 2) 3 day graft, 3) 7 day graft


title: Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2

overall_design: Refer to individual Series

first: Filiz

first: Theresa

last: Hauck

first: Bikash

last: Adhikari

first: Anton

last: Hamann

first: Yamila

last: Rocca

first: Lara

last: Grysczyk

first: Benedikt

last: Martin

last: Gebhardt-Wolf

last: Wiegering


title: Discovering specificity determinants in Gln3 and their regulation

city: Rehovot

first: Naama

last: Barkai

state: --- Select a state ---

address: Herzl St 234

department: Molecular Genetics

institute: Weizmann Institute

summary: Profiling of Gln3 IDR sequence to reveal specificity determinants and their regulation

overall_design: >70 IDR variants were designed and constructed using CRIPSR and profiled ChEC-Seq.

first: Felix

last: Jonas

first: Bohdana

last: Hurieva

first: Divya

last: Kumar

first: Rotem

last: Morag

first: Offir

last: Lupo


title: The Ric-8b protein (resistance to inhibitors of cholinesterase 8b) is key to preserving contractile function in the adult heart.

city: London

first: Alison

last: Thomas

address: Charterhouse Square, William Harvey Heart Centre, 1st Floor

department: Clinical Pharmacology and Precision Medicine

institute: QMUL

summary: Resistance to inhibitors of cholinesterases (Ric-8 proteins) are involved in modulating G-protein function but little is known of their potential physiological importance in the heart.  In the present study, we assessed the role of resistance to inhibitors of cholinesterase 8b (Ric-8b) in determining cardiac contractile function using a mouse model. Deletion of Ric-8b in cardiac tissue  led to severely reduced contractility as measured using echocardiography days after administration of tamoxifen. Histological analysis of the ventricular tissue showed highly variable myocyte size, prominent fibrosis and an increase in cellular apoptosis. RNA sequencing revealed transcriptional remodelling in response to cardiac Ric-8b deletion involving the extracellular matrix and inflammation. Phosphoproteomic analysis revealed substantial downregulation of phosphopeptides related to myosin light chain 2. At the cellular level, the deletion of Ric-8b led to loss of activation of the L-type calcium channel through the β-adrenergic pathways. Using fluorescence resonance energy transfer-based assays in heterologous expression systems we showed Ric-8b protein selectively interacts with the stimulatory G-protein, Gαs. We explored if deletion of Gnas (the gene encoding Gαs) in cardiac tissue using a similar approach in the mouse led to an equivalent phenotype. The conditional deletion of the Gαs gene in the ventricle led to comparable effects on contractile function and cardiac histology. We conclude that Ric-8b is essential to preserve cardiac contractile function likely through an interaction with the stimulatory G-protein and downstream phosphorylation of myosin light chain 2.

overall_design: We developed a murine model in which it was possible to conditionally delete Ric-8b in cardiac tissue in the adult animal after the addition of tamoxifen. Ric-8b (flx/flx) mice were combined with the cardiac muscle specific tamoxifen inducible cre to create “Ric-8b (flx/flx) MCM” mice. RNA-sequencing was carried out on hearts of Ric-8b (flx/flx) MCM and control mice to compare the differences within the transcriptome when Ric-8b is knocked out.

first: Elena

last: Tsisanova

first: Muriel

last: Nobles

first: Sonia

last: Sebastian

first: Keat-Eng

last: Ng

first: Lee

last: Weinstein

first: Patricia

last: Munroe

first: Andrew

last: Tinker


title: Palovarotene action against heterotopic ossification includes the inhibition of local participating activin A-expressing cell populations

city: Shenyang

first: Lutian

last: Yao

address: 155 Nanjing st

department: Department of Orthopedic

institute: the First Affiliated Hospital of China Medical University

summary: Heterotopic ossification (HO) consists of endochondral bone forming at extraskeletal anatomical sites. One form of HO is acquired and instigated by traumas or invasive surgery, and another form is genetic and severe and characterizes patients with Fibrodysplasia Ossificans Progressiva (FOP). Previously, we showed that the synthetic retinoid agonist Palovarotene inhibits both acquired and genetic HO, and we and others subsequently showed that systemic administration of a neutralizing activin A antibody reduces both HO forms as well. Here, we asked whether Palovarotene’s action includes an interference with endogenous activin A expression and/or participation in HO. Using a standard mouse model of acquired HO, we found that activin A protein and encoding RNA (Inhba) were prominent in chondrogenic cells within developing HO masses in control untreated mice. Single cell RNAseq (scRNAseq) assays verified that Inhba expression characterized chondroprogenitors and chondrocytes in control HO, in addition to its expected expression in inflammatory cells and macrophages particularly. Palovarotene administration (4 mg/kg/day/gavage) caused a sharp inhibition of both HO and amounts of activin A and Inhba transcripts. Bioinformatic analyses of scRNAseq datasets indicated that the drug had reduced interactions and crosstalk amongst local cell populations. To determine if Palovarotene inhibited Inhba expression directly, we tested it in primary cultures of growth plate chondrocytes. Drug treatment inhibited cartilage phenotypic expression but not Inhba expression. Our data reveal that Palovarotene intersects with activin A during its anti-HO action and markedly reduces the number of local participating Inhba-expressing cell populations. The data broaden the spectrum of HO culprits against which Palovarotene acts, accounting for its therapeutic effectiveness.

