title: The yes-associated protein-1 (YAP1) inhibitor celastrol suppresses the ability of transforming growth factor β to activate human gingival fibroblasts

city: Saskatoon

first: Andrew

last: Leask

state: Saskatchewan

address: 105 wiggins road

department: Dentistry

institute: university of saskatchewan

summary: Background: Fibrosis, defined by the excessive accumulation of extracellular matrix (ECM) proteins, including type I collagen, results in a stiff scar tissue that impairs organ function and can cause death. The effector cells of fibrosis are myofibroblasts, an activated form of fibroblast. In gingiva (gums), fibrotic responses are predominantly hyperproliferative (hyperplasia), where transforming growth factor (TGF)-β is overexpressed, and via canonical signaling pathway acts on gingival fibroblasts to induce fibroproliferative genes such as cellular communications network factor 2 (CCN2). Targeting of TGFβ for therapeutic gain however is challenging due its pleotropic nature, and therefore identifying new and targetable alternatives is warranted. We have shown that mechanical tension as a result of stiff ECM increases TGFβ-driven expression of CCN2 in gingival fibroblasts. We further identified yes-activated protein (YAP1), a mechanosensitive transcription (co)factor that translocate to nucleus in response to mechanical tension in gingival fibroblasts. To test if YAP is the perpetuator of TGFβ-mediated fibroproliferative gene induction, I used celastrol, a recently identified YAP/TAZ (transcriptional co-activator PDZ-binding motif) inhibitor, whose anti-fibrotic potential is not yet elucidated -- projecting it as a potential treatment option for gingival hyperplasia.  Methods: Human gingival fibroblasts (HGFs) in culture were serum starved overnight and pretreated with 500nM Celastrol or DMSO for 45 minutes followed by stimulation with or without TGFβ1 for 6 hours (RT-PCR) and 24 hours (Western blot). mRNA samples collected at 6 hours were also analysed using BulkRNAsequencing and functional cluster analysis. Celastrol’s capacity to prevent the TGFβ induced proliferation of HGFs was investigated with BrdU cell proliferation assay. Celastrol’s ability to block TGFβ-mediated YAP nuclear localization in HGFs was assessed using immunofluorescent staining of YAP protein.  Results: In response to added TGFβ1, the gene and protein expression of fibrotic marker and mediator CCN2 (a known YAP1 target) was significantly downregulated in the presence of Celastrol. Similarly, of the 1364 genes induced by TGFβ1 (>2-fold), 657 genes showed a >50% decrease in the presence of Celastrol including genes involved in fibrotic responses such as type 4 collagen, interleukin 11, etc., Functional cluster analysis revealed ECM, Wnt/cytokine signaling, focal adhesion clusters induced by added TGFβ1, were downregulated in a Celastrol sensitive fashion. Also, basal level proliferation of HGFs was repressed in the presence of Celastrol. Additionally, Celastrol also successfully blocked TGFβ-mediated nuclear translocation of YAP as seen with immunofluorescence microscopy.  Conclusion: These results are consistent with our hypothesis that targeting YAP/TAZ may be useful in blocking the profibrotic effects of TGFβ and that celastrol or other YAP inhibitors are eligible candidates as a treatment option for gingival hyperplasia.

overall_design: To investigate the effect of Celastrol on TGFβ1 mediated gene expression in human gingival fibroblasts

first: Angha

last: Naik

first: Pratyusha

last: Chitturi

first: John

last: Nguyen

first: David

last: Carter


title: Activation of imprinted gene Pw1 promotes cardiac fibrosis after ischemic injury [control_and_Pw1 CKO]

status: Public on Dec 04 2024

city: shanghai

first: Shan

last: Kou

address: No.393 Middle Huaxia Road

department: Life Science and Technology

country: China

institute: ShanghaiTech University

type: Expression profiling by high throughput sequencing

summary: The activation of fibroblasts and production of extracellular matrix (ECM) after cardiac injury has been the focus of research on cardiac fibrosis. Whether imprinted genes play a role in the fibrogenesis or extracellular matrix production after heart damage has not been studied. Here, we show that the imprinted gene Pw1 directed ECM production by regulating de novo purine synthesis (DNPB) after myocardial infarction (MI). After MI, the DNA methylation of the imprinting control region (ICR) of Pw1 gene in the injured area was reduced and both alleles of Pw1 in fibroblasts are activated. Further, loss of Pw1 in fibroblasts can enhance cardiac function, reduce cardiac fibrosis and extracellular matrix secretion after cardiac injury. To screen the targets of PW1, we contrasted chromatin immunoprecipitation (ChIP)-seq and RNA-seq using the injured left ventricles at 7 days post-MI, we next found PW1 binds directly upstream of the Pfas gene that encodes one of the DNPB enzymes phosphoribosylformylglycinamidine synthase (PFAS) in heart after injury. Similarly, specific knockout of pfas in fibroblasts also improves cardiac function and reduces ECM production. Moreover, de novo purine biosynthesis is necessary for ECM production in activated fibroblasts. These observations implicate understanding the mechanism of Pw1 in the repair of cardiac injury will provide new ideas for the treatment of cardiovascular diseases.

overall_design: We studied the regulatory effects of Pw1 on ECM genes in cardiac fibroblasts isolated from the injured left ventricles of Pw1 control or CKO mice at 3 days post-MI.Taking advantage of total RNA purified from the fibroblasts, we performed transcriptomic RNA-seq.

first: Zhengkai

last: Lu

first: Defang

last: Deng

first: Min

last: Ye

first: Yu

last: Sui

first: Lieyang

last: Qin

first: Teng

last: Feng

first: Zhen

last: Jiang

first: Jufeng

last: Meng

first: Chao-Po

last: Lin

first: Xiajun

last: Li

first: Chen

last: Liu

first: Juan

last: Tang

first: Hui

last: Zhang


title: Activation of imprinted gene Pw1 promotes cardiac fibrosis after ischemic injury [control_and_Pfas CKO RNA-seq]

overall_design: To investigate the role of Pfas in fibroblasts in vivo, we employed the Pfas CKO mice, and performed MI surgery in them by permanent ligation of the left anterior descending coronary artery.We isolated cardiac fibroblasts from the Pfas control and CKO mice, performed RNA-seq.


title: Activation of imprinted gene Pw1 promotes cardiac fibrosis after ischemic injury [sham_and_MI]

overall_design: We performed RNA-seq using the left ventricles sheared from the injured or sham-operated hearts at 7 days post-surgery, and found 7614 DEGs in the injured left ventricles


title: Activation of imprinted gene Pw1 promotes cardiac fibrosis after ischemic injury