overall_design:
Single cell RNA seq of cells from Ectopic masses were harvested on day 5  from mice implanted with Matrigel/rhBMP2 mixture, with the latter mice treated systemically with Palovarotene
Single cell RNA seq of cells from Ectopic masses were harvested on day 5  from mice implanted with Matrigel/rhBMP2 mixture, with the mice treated systemically with Palovarotene
Associated previously published scRNA seq files can be found in GSE157679

first: Maurizio

last: Pacifici


title: DNA methylation in developing fetal brain of pig

city: Columbia

first: SUSANTA

last: BEHURA

state: MO

address: 920 East Campus Dr

institute: University of Missouri

type: Methylation profiling by high throughput sequencing

summary: DNA methylation was profiled in a genome-wide manner by Enzymatic Methyl-seq (EM-seq) in fetal brain of both sexes at three gestation days 45, 60 and 90. The analysis showed nearly 3-fold fewer differentially methylated sites (DMSs) between GD90 and GD60 (n=54) compared to that between GD60 and GD45 (n=150).  Comparative analysis of the methylation data with the RNA-seq data previously generated from the same gestation times showed an opposition pattern where more number of differentially expressed genes (DEGs) were observed between GD90 and GD60 compared to GD60 and GD45.

overall_design: DNA methylation profiling of all 12 samples (6 fetal brain samples and 6 adult blood samples) by enzymatic methyl-seq (EM-seq).

first: Monika

last: Strawn

first: Timothy

last: Safranski

first: Susanta

last: Behura


title: The SW480 cell line as a model of resident and migrating colon cancer stem cells

status: Public on May 31 2024

city: Rotterdam

first: Mathijs

last: Verhagen

address: Doctor Molewaterplein 40

country: Netherlands

institute: Erasmus Medical Center

summary: Gene expression profiling of the SW480 sphere and bulk subpopulations.

overall_design:
RNAseq profiles of subpopulations from the SW480 colon cancer cell line. N = 4 replicates per group.

Please note that the current study explores an additional subpopulation in the SW480 colon cancer cell line (i.e. "spheres"). For reference, the normalized data (with similar preprocessing) of other two subpopulations that have been sequenced in the same lab (GSM4683947-GSM4683954 in GSE154927) are included in the 'SW480_vst.xlsx'.

first: Andrea

last: Sacchetti

first: Tong

last: Xu

first: Roberto

last: Stabile

first: Riccardo

last: Fodde


title: Evolved differences in RNA and DNA methylation in the burying beetle

city: CAMBRIDGE

first: Rahia

last: Mashoodh

state: Cambridgeshire

address: Downing Street, Zoology, University of Cambridge

department: Zoology

institute: University of Cambridge

summary: DNA methylation and gene expression in response to the loss of care for 30 generations in N. Vespilloides

overall_design: FC and NC populations that were either exposed to native or reciprocal conditions.

first: R

first: J

last: Westoby


title: Nonsense-mediated mRNA decay counteracts TRP53 to maintain the progenitor cell pool for brain development (snRNA-seq_e15invivo)

city: Riverside

first: Sika

last: Zheng

address: University Ave 900

department: School of Medicine

institute: University of California, Riverside

summary: To examine the transcriptomic changes in neocorices from E13.5 Emx1-Upf2cKO and their control littermates at a single cell level, we performed snRNA-seq using the 10x Chromium platform.

overall_design: Comparative gene expression profiling analysis of snRNA-seq data for neocorices from E15.5 Emx1-Upf2cKO and their control littermates.

first: Zheng

last: Sika

first: Zhao

last: Jing Rong

first: Kubota

last: Naoto

first: Chen

last: Liang


title: Transcriptomic change of whole blood cells from mice treated with CPI-0209

city: Boston

first: Jike

last: Cui

address: 470 Atlantic Ave

institute: MorphoSys AG

summary: Recurrent mutations in ARID1A occur frequently in advanced urothelial carcinoma, endometrial cancers and ovarian clear cell carcinoma, and may create an alternative chromatin state that can be exploited therapeutically. Previously, the histone methyltransferase EZH2 was identified as a targetable vulnerability in the context of ARID1A mutations.  Here we describe the discovery of tulmimetostat (CPI-0209), a novel, orally available, clinical stage EZH2 inhibitor with remarkably long residence time and elucidate aspects of its application potential in ARID1A mutant tumors. Tulmimetostat administration as a single agent achieves in vivo efficacy in multiple ARID1A mutant bladder, ovarian, and endometrial tumor models and improves cisplatin response in chemotherapy-resistant models. Consistent with its potency and the comprehensive and durable level of target coverage, tulmimetostat demonstrates greater efficacy than other PRC2-targeted inhibitors at comparable or lower exposures in a bladder cancer xenograft mouse model due to greater changes in gene expression and a profound reduction of H3K27me3 levels in the tumors. Importantly, we report the identification of a tulmimetostat controlled gene expression signature in whole blood from a cohort of 32 cancer patients, whereby the number of genes altered in expression as well as the magnitude of expression changes exquisitely correlate with tulmimetostat exposure, thus representing a novel pharmacodynamic marker for the assessment of target coverage for PRC2-targeted agents in the clinic.  Collectively, our data suggest tulmimetostat may unlock the full potential of EZH2 inhibition as a cancer therapeutic mechanism with potential applications in hematologic malignancies and in solid tumors, including in an ARID1A mutant setting.

overall_design: CB17-SCID with HT1376 cell line derived xenograft mice were treated 11 days with vehicle or CPI0209 at 35mpk PO QD dose, respectively. Whole blood was collected at 1 hr, 6 hr or 24 hr after last dose. RNA-seq was performed on the whole blood samples.

first: P

last: Keller

first: E

last: Adams

last: Wu

last: Trojer


title: A genetics screen uncovers the role of RNA-binding protein RBP45D in flowering time under heat and DCL3-independent RNA-directed DNA methylation II

city: Planegg

first: Tatjana

last: Kleine

address: Großhaderner Straße 2

department: Biologie I, Botanik

institute: Ludwig-Maximilians-Universität München

summary: In the canonical RdDM, small interfering RNAs (siRNAs) are produced through the slicer activity of DICER-LIKE 3 (DCL3). Here, we describe the Arabidopsis thaliana prors1 (LUC) transgenic system in which transcriptional gene silencing (TGS) through RdDM is independent of DLC3. To identify players in the DCL3-independent pathway, a forward genetics screen was performed with the prors1 (LUC) system to identify suppressor mutants in which TGS is released. Among already known components of RdDM, we identified RNA-binding protein RBP45D. RBP45D promotes DNA methylation changes, and a lack of RBP45D results in a late flowering phenotype especially under heat, presumably mediated by elevated FLC levels. A lack of RBP45D did not influence the pre-mRNA splicing of known RdDM genes, suggesting that RBP45D involvement in the RdDM might be direct. RBP45D is localized to the nucleus where it is associated with snRNAs and snoRNAs. RBP45D promotes siRNA production originating from the transgene and we hypothesize that this function is direct and not indirect.

overall_design: Examination of small RNA accumulation in the Arabidopsis prors1 (LUC) and rls6 mutants in comparison to Col-0 (LUC)


title: The HtrA chaperone monitors sortase-assembled pilus biogenesis in E. faecalis

city: Genève

first: Kimberly

last: Kline

address: 1 Rue Michel Servet

institute: University of Geneva

summary: Sortase-assembled pili contribute to virulence in many Gram-positive bacteria. In Enterococcus faecalis, the endocarditis and biofilm-associated pilus (Ebp) is polymerized on the membrane by sortase C (SrtC) and attached to the cell wall by sortase A (SrtA). In the absence of SrtA, polymerized pili remain anchored to the membrane (i.e. off-pathway). Here we show that the high temperature requirement A (HtrA) bifunctional chaperone/protease of E. faecalis is a quality control system that clears aberrant off-pathway pili from the cell membrane. In the absence of HtrA and SrtA, accumulation of membrane-bound pili leads to cell envelope stress and partially induces the regulon of the ceftriaxone resistance-associated CroRS two-component system, which in turn causes hyper-piliation and cell morphology alterations. Inactivation of croR in the ∆srtA∆htrA background partially restores the observed defects of the ∆srtA∆htrA strain, supporting a role for CroRS in the response to membrane perturbations. Moreover, absence of SrtA and HtrA decreases basal tolerance of E. faecalis against cephalosporins and daptomycin. The link between HtrA, pilus biogenesis and the CroRS two-component system provides new insights into the E. faecalis response to endogenous membrane perturbations.