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: Nanobody-based in vivo blockade of the P2X7 ion channel uncovers a hitherto unknown large subpopulation of parenchymal kidney TRM and NKT cells

city: Hamburg

first: Nicola

last: Wanner

address: Martinistr

country: Germany

institute: UKE Hamburg-Eppendorf

summary: The P2X7 ion channel, a key sensor of sterile inflammation, has been implicated as a therapeutic target in glomerulonephritis and P2X7-antagonistic nanobodies can attenuate experimental glomerulonephritis. However, little is known about the expression of P2X7 on renal immune cells. We used i.p. injection of nanobodies in mice followed by flow cytometry analysis of parenchymal T cells and RNA-sequencing to elucidate the expression and function of P2X7 on parenchymal and vascular immune cells in the mouse kidney. We show that parenchymal T cells, including a large subset NKT cells and tissue-resident memory T cells, display much higher cell surface levels of P2X7 than vascular T cells. These parenchymal T cells are highly sensitive to NAD+-induced cell death unless injection of P2X7-antagonistic nanobodies was performed before cell preparation. Remarkably, within 30 min after a single intraperitoneal injection of P2X7-blocking nanobodies, P2X7 is fully occupied by the injected nanobodies on parenchymal T cells in the kidney. This results in an effective protection of these cells from NAD+-induced cell death. Conversely, systemic injection of NAD+ that mimics sterile inflammation results in the selective depletion of P2X7hiCD69hi T cells from the kidney parenchyma. In summary, our study uncovers a novel purinergic regulatory mechanism affecting kidney resident T cell populations.

overall_design: T cell subpopulations (vascular CD4+, parenchymal NKT CD1dtet+/CD69+, TRM CD1dtet-/CD4+/CD69+ and non TRM CD1dtet-/CD69-) were sorted by FACS and total RNA was extracted using the Qiagen RNeasy Micro Kit. RNA sequencing was performed by the core facility of the Leibniz Institute of Virology,

first: Marten

last: Junge

first: Nicole

last: Schwarz

first: Carolina

last: Pinto-Espinoza

first: Nastassia

last: Liaukouskaya

first: Björn

last: Rissiek

first: Christian

last: Krebs

first: Hans-Willi

last: Mittrücker

first: Annette

last: Nicke

first: Friedrich

last: Haag

first: Cathy

last: Meyer-Schwesinger

last: Koch-Nolte


title: DDX50 cooperates with STAU1 to effect stabilization of pro-differentiation RNAs

city: Stanford

first: Douglas

last: Porter

state: CA

address: 269 Campus Drive

department: Dermatology

country: USA

institute: Stanford

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: YTHDC1 cooperates with the THO complex to prevent RNA damage-induced DNA breaks

status: Public on Dec 03 2024

city: Saint Louis

first: Ning

last: Tsao

state: MO

address: 660 S Euclid Ave

institute: Washington University in St Louis

summary: Certain environmental toxins are nucleic acid damaging agents, as are many chemotherapeutics used for cancer therapy. These agents induce various adducts in DNA as well as RNA. Indeed, most of the nucleic acid adducts (>90%) formed due to these chemicals, such as alkylating agents, occur in RNA. However, compared to the well-studied mechanisms for DNA alkylation repair, the biological consequences of RNA damage are largely unexplored. Here, we demonstrate that RNA damage can directly result in loss of genome integrity. Specifically, we show that a human YTH domain-containing protein, YTHDC1, regulates alkylation damage responses in association with the THO complex (THOC). In addition to its established binding to N6-methyladenosine (m6A)-containing RNAs, YTHDC1 binds to N1-methyladenosine (m1A)-containing RNAs upon alkylation. In the absence of YTHDC1, alkylation damage results in increased alkylation damage sensitivity and DNA breaks. Such phenotypes are fully attributable to RNA damage, since an RNA-specific dealkylase can rescue these phenotypes. These RNA damage-induced DNA breaks (RDIBs) depend on R-loop formation and the processing is mediated by the factors involved in the transcription-coupled nucleotide excision repair. Strikingly, in the absence of YTHDC1 or THOC, an RNA m1A methyltransferase targeted to the nucleus is sufficient to induce DNA breaks. Our results uncover a unique role of YTHDC1-THOC in base damage responses by preventing RDIBs, providing definitive evidence for how damaged RNAs can impact genomic integrity.

overall_design: For CLIP-seq, Flag-YTHDC1 expressing HeLa cells were non-treated or treated with alkylating agent MMS for 1 h, then processed with CLIP experiment. The purified RNAs were subjected to library preparation and sequencing. For m1A-DIP-seq, genomic DNAs extracted from control or YTHDC1 KD HeLa cells (treated with MMS) were subjected to m1A immunoprecipitation with m1A antibody. The purified DNAs were processed with library preparation and sequencing. For MapR, MMS-treated control and YTHDC1 KD HeLa cells were harvested for MapR assay incubating with GST-Mnase or GST-Mnase-dRNH. The purified DNAs were processed with library preparation and sequencing.

first: Patrick

middle: M

last: Lombardi

first: Ajin

last: Park

first: Jennifer

last: Olabode

first: Rebecca

last: Rodell

first: Hua

last: Sun

first: Shilpa

last: Padmanaban

first: Joshua

middle: R

last: Brickner

first: Miaw-Sheue

last: Tsai

first: Elizabeth

middle: A

last: Pollina

first: Chun-Kan

last: Chen

first: Nima

last: Mosammaparast


title: Transcriptomic signature associated with education in uterine Natural Killer cells during pregnancy

city: Seattle

first: Delphine

last: Depierreux

state: WA

address: 1000 Fairview Ave N

department: Human Biology

institute: Fred Hutchinson Cancer Center

summary: Uterine natural killer (uNK) cells regulate vascular changes crucial for placental function and fetal growth. The functional capacity of uNK cells is shaped by their education status.  To explore the mechanisms of uNK cell education, we isolated NKG2A+ and Ly49C/I+ educated uNK cells and compared their transcriptomes to uneducated NKG2A-Ly49C/I- uNK cells.

overall_design: At gestation days 9.5–10.5, pregnant mouse uteri were enzymatically and mechanically dissociated into single-cell suspensions. NK cells were isolated based on the expression of educating receptors Ly49C/I and NKG2A into three populations: uneducated (Ly49C/I⁻NKG2A⁻) and educated (Ly49C/I⁺NKG2A⁻, Ly49C/I⁻NKG2A⁺). Double-positive cells (Ly49C/I⁺NKG2A⁺) were excluded due to limited sample size. Bulk RNA-seq was performed to compare transcriptomic profiles between these populations, identifying differential gene expression associated with uNK cell education.