overall_design: To investigate the relationship between HtrA, pilus biogenesis, and the CroRS two-component system in E. faecalis, we created a panel of clean deletion mutant strains of htrA, srtA, htrA srtA, croR::tn htrA srtA, and srtA htrA ebpABC, and performed RNA-Seq on exponentially grown cells in TSB supplemented with 0.25% glucose. We also performed RNA-Seq of wild-type OG1RF treated with the cell envelope-targeting antibiotic daptomycin and compared it to untreated. In this case, cells were grown in BHI media.

first: Cristina

last: Colomer-Winter

first: Adeline

last: Yong

first: Kelvin

last: Chong

first: Mark

last: Veleba

first: Pei

last: Choo

first: Iris

last: Gao

first: Foo

last: Ho

first: Artur

last: Matysik

first: Swaine

last: Chen


title: Detection of allele-specific expression in spatial transcriptomics with spASE

first: Luli

last: Zou

address: 677 Huntington Ave

institute: Harvard

summary: Spatial transcriptomics technologies permit the study of the spatial distribution of RNA at near-single-cell resolution genome-wide. However, the feasibility of studying spatial allele-specific expression (ASE) from these data remains uncharacterized. Here, we introduce spASE, a computational framework for detecting and estimating spatial ASE. To tackle the challenges presented by cell type mixtures and a low signal to noise ratio, we implement a hierarchical model involving additive mixtures of spatial smoothing splines. We apply our method to allele-resolved Visium and Slide-seq from the mouse cerebellum and hippocampus and report new insight into the landscape of spatial and cell type-specific ASE therein.

overall_design: We used female CAST/EiJ x 129S1/SvImJ obtained from Jackson Laboratories. Slide-seqV2 was performed as described previously on 10um-thick coronal slices of the hippocampus from three mice (Mice 1-3) and cerebellum (Mouse 3). For Mice 1 and 3, two serial sections were sequenced and reads were aggregated downstream, while for Mouse 2, only one section was sequenced. Slide-seq libraries were processed with the slideseq-tools pipeline (https://github.com/MacoskoLab/slideseq-tools) and then re-aligned to a custom transcriptome. For longer read libraries (Mouse 3 Slide-seq), we used Atropos to trim adapter sequences and low quality bases. 10x Genomics Visium was performed on two mice (Mice 4,5) on 10umn-thick coronal slices of the cerebellum. Visium libraries were processed with the 10x Genomics Space Ranger 1.1.0 pipeline and then re-aligned to a custom transcriptome. We generated a pooled CASTx129 transcriptome using the command create-hybrid from the EMASE software on the CAST and 129 transcript fasta files downloaded from ftp://churchill-lab.jax.org/software/g2gtools/mouse/R84-REL1505/. We then aligned reads to this pooled transcriptome with bowtie2 using the parameters -k 100 -p 16 --very-sensitive. This method of alignment ensures there is no reference bias. The multi-mapping parameter k=100 was chosen to report a large amount of multi-maps as bowtie2 randomly reports multi-mapping locations (in order of increasing number of mismatches). We used a custom script (https://github.com/lulizou/spASE/blob/master/scripts/processBowtie2.py) for processing the aligned BAM file to create a gene UMI count matrix only from reads that uniquely aligned to one gene and one allele. We conservatively restricted attention to alignments with 3 or fewer mismatches and only considered alignments that had the fewest number of mismatches for that read. We merged the reads from samples that had two serial sections: Mouse 1 hippocampus, Mouse 3 hippocampus, and Mouse 3 cerebellum.


title: Crosstalk between Regnase-1 and -3 shapes mast cell survival and cytokine expression

city: Bellinzona

last: Leoni

state: Ticino

address: Via F. Chiesa 5

department: Molecular Immunology

institute: Institute for Research in Biomedicine

summary: Dysregulation of mast cell activation can lead to allergic and anaphylactic reactions. Post-transcriptional regulation of immune-related transcripts by RNA-binding proteins (RBPs) is critical in controlling immune cell responses, including mast cell functionality. The Regnase family of RBPs plays a central role in regulating gene expression and controlling immune responses in both lymphoid and myeloid cells. However, what is the functional role of Regnase proteins in mast cells remains to be understood. Using different approaches of Regnase proteins depletion and deletion as well as overexpression by mRNA delivery, we found that Regnase-1 is a powerful negative regulator of mast cell proliferation, survival and ability to produce inflammatory cytokines. On the other hand, Regnase-3 was essential to modulate Regnase-1 expression in mast cells by direct targeting of the 3’-untranslated region of Regnase-1, leading to the destabilization of the Zc3h12a mRNA. Regnase-1 deletion had widespread effect on mast proliferation and survival even in the absence of stimulation and was at least in part linked to specific nuclear functions of Regnase-1 in modulating the ability of mast cells to withstand DNA damage. Overall, we describe the importance of Regnase proteins in modulating mast cell homeostatic and inflammatory responses and we describe the impact of Regnase-1 in the maintenance of genome stability.

overall_design: Bone marrow-derived mast cells (BMMCs) were in vitro differentiated from 4 mice and transfected with Zc3h12a (encoding for Regnase-1) or scrambled (non-targeting) gRNA-Cas9 RNPs. Total RNA was extracted from each sample and used for mRNA sequencing.

first: Marian

last: Bataclan

first: Silvia

last: Monticelli


overall_design: Bone marrow-derived mast cells (BMMCs) were in vitro differentiated from 3 mice and transfected with siRNAs targeting Zc3h12a, Zc3h12c, or combined. After 48 hours, cells were stimulated with IgE and antigen complexes for 2 hours. Total RNA was extracted from each sample and used for gene expression profiling using the Nanostring nCounter® Myeloid Innate Immunity Panel.


title: Epigenetic and transcriptional regulation of dendritic cell development and anti-tumor function by HDAC1 (RNA-Seq)

status: Public on May 30 2024

city: Vienna

first: Cristiano

last: de Sá Fernandes

address: Borschkegasse 8a

department: Center of Cancer Research

country: Austria

institute: MedicalUniversity of Vienna

summary: DC progenitors adapt their transcriptional program during development generating different subsets. How chromatin modifications modulate these processes is unclear. Here we investigate the impact of histone deacetylation on DCs by genetically deleting HDAC1 or HDAC2 in hematopoietic progenitors and CD11c-expressing cells. While HDAC2 isn’t critical for DC development, HDAC1 deletion impairs pro- and mature pDC generation and affects ESAM+cDC2 differentiation from tDC and pre-cDC2, whereas cDC1s are unchanged. HDAC1 knock-down in human hematopoietic cells also impairs cDC2 development highlighting its crucial role across species. Multi-omics analyses reveal that HDAC1 controls expression, chromatin accessibility and histone-acetylation of the transcription factors IRF4, IRF8 and SPIB required for efficient development of cDC2 subsets. Without HDAC1, DCs switch immunologically enhancing tumor surveillance through increased cDC1 maturation and IL-12 production driving Th1-mediated immunity and CD8+T-cell recruitment. Our study reveals the importance of histone-acetylation in DC development and anti-tumor immunity suggesting DC-targeted therapeutic strategies for immuno-oncology.

overall_design: Comparative transcriptional analysis of the three main DC subsets from HDAC1 control and HDAC1-Mx1Cre mice.

first: Maria

last: Sibilia


title: Development of a Cellular Assay as a Personalized Model for Testing Chronic Wound Therapeutics

first: Barbara

last: Wolff-Winiski

state: Other (Non US)

address: Dr. Bohr-Gasse 7

institute: Akribes Biomedical

summary:
This SuperSeries is composed of the SubSeries listed below:
GSE268638
GSE268639


title: Molecular dialogue between Phaseolus vulgaris seed and Xanthomonas (Xcf) bacteria interactions during seed transmission

city: Angers

first: Jerome

last: Verdier

address: Rue Georges Morel

institute: INRAE / IRHS

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

sample_organism:
Phaseolus vulgaris
Xanthomonas citri pv. fuscans


title: Epigenetic and transcriptional regulation of dendritic cell development and anti-tumor function by HDAC1 (CUT&RUN)

overall_design: Comparative H3K27Ac levels from splenic cDC2 DC from HDAC1 control and HDAC1-Mx1Cre mice.

first: Davide

last: Seruggia


title: Development of a Cellular Assay as a Personalized Model for Testing Chronic Wound Therapeutics (NS79)

summary: Exudates of non-healing wounds contain drivers of pathogenicity. We utilized >800 exudates from non-healing and healing wounds of diverse etiologies, collected by three different methods, to develop a wound-specific, cell-based functional biomarker assay. Human dermal fibroblast proliferation served as readout to a) to differentiate between healing and non-healing wounds, b) follow the healing process of individual patients, and c) assess the effects of therapeutics for chronic wounds ex vivo. We observed a strong correlation between wound chronicity and inhibitory effects of individual exudates on fibroblast proliferation, with good diagnostic sensitivity (76-90%, depending on the sample collection method). Transition of a clinically non-healing to a healing phenotype restored fibroblast proliferation and extracellular matrix formation while reducing inflammatory cytokine production. Transcriptional analysis of fibroblasts exposed to ex vivo non-healing wound exudates revealed an induction of inflammatory cytokine- and chemokine pathways and the unfolded protein response, indicating that these changes may contribute to the pathology of non-healing wounds. Testing the wound therapeutics platelet derived growth factor and silver sulfadiazine yielded responses in line with clinical experience and indicate the usefulness of the assay to search for and profile new therapeutics.