first: Francesco

last: Colucci


title: CAR T cells, CAR NK cells and CAR macrophages exhibit distinct traits in glioma models but are similarly enhanced when combined with cytokines

city: Freiburg

first: Roman

last: Sankowski

address: Breisacher Str. 64

department: Institute of Neuropathology

institute: University of Freiburg Medical Center

summary: Chimeric antigen receptor (CAR) T cell therapy is a promising immunotherapy against cancer. Although there is a growing interest in other cell types, a comparison of CAR immune effector cells in challenging solid tumor contexts is lacking. Here, we compare mouse and human NKG2D-CAR expressing T cells, NK cells and macrophages against glioblastoma, the most aggressive primary brain tumor. In vitro we show that T cell cancer killing is CAR-dependent, whereas intrinsic cytotoxicity overrules CAR-dependence for NK cells and CAR macrophages reduce glioma cells in co-culture assays. In orthotopic immunocompetent glioma mouse models, systemically administered CAR T cells demonstrate superior accumulation in the tumor and each immune cell type induces distinct changes in the tumor microenvironment. An otherwise low therapeutic efficacy is significantly enhanced by co-expression of pro-inflammatory cytokines in all CAR immune effector cells, underscoring the necessity for multifaceted cell engineering strategies to overcome the immunosuppressive solid tumor microenvironment.

overall_design: Mice bearing Gl261 syngeneic tumors were injected with different CAR cells and analyzed using single-cell RNA-Sequencing. Biological replicates were multiplexed with barcoded lipids. Library preparation was conducted using 10X 3' mRNA-Sequencing reagents.

first: Thomas

last: Look

first: Tobias

last: Weiss


title: DEPLETION OF TP53 IN HUMAN PLURIPOTENT STEM CELLS TRIGGERS MALIGNANT-LIKE BEHAVIOUR

city: Utrecht

first: Joaquin

last: Montilla Rojo

address: Yalelaan 1

country: Netherlands

institute: Utrecht University

summary: Human pluripotent stem cells (hPSCs) tend to acquire genetic aberrations upon culture in vitro. Common aberrations are mutations in the tumor suppressor TP53, suspected to confer a growth-advantage to the mutant cells. However, their full impact in the development of malignant features and safety of hPSCs for downstream applications is yet to be elucidated. Here, we knock-out TP53 in hPSCs using CRISPR-Cas9 and compare them with isogenic wild-type hPSCs and human germ cell tumor lines as models of malignancy. While no major changes in proliferation, pluripotency and transcriptomic profiles are found, mutant lines display aberrations in some of the main chromosomal hotspots for genetic abnormalities in hPSCs. Additionally, enhanced clonogenic and anchorage-free growth, alongside resistance to chemotherapeutic compounds is observed. Our results indicate that common TP53-depleting mutations in hPSCs, although potentially overlooked by standard analyses, could impact their behavior and safety in a clinical setting.

overall_design: To explore the impact of TP53-depleting mutations, we generated Tp53-KO lines of an embryonic (H9) and induced pluripotent (LU07) stem cell lines using CRSIPR-Cas9 technology. To deepen into the impact of these mutations in the transcriptome of the different cell lines, bulk RNAseq of the lines was performed. We compared the gene expression profiles of the different cell lines and their respective KO isogenic counterparts

last: Montilla-Rojo

middle: F

last: Eleveld

first: Marnix

last: van Soest

first: Sanne

last: Hillenius

first: Dennis

last: Timmerman

first: Ad

middle: J

last: Gillis

first: Bernard

last: Roelen

first: Christine

middle: L

last: Mummery

first: Leendert

middle: H

last: Looijenga

first: Daniela

middle: C

last: Salvatori


title: DNA hypomethylation promotes UHRF1- and SUV39H1/H2-dependent crosstalk between H3K18ub and H3K9me3 to reinforce heterochromatin states [EPIC]

city: Grand Rapids

first: Scott

last: Rothbart

state: MI

address: 333 Bostwick Avenue NE

institute: Van Andel Research Institute

summary: Mono-ubiquitination of lysine 18 on histone H3 (H3K18ub), catalyzed by UHRF1, is a DNMT1 docking site that facilitates replication-coupled DNA methylation maintenance. Its functions beyond this are unknown. Here, we genomically map simultaneous increases in UHRF1-dependent H3K18ub and SUV39H1/H2-dependent H3K9me3 following DNMT1 inhibition. Mechanistically, transient accumulation of hemi-methylated DNA at CpG islands facilitates UHRF1 recruitment and E3 ligase activity towards H3K18. Notably, H3K18ub enhances SUV39H1/H2 methyltransferase activity and, in colon cancer cells, nucleates new H3K9me3 domains at CpG island promoters of DNA methylation-silenced tumor suppressor genes. Disrupting UHRF1 enzyme activity prevents H3K9me3 accumulation while promoting PRC2-dependent H3K27me3 as a tertiary layer of gene repression in these regions. In contrast, disrupting H3K18ub-dependent SUV39H1/H2 activity enhances the transcriptional activating and antiproliferative effects of DNMT1 inhibition. Collectively, these findings reveal roles for UHRF1 and H3K18ub in regulating a hierarchy of repressive histone methylation signaling and rationalize a combination strategy for epigenetic cancer therapy.

overall_design: EPIC array analysis in WI-38 cells following DMSO control treatment or DNMT1 inhibition to profile DNA methylation changes following indicated treatment.

first: Yanqing

last: Liu

first: Rochelle

last: Tiedemann

middle: B


title: DNA hypomethylation promotes UHRF1- and SUV39H1/H2-dependent crosstalk between H3K18ub and H3K9me3 to reinforce heterochromatin states [RNA-Seq]

overall_design: RNA-seq in RKO cells with SUV39H2 KO and dox-inducible SUV39H1 shRNA KD. There are four treatment groups: cells were treated with 1) DMSO control, 2) 20ng/mL doxycycline (SUV39H1 KD), 3) 1uM GSK3484862(DNMT1 inhibition), and 4) doxycycline+GSK3484862 (SUV39H1 KD + DNMT1 inhibition) for 5 days before RNA collection.


title: DNA hypomethylation promotes UHRF1- and SUV39H1/H2-dependent crosstalk between H3K18ub and H3K9me3 to reinforce heterochromatin states [ChIP-Seq]

overall_design: Chromatin immunoprecipitation sequencing (ChIP-seq) for H3K9me3 in WI-38 cells following DMSO control treatment, UHRF1 knockdown, DNMT1 inhibition, and UHRF1 KD+DNMT1 inhibition.

first: Bradley

last: Dickson


title: Interplay between Netrin-1 and Norrin controls arteriovenous zonation of blood–retina barrier integrity

city: New Haven

first: Jean-Leon

last: Thomas

state: CT

address: 800 Howard Ave

institute: Yale University

summary: The integrity of the blood–retina barrier (BRB) is crucial for phototransduction and vision, by tightly restricting transport of molecules between the blood and surrounding neuronal cells. Breakdown of the BRB leads to the development of retinal diseases. Here, we show that Netrin-1/Unc5b and Norrin/Lrp5 signaling establish a zonated endothelial cell gene expression program that controls BRB integrity. Using single-cell RNA sequencing (scRNA-seq) of postnatal BRB-competent mouse retina endothelial cells (ECs), we investigate >100 BRB genes encoding Wnt signaling components, tight junction proteins, and ion and nutrient transporters. We find that BRB gene expression is zonated across arteries, capillaries, and veins and regulated by opposing gradients of the Netrin-1 receptor Unc5b and Lrp5-β-catenin signaling between retinal arterioles and venules. Mice deficient for Ntn1 or Unc5b display more BRB leakage at the arterial end of the vasculature, while Lrp5 loss of function causes predominantly venular BRB leakage. ScRNA-seq of Ntn1 and Unc5b mutant ECs reveals down-regulated β-catenin signaling and  BRB gene expression that is rescued by Ctnnb1 overactivation, along with BRB integrity. Mechanistically, we demonstrate that Netrin-1 and Norrin additively enhance β-catenin transcriptional activity and Lrp5 phosphorylation via the Discs large homologue 1 (Dlg1) scaffolding protein, and endothelial Lrp5-Unc5b function converges in protection of capillary BRB integrity. These findings explain how arteriovenous zonation is established and maintained in the BRB and reveal that BRB gene expression is regulated at the level of endothelial subtypes.

overall_design: Mutant and corresponding control mice were injected with TAM P6-P8 and sacrified at P12. Retinas were dissected, pooled according to genotype and cells were isolated with endothelial cell enrichment.

first: Jessica

last: Furtado

first: Luiz

last: Geraldo

first: Felipe

middle: S

last: Leser

first: Bartlomiej

last: Bartkowiak

first: Mathilde

last: Poulet

first: Hyojin

last: Park

first: Mark

last: Robinson

first: Laurence

last: Pibouin-Fragner

first: Anne

last: Eichmann

first: Kevin

last: Boye


title: Monoclonal antibodies that block Roundabout 1 and 2 signaling target pathological ocular neovascularization through myeloid cells

summary: Roundabout (ROBO) 1 and 2 are transmembrane receptors that bind secreted SLIT ligands through their extracellular domains (ECD), and signal through their cytoplasmic domains to modulate the cytoskeleton and regulate cell migration, adhesion, and proliferation. SLIT-ROBO signaling was discovered as a guidance cue for axons in the developing nervous system, but ROBO receptors are expressed by many additional cell types and more broadly regulate organ morphogenesis, as well as cancer and pathological ocular neovascularization.  As pharmacological tools to prevent  SLIT-ROBOR signaling are lacking, herein we developed human monoclonal antibodies (mAb ) against the ROBO1 and 2 ECD. One antibody that inhibited in vitro  SLIT2 signaling through ROBO1/2 reduced ocular neovascularization in two pathological models in vivo . Single cell RNA sequencing revealed that antibody treatment affected several cell types relevant to angiogenesis, including endothelial cells, pericytes, and a heterogeneous population of myeloid cells. mAb treatment improved Blood-Retina Barrier integrity and prevented pathological pericyte activation . SLIT-ROBO signaling inhibition prevented pathological activation of myeloid cells, which was phenocopied by both myeloid cell specific ablation of Robo1 and 2 and by knockout of the downstream effector  Pi3kγ . Anti-ROBO1/2 blocking antibodies may thus provide a new promising strategy to combat inflammation in blinding eye diseases.

overall_design: Retinal cells dissected from P17 mouse pups following oxygen induced retinopathy (OIR) and treated with either Anti-Robo1/2 antibodies or IgG control antibodies. Endothelial and immune cells were isolated by CD31 and CD45 microbeads respectively. Enriched cell populations were subjected to single cell RNA-sequencing using the 10x platform.


title: SLIT2 treatment effect on transcription in bone marrow derived macrophages

summary: Roundabout (ROBO) 1 and 2 are transmembrane receptors that bind secreted SLIT ligands through their extracellular domains (ECD), and signal through their cytoplasmic domains to modulate the cytoskeleton and regulate cell migration, adhesion, and proliferation. SLIT-ROBO signaling was discovered as a guidance cue for axons in the developing nervous system, but ROBO receptors are expressed by many additional cell types and more broadly regulate organ morphogenesis, as well as cancer and pathological ocular neovascularization. As pharmacological tools to prevent SLIT-ROBOR signaling are lacking, herein we developed human monoclonal antibodies (mAb ) against the ROBO1 and 2 ECD. One antibody that inhibited in vitro SLIT2 signaling through ROBO1/2 reduced ocular neovascularization in two pathological models in vivo. Single cell RNA sequencing revealed that antibody treatment affected several cell types relevant to angiogenesis, including endothelial cells, pericytes, and a heterogeneous population of myeloid cells. mAb treatment improved Blood-Retina Barrier integrity and prevented pathological pericyte activation. SLIT-ROBO signaling inhibition prevented pathological activation of myeloid cells, which was phenocopied by both myeloid cell specific ablation of Robo1 and 2 and by knockout of the downstream effector Pi3kγ. Anti-ROBO1/2 blocking antibodies may thus provide a new promising strategy to combat inflammation in blinding eye diseases.

overall_design: Bulk RNA-sequencing of bone marrow derived macrophages (BMDM) treated with SLIT2 or vehicle control.


title: Glutathione accelerates the cell cycle and cellular reprogramming in plant regeneration [scRNA-seq]

city: New York

first: Kenneth

last: Birnbaum

state: NY

address: 12 Waverly Place

department: Biology Department

institute: New York Univerisity

summary: The plasticity of plant cells underlies their wide capacity to regenerate, with increasing evidence in plants and animals implicating cell cycle dynamics in cellular reprogramming. To investigate the cell cycle during cellular reprogramming, we developed a comprehensive set of cell cycle phase markers in the Arabidopsis root. Using single-cell RNA-seq profiles and live imaging during regeneration, we found that a subset of cells near an ablation injury dramatically increases division rate by truncating G1 phase. Cells in G1 undergo a transient nuclear peak of glutathione (GSH) prior to coordinated entry into S phase followed by rapid divisions and cellular reprogramming. A symplastic block of the ground tissue impairs regeneration, which is rescued by exogenous GSH. We propose a model in which GSH from the outer tissues is released upon injury licensing an exit from G1 near the wound to induce rapid cell division and reprogramming.

overall_design: Seedlings were grown vertically on sterile mesh on top of MS media and then transferred to media supplemented with 2 micromolar hydroxyurea for the indicated amount of time to enrich for different cell cycle phases and then protoplasted and profiled with 10X genomics V3 to produce single cell RNA-seq libraries.