overall_design: Independent of wound etiology, wound exudates from chronic wounds have inhibitory effects on proliferation of skin fibroblasts after incubation for 72h. To understand the early transcriptional response and the underlying molecular mechanism(s) leading to this inhibitory effect, primary skin fibroblasts were cultured for 6 hours with selected wound exudates from three patients with wounds of different etiologies (diabetic foot ulcer, mixed venous/arterial ulcer, and trauma/surgery). Wound exudates were used at concentrations that reduced fibroblast proliferation to 30% of control cells after 72h.

first: Petra

last: Doerfler

first: Nicole

last: Schoefmann

first: Gabriela

last: Cabral

first: Wolfgang

last: Bauer

first: Martin

middle: C

last: Berli

last: Binder

first: Carina

last: Borst

first: Sander

last: Botter

first: Lars

last: French

first: Tobias

last: Goerge

first: Juerg

last: Hafner

first: Daniela

last: Hartmann

first: Annette

last: Høgh

first: Wolfram

last: Hoetzenecker

first: Judith

last: Holzer-Geissler

last: Kamolz

first: Katrin

last: Kofler

first: Thomas

last: Luger

first: Sebastian

last: Nischwitz

first: Michael

last: Popovits

first: Klemens

last: Rappersberger

first: Gaetana

last: Restivo

first: Justin

middle: G

last: Schlager

first: Matthias

last: Schmuth

first: Georg

last: Stingl

first: Theresa

last: Stockinger

first: Anke

last: Stroelin

first: Anton

last: Stuetz

first: Julian

last: Umlauft

last: Weninger


title: Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2 (ChIP-Seq)

city: Kiel

first: Elmar

last: Wolf

address: Rudolf-Höber-Str. 1

institute: Christian-Albrechts-University Kiel

summary:
Objective:
The hallmark oncogene MYC drives the progression of most tumorstumours, but direct inhibition of MYC by a small-molecule drug has not reached clinical testing. MYC is a transcription factor that depends on several binding partners to function. We therefore explored the possibility of targeting MYC via its interactome in pancreatic ductal adenocarcinoma (PDAC).

Design:
To identify the most suitable targets among all MYC binding partners, we constructed a targeted shRNA library and performed screens in cultured PDAC cells and tumorstumours in mice.Results:Unexpectedly, many MYC binding partners were found to be important for cultured PDAC cells but dispensable in vivo. However, some were also essential for tumorstumours in their natural environment and, among these, the ATPases RUVBL1 and RUVBL2 ranked first. Degradation of RUVBL1 by the auxin-degron system led to the arrest of cultured PDAC cells but not untransformed cells and to complete tumortumour regression in mice, which was preceded by immune cell infiltration. Mechanistically, RUVBL1 was required for MYC to establish oncogenic and immuno-evasive gene expression identifying the RUVBL1/2 complex as a druggable vulnerability in MYC-driven cancer.

Conclusion:
One implication of our study is that PDAC cell dependencies are strongly influenced by the environment, so genetic screens should be performed in vitro and in vivo. Moreover, the auxin-degron system can be applied in a PDAC model, allowing target validation in living mice. Finally, by revealing the nuclear functions of the RUVBL1/2 complex, our study presents a pharmaceutical strategy to render pancreatic cancers potentially susceptible to immunotherapy.

overall_design: KPC-AID-Ruvbl1 cells were treated with 5-Ph-IAA or DMSO for 15 h (depletion of RUVBL1). A375-MYC-AID cells were treated with 5-Ph-IAA or DMSO for 3 h (depletion of MYC).

first: Markus

last: Vogt

first: Nevenka

last: Dudvarski Stankovic

first: Yiliam

last: Cruz Garcia

first: Julia

last: Hofstetter

first: Katharina

last: Schneider

first: Filiz

last: Kuybu

last: Hauck

first: Bikash

last: Adhikari

last: Hamann

first: Yamila

last: Rocca

first: Lara

last: Grysczyk

first: Benedikt

last: Martin

first: Anneli

last: Gebhardt-Wolf

first: Armin

last: Wiegering

last: Diefenbacher

last: Gasteiger

first: Stefan

last: Knapp

first: Dieter

last: Saur

first: Martin

last: Eilers

first: Mathias

last: Rosenfeldt

first: Florian

last: Erhard

first: Seychelle

last: Vos


title: Epigenetic and transcriptional regulation of dendritic cell development and anti-tumor function by HDAC1 (ATAC-Seq)

first: Cristiano

last: de Sá Fernandes

overall_design: Comparative chromatin accessibility analysis of cDC1 and cDC2 DC subsets from HDAC1 control and HDAC1-Mx1Cre mice.