first: Lee

last: Laura

first: Birnbaum

last: Kenneth


title: Analysis of heterogeneity within genetic murine models of HCC

city: Glasgow

first: Thomas

middle: G

last: Bird

address: Switchback Road, Bearsden

department: CRUK Scotland Institute

country: United Kingdom

institute: Beatson Institute

summary: Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is a leading cause of cancer related mortality worldwide1,2. HCC occurs typically from a background of chronic liver disease, caused by a spectrum of predisposing conditions. Tumour development is driven by the expansion of clones that accumulated progressive driver mutations3, with hepatocytes the most likely cell of origin2. However, the landscape of driver mutations in HCC is independent of the underlying aetiologies4.  Despite an increasing range of systemic treatment options for advanced HCC outcomes remain heterogeneous and typically poor. Emerging data suggest that drug efficacies depend on disease aetiology and genetic alterations5,6. Exploring subtypes in preclinical models with human relevance will therefore be essential to advance precision medicine in HCC7. We generated over twenty-five new genetically-driven in vivo and in vitro HCC models. Our models represent multiple features of human HCC, including clonal origin, histopathological appearance, and metastasis to distant organs. We integrated transcriptomic data from the mouse models with human HCC data and identified four common human-mouse subtype clusters. The subtype clusters had distinct transcriptomic characteristics that aligned with histopathology.  In a proof-of-principle analysis, we verified response to standard of care treatment and used a linked in vitro-in vivo pipeline to identify a promising therapeutic candidate, cladribine, that has not been linked to HCC treatment before. Cladribine acts in a highly effective subtype-specific manner in combination with standard of care therapy.

overall_design:
To investigate the gene expression profile of liver tumors in mouse models containing heterozygous Ctnnb1 tm1Mmt alleles and the human MYC gene, we employed an adeno-associated viral (AAV) vector system. This system was used to express the human MYC gene in Cre-expressing cells, thereby inducing liver tumors. Additionally, we examined gene expression variation in response to high AAV concentrations by comparing control and tumor tissues.
Tumor samples (13) and control non-tumor tissue samples (6) were collected. The control non-tumor tissue samples included three from standard AAV levels and three from high AAV levels.
We then preformmed bulk RNA-Seq on the tumour and non-tumour samples to determine differential gene expression.

first: Miryam

last: Müller

first: Stephanie

last: May

first: Holly

last: Hall

first: Timothy

last: Kendall

first: Lynn

last: McGarry

first: Lauriane

last: Blukacz

first: Sandro

last: Nuciforo

first: Anastasia

last: Georgakopoulou

last: Jamieson

first: Narisa

last: Phinichkusolchit

first: Sandeep

last: Dhayade

first: Toshiyasu

last: Suzuki

first: Júlia

last: Huguet-Pradell

first: Ian

last: Powley

first: Leah

last: Officer-Jones

first: Rachel

last: Pennie

first: Roger

last: Esteban-Fabró

first: Albert

last: Gris-Oliver

first: Roser

last: Pinyol

first: George

last: Skalka

first: Jack

last: Leslie

first: Matt

last: Hoare

first: Joep

last: Sprangers

first: Gaurav

last: Malviya

first: Agata

last: Mrowinska

first: Emma

last: Johnson

first: Misti

last: McCain

first: John

last: Halpin

first: Christos

last: Kiourtis

first: Colin

last: Nixon

first: Graeme

last: Clark

first: William

first: Robin

last: Shaw

first: Ann

last: Hedley

last: Drake

first: Ee

last: Tan

last: Neilson

first: Daniel

last: Murphy

first: David

last: Lewis

first: Helen

last: Reeves

last: Le Quesne

first: Derek


title: Glutathione accelerates the cell cycle and cellular reprogramming in plant regeneration [bulk RNA-seq]

first: Kenneth

last: Birnbaum

overall_design: Cells were sorted via FACS using ploidy as a proxy for cell cycle phase and then profiled via bulk RNA-seq

first: Lee

last: Laura

first: Birnbaum

last: Kenneth


title: Deep learning of enhancer codes highlights similarities between mammalian and avian telencephalon cell types [mpra]

city: Leuven

first: Gert

last: Hulselmans

address: Herestraat 49 PO Box 602

department: Center for Brain and Disease Research

country: Belgium

institute: VIB

summary: Combinations of transcription factors govern the identity of cell types, which is reflected by genomic enhancer codes. We utilized deep learning to characterize these enhancer codes and devised three novel metrics to compare cell types in the telencephalon between mammals and birds. To this end, we generated single-cell multiome and spatially-resolved transcriptomics data of the chicken telencephalon. Enhancer codes of orthologous non-neuronal and GABAergic cell types show a high degree of similarity across vertebrates, while excitatory neurons of the mammalian neocortex and avian pallium exhibit varying degrees of similarity. Enhancer codes of avian mesopallial neurons are most similar to those of mammalian deep layer neurons. With this study, we present generally applicable deep learning approaches to characterize and compare cell types solely based on genomic sequences.

overall_design: A saturation mutagenesis library of the FIRE enhancer is cloned in a lentiviral MPRA reporter. Mouse BV2 cells are transduced with the virus and RNA and genomic DNA are harvested after 48 hours. The reporter barcodes are sequenced to estimate enhancer activity of each sequence.

first: Nikolai

last: Hecker

first: Niklas

last: Kempynck

last: Mauduit

first: Darina

last: Abaffyova

first: Ioannis

last: Sarropoulus

first: Sam

last: Dieltens

first: Carmen

last: Bravo Gonzalez-Blas

first: Roel

last: Vandepoel

first: Elke

last: Leysen

first: Lynette

last: Lim

first: Joris

last: De Wit

first: Valerie

last: Christiaens

first: Suresh

last: Poovathingal

first: Aerts

last: Stein


title: Transcriptome immune-regulatory differences between leprosy patients and type 1 reaction patients, before onset of symptoms

city: Montreal

first: Wilian

last: Macedo

state: Quebec

address: 1001 Decarie Blvd

country: Canada

institute: McGill-MUHC

summary: Leprosy is a chronic disease of the skin and peripheral nerves caused by Mycobacterium leprae. A major public health and clinical problem are leprosy reactions, which are inflammatory episodes that often contribute to nerve damage and disability. Type I reversal reactions (T1R) can occur after microbiological cure of leprosy and affect up to 50% of leprosy patients. Early intervention to prevent T1R and, hence, nerve damage, is a major focus of current leprosy control efforts. In this study, we compared transcript (i.e. isoform) expression and usage profiles from leprosy patients free of T1R at enrollment (who eventually developed T1R) against T1R-free leprosy patients. Our results showed that, at baseline, cells from T1R-destined and T1R-free subjects had no main difference in their transcripts expression and usage. However, the cells of T1R patients displayed a transcriptomic immune response to M. leprae antigens that was significantly different from the one of cells from leprosy patients who remained T1R-free. Transcripts with significantly higher upregulation in the T1R-destined group, compared to the cells from T1R-free patients, were enriched for pathways and GO terms involved in response to intracellular pathogens, apoptosis regulation and inflammatory processes. Similarly, transcript usage analysis pinpointed different transcript proportions in response to the in-vitro challenge of cells from T1R-destined patients. Hence, transcript usage in concert with transcript expression suggested a dysregulated inflammatory response including increased apoptosis regulation in the peripheral blood cells of T1R-destined patients before the onset of T1R symptoms. Combined, these results provided detailed insight into the pathogenesis of T1R.