first: Maria

last: Sibilia


title: The roles of nuclear orphan receptor NR2F6 in anti-viral innate immunity [ChIP-seq]

city: Wuhan

first: Chen-Yu

last: Wang

state: Hubei

address: 299 Bayi Road, Wuchang District

institute: Wuhan University

summary: The regulation of proper gene transcription is critical for anti-viral defense. Dynamics of enhancer activity play important roles in many biological processes, and epigenomic analysis is widely used to determine the involved enhancers and transcription factors. To determine new transcription factors in anti-DNA-virus response, we perform H3K27ac ChIP-Seq analysis to profile the active enhancer pattern in HSV-1-infected THP-1 cells. We have identified three transcription factors, NR2F6, MEF2D and MAFF, and experimentally verified their roles in promoting HSV-1 replication. NR2F6 promotes HSV-1 replication and gene expression in vitro and in vivo, which is not related with the classical cGAS/STING pathway. NR2F6 binds to the promoter of MAP3K5 and activate AP-1/c-Jun pathway, which is critical for DNA virus replication. On the contrary, NR2F6 expression is transcriptionally repressed by c-Jun upon HSV-1 infection, which forms a negative feedback loop. Meanwhile, cGAS/STING innate immunity signaling represses NR2F6 through STAT3. Taken together, we have identified new transcription factors involved in the interactive network between DNA viruses and host cells, and revealed the underlying mechanisms for anti-viral research.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone modifications H3K27ac in THP-1 cells.

first: Yang

last: Chen

first: Wang

last: Chenyu

first: Long

last: Qiaoyun

first: Wei

last: Minglang

first: Tang

last: Shanbo

first: Xiao

last: Qiong

first: Wu

last: Min

first: Li

last: Lianyun

first: Chen

last: Mingkai

last: Yong


title: Oncogenic Calreticulin Induces Immune Escape by Stimulating TGF-β Expression and Regulatory T Cell Expansion in the Bone Marrow Microenvironment - scRNAseq

city: Freiburg

first: Geoffroy

last: Andrieux

address: Breisacherstr 153

institute: University clinics Freiburg

summary: We established a model for myeloproliferative disease in mice, that is based on primary bone marrow expressing human Thrombopoietin receptor and human mutant Calrdel52. To learn more about cellular and transcriptional rearrangements in the bone marrow we performed single cell RNA sequencing comparing bone marrow cells of empty vector control mice with mice that received MPL-Calrdel52 bone marrow.

overall_design: A total of two mice for each group were analyzed. Two Mice received empty vector control BM. Two mice received MPL-Calr(del52) BM. Vector included an IRES-GFP sequence as transduction/engraftment control. Engraftment and disease phenotype was checked before analysis.

first: Melanie

last: Boerries

first: Dominik

last: Schmidt

first: Robert

last: Zeiser


title: Transcriptomic profile at single cell level of OCT2fl/fl and Ncr(+)/OCT2fl/fl mice [scRNA-seq]

city: Osaka

first: Xin

last: Sun

address: 3-1 Yamadaoka, Suita

institute: IFReC, Osaka University

summary: We used single cell RNA sequencing (scRNA-seq) to investigate the role of OCT2 in the regulation of Regnase-1.

overall_design: Freshly sorted splenic NK cells from 8-week-old OCT2fl/fl and Ncr(+)/OCT2fl/fl mice were analyzed using scRNAseq


title: Development of a Cellular Assay as a Personalized Model for Testing Chronic Wound Therapeutics (NS18)

first: Barbara

last: Wolff-Winiski

first: Petra

last: Doerfler

first: Nicole

last: Schoefmann

first: Gabriela

last: Cabral

first: Wolfgang

last: Bauer

first: Martin

last: Berli

last: Binder

first: Carina

last: Borst

first: Sander

last: Botter

first: Lars

last: French

first: Tobias

last: Goerge

first: Juerg

last: Hafner

first: Daniela

last: Hartmann

first: Annette

last: Høgh

first: Wolfram

last: Hoetzenecker

first: Judith

last: Holzer-Geissler

last: Kamolz

first: Katrin

last: Kofler

first: Thomas

last: Luger

first: Sebastian

last: Nischwitz

first: Michael

last: Popovits

first: Klemens

last: Rappersberger

first: Gaetana

last: Restivo

first: Justin

last: Schlager

first: Matthias

last: Schmuth

first: Georg

last: Stingl

first: Theresa

last: Stockinger

first: Anke

last: Stroelin

first: Anton

last: Stuetz

first: Julian

last: Umlauft

last: Weninger


title: Oncogenic Calreticulin Induces Immune Escape by Stimulating TGF-β Expression and Regulatory T Cell Expansion in the Bone Marrow Microenvironment - microarray

first: Geoffroy

last: Andrieux

summary: To understand transcriptional aberrations induced by oncogenic Calreticulin (Calr) in myeloproliferative neoplasms we used a cell line – 32D, generated from the murine myeloid precursor cells, to overexpress human thrombopoietin receptor (MPL) together with human Calr. Cells expressing oncogenic Calr show increased JAK/STAT signaling independent of extrinsic stimuli like IL3.