overall_design: In a prospective study, we enrolled and collected samples from 32 leprosy patients before the onset of T1R. Whole blood aliquots were challenged with M. leprae sonicate or media and total RNA was extracted. After a three-year follow-up, the transcriptomic response was compared between cells from 22 patients who remained T1R-free and 10 patients who developed T1R during that period. Our analysis focused on differential transcript expression and usage.

last: Correa-Macedo

first: Erwin

last: Schurr


title: Transcription factor 21 regulates cardiac myofibroblast formation and fibrosis [ChIP-Seq]

city: Cincinnati

first: Jeffery

middle: D

last: Molkentin

state: Ohio

address: 240 Albert Sabin Way MLC 7020

department: Molecular Cardiovascular Biology

institute: Cincinnati Children's Hospital Medical Center

summary: Transcription factor 21 (TCF21) is a basic helix-loop-helix protein required for developmental specification of cardiac fibroblasts from epicardial progenitor cells that normally surround and invade the heart. In the adult heart, TCF21 is expressed in tissue resident fibroblasts but is downregulated in response to injury or stimuli leading to myofibroblast differentiation. These findings led to the hypothesis that Tcf21 could be a regulator of fibroblast cell-fate in the adult mammalian heart and contribute to cardiac fibrosis. Here, chromatin immunoprecipitation followed by sequencing was used to identify TCF21 genome occupancy in adult cardiac fibroblasts.

overall_design:
Col1a1-TCF21 mice were generated that express TCF21 upon Cre mediated recombination. TCF21 genome-wide occupancy was evaluated by chromatin immunoprecipitation and sequencing (ChIP-seq) in isolated cardiac fibroblasts isolated from Col1a1-TCF21 hearts. Fibroblasts were infected with an adenovirus encoding Cre recombinase to induce expression of TCF21 and expanded for 5 days. Transforming growth factor b1 was added in the last 24 hours to further promote fibroblast activation and ChIP-seq was performed.
ChIP-seq data were analyzed with the Trim Galore Chip-seq paired-end reads pipeline (https://github.com/datirium/workflows/blob/master/workflows/trim-chipseq-pe.cwl) in the Scientific Data Analysis Platform, SciDAP (https://scidap.com/) (Datirium). Adapters were trimmed using Trim Galore (https://github.com/FelixKrueger/TrimGalore) and the reads were aligned to the mouse genome assembly mm10/GRCm38 using Bowtie17. Peak-calling was performed with MACS218.  The UCSC Genome Browser's bedGraphToBigWig tool was used to produce a bigWig file. Genome browser tracks were created with the genome coverage in BEDTools19 and normalized to total mapped read number and displayed as coverage on IGV browser20.

first: Anne-Katrine

last: Johansen

first: Adenike

last: Shittu

first: Artem

last: Barski


title: Transcription factor 21 regulates cardiac myofibroblast formation and fibrosis [scRNA-Seq]

state: OH

address: 3333 Burnet Ave

institute: Cincinnati Children’s Hospital Medical Center

summary: Transcription factor 21 (TCF21) is a basic helix-loop-helix protein required for developmental specification of cardiac fibroblasts from epicardial progenitor cells that normally surround and invade the heart.  In the adult heart, TCF21 is expressed in tissue-resident fibroblasts but is downregulated in response to injury or stimuli leading to myofibroblast differentiation. These findings led to the hypothesis that Tcf21 could be a regulator of fibroblast cell fate in the adult mammalian heart and contribute to cardiac fibrosis. Here, single-cell RNA-sequencing was used to study the effect of loss of Tcf21 in cardiac fibroblasts in adult mouse hearts at baseline and after an ischemic cardiac injury (myocardial infarction).

overall_design: To investigate the function of TCF21 in vivo, TCF21 was conditionally deleted in cardiac fibroblasts. Mice were either studied in the absense of injury or after 3 days of myocardial infarction. Cardiac tissue was digested to obtain a single cell suspension and live nucleated cells were sorted for single cell RNA sequencing.

first: Anne Katrine

middle: K

first: Jeffrey


title: Transcription factor 21 regulates cardiac myofibroblast formation and fibrosis [RNA-Seq]

summary: Transcription factor 21 (TCF21) is a basic helix-loop-helix protein required for developmental specification of cardiac fibroblasts from epicardial progenitor cells that normally surround and invade the heart.  In the adult heart, TCF21 is expressed in tissue resident fibroblasts but is downregulated in response to injury or stimuli leading to myofibroblast differentiation. These findings led to the hypothesis that Tcf21 could be a regulator of fibroblast cell-fate in the adult mammalian heart and contribute to cardiac fibrosis. Here, bulk RNA sequencing was used to determine the effect of loss of TCF21 and enforced TCF21 expression in adult cardiac fibroblasts.

overall_design: RNA-sequencing was performed on cardiac fibroblasts isolated by flow cytometry from adult genetically engineered mouse models with loss or gain of function of TCF21 in the healthy heart or after an injury. Tcf21fl/fl mice were crossed with Tcf21-MerCreMer (Tcf21MCM) mice to permit ablation of Tcf21 upon exposure to tamoxifen. Tcf21MCM mice were used as controls. Col1a1-TCF21 mice were crossed with platelet-derived growth factor receptor, alpha polypeptide (Pdgfra) gene (Pdgfra-CreERT2) mice to permit overexpression of TCF21 upon exposure to tamoxifen. Pdgfra-CreERT2 mice were used as controls. All mice contained a Rosa26-loxP-STOP-loxP-enhanced green fluorescent protein for lineage tracing and sorting fibroblasts. To study the baseline effects of loss of Tcf21 in cardiac fibroblasts, tamoxifen was administered to adult mice and fibroblasts were isolated two weeks later by flow cytometry for RNA-sequencing. To study the effects of loss and gain of function of Tcf21 in the presence of an injury, tamoxifen was administered to mice to induce genetic recombination. Mice were then exposed to a continuous infusion of the fibrotic agonists, angiotensin II and phenylephrine for one week before isolation and sorting of fibroblasts by flow cytometry for RNA-sequencing.

first: Anne


title: BRAF inhibitors promote erythropoiesis and hematopoiesis via paradoxical MAPK activation (RNA-Seq)

city: Beijing

first: Yuelin

last: Deng

state: Beijing

address: Beijing, China

institute: Peking University

summary: Erythropoiesis is a crucial part in hematopoietic system, while facing disruptions from various diseases conditions. Anemia and blood shortages has become global challenges, with current finite pharmacological options like EPO or glucocorticoids having limitations, especially in genetic anemias like Diamond-Blackfan anemia (DBA), calling for novel candidates in stimulating the erythropoiesis. We designed the primary human hematopoietic stem and progenitor cells (HSPCs) chemicals screening system in erythropoiesis and unexpectedly discovered a BRAF inhibitor, effectively against BRAFV600E mutant cancers, delaying erythroid differentiation while promoting progenitors’ proliferation. Further research demonstrated its efficacy in cytokine-restricted conditions and in samples from erythroid disorder patients. Mechanistically, BRAF inhibitors paradoxically activated the RAF-MEK-ERK/MAPK pathway. Unlike oncogenic BRAFV600E impaired hematopoiesis and erythropoiesis via AP-1 hyperactivation, BRAF inhibitors minimally affected HSPCs self-renewal and differentiation. In vivo studies further exhibited BRAF inhibitors' potential to enhance human hematopoiesis and erythropoiesis in severe immunodeficient mouse models and alleviate anemia symptoms in Rpl11 haploinsufficiency DBA models and other anemia models. This discovery sheds light on the MAPK pathway's role in hematopoiesis, making BRAF inhibitors a novel clinically therapeutic choice in improving hematopoietic reconstitution and the recovery of anemias like DBA.

overall_design: CD34+ cells were resuscitated in HSPCs (CD34+) retention culture medium and treated with lentivirus or GDC after 12h. GFP+ cell sorting was performed in the second day, and samples were collected on day6 for RNAseq and ATACseq.


title: BRAF inhibitors promote erythropoiesis and hematopoiesis via paradoxical MAPK activation (ATAC-Seq)


title: DNA hypomethylation promotes UHRF1- and SUV39H1/H2-dependent crosstalk between H3K18ub and H3K9me3 to reinforce heterochromatin states

first: Scott

last: Rothbart

type: Methylation profiling by genome tiling array

summary: Mono-ubiquitination of lysine 18 on histone H3 (H3K18ub), catalyzed by UHRF1, is a DNMT1 docking site that facilitates replication-coupled DNA methylation maintenance. Its functions beyond this are unknown. Here, we genomically map simultaneous increases in UHRF1-dependent H3K18ub and SUV39H1/H2-dependent H3K9me3 following DNMT1 inhibition. Mechanistically, transient accumulation of hemi-methylated DNA at CpG islands facilitates UHRF1 recruitment and E3 ligase activity towards H3K18. Notably, H3K18ub enhances SUV39H1/H2 methyltransferase activity and, in colon cancer cells, nucleates new H3K9me3 domains at CpG island promoters of DNA methylation-silenced tumor suppressor genes. Disrupting UHRF1 enzyme activity prevents H3K9me3 accumulation while promoting PRC2-dependent H3K27me3 as a tertiary layer of gene repression in these regions. In contrast, disrupting H3K18ub- dependent SUV39H1/H2 activity enhances the transcriptional activating and antiproliferative effects of DNMT1 inhibition. Collectively, these findings reveal roles for UHRF1 and H3K18ub in regulating a hierarchy of repressive histone methylation signaling and rationalize a combination strategy for epigenetic cancer therapy.

overall_design: EPIC array analysis in RKO cells following DMSO control treatment, UHRF1 knockdown, DNMT1 inhibition, and UHRF1 KD+DNMT1 inhibition to profile DNA methylation changes following indicated treatment.

first: Yanqing

last: Liu

first: Rochelle

last: Tiedemann


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

overall_design: Refer to individual Series


title: Oxygen-dependent alternative mRNA splicing and a cone-specific motor protein revealed by single-cell RNA sequencing in hypoxic retinas

city: Basel

first: Duygu

last: Karademir Sigurdsson

address: Klybeckstrasse 200

country: Switzerland

institute: Institute of Molecular and Clinical Ophthalmology Basel

summary: Restricted oxygen supply from the choroid may lead to hypoxic conditions in the outer retina of the ageing eye and contribute not only to physiological aging but also to nonhereditary degenerative retinal diseases. To understand the hypoxic response of specific retinal cell types, we performed droplet-based single-cell RNA sequencing (scRNA-Seq) of retinas isolated from mice exposed to hypoxia. Except for ganglion cells, we identified all retinal cell types and a small RPE cluster among the 9410 normoxic and 9033 hypoxic cells in the analysis. Expression of hypoxic marker genes including Bnip3, Higd1a, and Egln1 was significantly upregulated in hypoxic clusters, confirming a general transcriptional response to hypoxia. Interestingly, RNA-binding proteins Rbm3 and Cirbp were differentially expressed across clusters and demonstrated isoform switching in hypoxia. The resulting short variants of these gene transcripts are connected to epitranscriptomic regulation, a notion supported by the differential expression of writers, readers and erasers of m 6 A RNA methylations in the hypoxic retina. By focusing on the hypoxic response in photoreceptors, we identified and confirmed a kinesin motor protein (Kif4) that was specifically and strongly induced in hypoxic cones. Retinal cells adapted to hypoxic conditions by adjusting their gene expression profile. Epitranscriptomic regulation of m 6 A RNA methylations may support the adaptational process. The cone-specific upregulation of Kif4, a motor protein with multiple functions, indicated a cone-specific response to hypoxia that may include cargo-delivery to specific intracellular locations, enrichment of transmembrane receptors or channels and/or modulation of chromatin structure.

overall_design: A male and a female wildtype mouse were exposed to 7% oxygen for 6 hours to induce hypoxic conditions in the retina. The retinas from hypoxic mice and a pair of male and female age-matched control mice were collected and dissociated for droplet-based single-cell RNA sequencing. The cell dissociates were fixed with methanol and the dissociates from the two animals under the same oxygen condition were combined. The two samples, "hypoxia" and "normoxia", were loaded onto separate lanes of a 10X Genomics chip and were prepared for sequencing using the v3 chemistry. The samples were then sequenced on an Illumina NovaSeq 6000.