overall_design: A total of 8 samples were prepared, split in two groups. 4 replicates from 32D cells transduced with MPL + Calr(WT) and 4 replicates from 32D cells transduced with MPL + Calr(ins5).

first: Dominik

last: Schmidt

first: Robert

last: Zeiser

first: Melanie

last: Boerries


title: Transcriptomic profile at single cell level of TIL-NK cells and tumor cells from  tumor-bearing Reg-1fl/fl and Reg-1ΔNK mice

first: Xin

last: Sun

summary: The innate immune system can recognize tumor cells and coordinate with the adaptive immune system to initiate an anti-tumor response within the tumor microenvironment. To investigate the role of NK cells and their interaction with other immune cells in the anti-tumor immunity in Reg-1ΔNK. We subcutaneously injected B16F10 cells into Reg-1fl/fl and Reg-1ΔNK, followed by scRNA-seq analysis of the whole tumor cell suspension and sorted TIL-NK cells.

overall_design: B16F10 cells were s.c. injected into Reg-1fl/fl and Reg-1ΔNK mice (Day 0). Tumor-bearing mice were sacrificed on Day 10 for Reg-1fl/fl mice and Day 17 for Reg-1ΔNK mice, respectively. Tumor suspension and sorted NK cells from tumor-bearing Reg-1fl/fl and Reg-1ΔNK mice were analyzed using scRNAseq


title: Transcriptomic profile at single cell level of splenic NK cells from Reg-1fl/fl and Reg-1ΔNK mice [CITE-Seq]

summary: Regnase-1 functions as an endoribonuclease protein. We used single cell RNA sequencing (scRNA-seq) to identify candidate genes that are regulated by Regnase-1-mediated mRNA decay

overall_design: Freshly sorted splenic NK cells from 8-week-old Reg-1fl/fl and Reg-1ΔNK mice were analyzed using scRNAseq


title: Identification of the genome-wide binding sites of OCT2 in Reg-1fl/fl- and Reg-1ΔNK-NK cells. [ChIP-Seq]

summary: The expression of OCT2 was also increased in NK cells from Reg-1fl/fl in response to IL-12/18 stimulation implying that OCT2 has an intrinsic function not only in Regnase-1-deleted NK cells but also in wild-type NK cells. To identify the genome-wide binding sites of OCT2, we performed chromatin immunoprecipitation sequencing (ChIP-seq) for OCT2 in Reg-1fl/fl- and Reg-1ΔNK-NK cells.

overall_design: Freshly sorted splenic NK cells from Reg-1fl/fl and Reg-1ΔNK were cultured and analyzed using ChIPseq.


title: ATAC-seq Profiling of Mixed Stage C. elegans Neuronal Nuclei

status: Public on May 29 2024

city: Stanford

first: Callista

last: Yee

state: California

address: 327 Campus Drive, Shen lab, Bass Biology 329

institute: Stanford University/HHMI

summary: Using ATAC-seq, we aimed to identify regions of open chromatin in the developing C. elegans nervous system. This dataset was used to determine regions of chromatin and compare it to ChIP-seq data of TFs of interest, to assist us in identifying ChIP-seq peaks which are solely expressed in neurons (vs. general, total worm binding). Specifically, since EGL-43 is expressed in many tissues, including both neuronal and the somatic gonad, ATAC-seq was particularly helpful in identifying neuronal-specific ChIP-seq peaks and NanoDam peaks of interest (GSE227552).

overall_design: We utilized a strongly expressed pan-neuronal nuclear GFP strain, and grew worms to density as a mixed stage population, with the majority being larval animals. Then, we used standard homogenizing procedures using a stainless steel dounce and ChIP-seq buffers to isolate neuronal nuclei. Nuclei were sorted on a FACSAria Fusion with DAPI to stain for nuclei. Neuronal nuclei were gated as GFP+ and DAPI+; Total Nuclei were gated as DAPI+, and 50,000 nuclei were used for each sample. Nuclei were subjected immediately to Tn5 transposition and amplification.

first: Kang

last: Shen