first: Lynn

last: Ebner

first: Christian

last: Grimm


overall_design: Chromatin immunoprecipitation sequencing (ChIP-seq) for H3K9me3, UHRF1, H3K18ub, and H3K27me3 in RKO cells following DMSO control treatment, UHRF1 knockdown, DNMT1 inhibition, and UHRF1 KD+DNMT1 inhibition.

first: Bradley

last: Dickson


title: Inhibition of Acyl-CoA Synthetase Long Chain (ACSL) Isozymes Decreases Multiple Myeloma Cell Proliferation, Mitochondrial Function and Affects Key Survival Pathways

city: Burlington

first: Princess

last: Rodriguez

state: Vermont

address: 89 Beaumont Avenue

department: Microbiology and Molecular Genetics

institute: University of Vermont

summary: Multiple myeloma (MM) is an incurable cancer of plasma cells that will cause ~12,590 deaths in the USA in 2023. Dysregulation of fatty acid (FA) metabolism is associated with MM development and progression but the underlying mechanisms remains unknown. The acyl-CoA synthetase long-chain family members (ACSLs) convert free long-chain fatty acids into fatty acyl-CoA esters, and play a key role in catabolic and anabolic fatty acid metabolism. Cancer Dependency Map data suggested that ACSL4 and ACSL3 are among the top 25% Hallmark Fatty Acid Metabolism genes that support MM fitness. Here, we show inhibition of the ACSLs in human myeloma cell lines using the pharmacological inhibitor Triascin C (TriC) caused apoptosis, and decreased proliferation in a dose- and time-dependent manner. As KRAS mutants are the most common mutation among MM patients, we used MM.1S cells to study the mechanisms of TriC toxicity. RNA-seq of MM.1S cells treated with 1.00 μM TriC for 24 hours had gene expression profiles significantly enriched in apoptosis, ferroptosis, and ER stress compared to vehicle treated cells. Proteomics of MM.1S cells treated with either 1.00 μM or 2.00 μM TriC for 48 hours revealed that mitochondrial dysfunction and oxidative phosphorylation were significantly enriched pathways of interest relative to vehicle treated cells. Indeed, metabolic flux analysis showed MM.1S cells treated with TriC after 24 hours had decreased mitochondrial ATP production rate. Flow cytometric analyses revealed concomitant decreases in number and mitochondrial membrane potential, and increases in mitochondrial superoxide.  Implications: Overall, our data support the hypothesis that suppression of ACSL in human MM cells inhibited their growth and viability, potentially indicating that ACSL proteins may be promising therapeutic targets in treating myeloma progression.

overall_design: To understand the transcriptional changes associated with triacsin C inhibition of the ACSL family, we treated the human multiple myeloma cell line, MM.1S cells that constitutively express GFP and luciferase (MM.1S GFP(+), Luc(+) ) with either vehicle (dimethyl sulfoxide, DMSO) or 1.00 μM triacsin C for 24 hours. We then performed gene expression profiling analysis using paired-end RNA seqencing for three biological replicates per treatment condition.

first: Connor

last: Murphy

first: Victoria

last: DeMambro

first: Samaa

last: Fadel

first: Heather

last: Fairfield

first: Carlos

last: Garter

first: Calvin

last: Vary

first: Michaela

last: Reagan


overall_design: RNA-seq for RKO cells following DMSO control treatment, UHRF1 knockdown, DNMT1 inhibition, and UHRF1 KD+DNMT1 inhibition to define transcriptome changes following indicated treatments.


title: Multi-omics approach to decipher Ewing sarcoma metastases using a spontaneous metastasis mouse model

city: Santiago de Compostela

first: Angel

last: Diaz-Lagares

state: A Coruña

address: C/ Choupana s/n

department: ONCOMET

country: Spain

institute: Health Research Institute of Santiago (IDIS)

summary: Ewing sarcoma (ES) is the second most common bone tumor affecting children and young adults, with dismal outcomes for patients with metastases at diagnosis. Mechanisms leading to metastasis remain poorly understood. To deepen our knowledge on ES progression, we have profiled 3 tumors and 5 metastases from our spontaneous metastasis ES mouse model using a multi-omics approach. Combining transcriptomics, proteomics and methylomics analyses, we identified signaling cascades and targets enriched in metastases that could be modulating aggressiveness in ES.

overall_design: We have profiled 3 tumors and 5 metastases from our spontaneous metastasis ES mouse model using a multi-omics approach. The DNA methylome of these samples was analyzed by EPIC arrays.

first: Oscar

last: M Tirado


title: RNA-seq data of metformin treated MDA-MB-231 cells

city: Houston

first: Chad

last: Creighton

state: TX

address: One Baylor Plaza, Mail Stop: BCM305

department: Biostatistics, Ducan Cancer Center

institute: Baylor College of Medicine

summary: Our previous studies in Triple-Negative Breast Cancers (TNBCs) have demonstrated high fatty acid Beta-oxidation (FAO) and Electron Transport Chain (ETC) dependency to activate Src kinase by the Y419 autophosphorylation of Src. Higher doses of metformin have been shown to directly inhibit complex I of ETC. Here, analysis of RNA-seq data upon treatment with high concentration of metformin reveals inhibition of various oncopathways including the Src kinase pathway. Our study identifies a clinically relevant therapeutic approach to better manage poorly-targetable TNBCs.

overall_design: two group comparison

first: Yiqun

last: Zhang

first: Benny

last: Kaipparettu


title: CD4+ T cell activation in absence of WNK1

city: London

first: Victor

last: Tybulewicz

address: 1 Midland Road

institute: Francis Crick Institute

summary: Signaling from the T cell antigen receptor (TCR) on CD4+ T cells triggers the adaptive immune response by inducing T cell activation, proliferation, and differentiation. However, TCR signaling pathways are incompletely understood. Unexpectedly, we demonstrate that WNK1, a kinase previously implicated in osmoregulation in the kidney, is required for T-dependent antibody responses. We show that WNK1-OXSR1-STK39-dependent water influx through AQP3 is required for TCR signaling in CD4+ T cells and for entry into cell cycle. Additionally, by preventing ATR activation this signaling pathway is required for T cells to progress through the G2 phase of the cell cycle. Thus, TCR signaling via WNK1, OXSR1, STK39 and AQP3 leads to water entry that is essential for CD4+ T cell proliferation and hence T cell-dependent antibody responses.

overall_design: Mouse CD4+ T cells of either of Wnk1+/- or Wnk1-/- genotype activated in vitro over a 24 hr timecourse

first: Joshua

last: Biggs O'May

first: Miriam

last: Llorian Sopena


title: High-throughput capture of transcription factor-driven chromatin dynamics using PHILO ChIP-seq [ChIP-seq 4]

status: Public on Dec 02 2024

city: Piscataway

first: Mark

last: Zander

address: 190 Frelinghuysen Rd

institute: Rutgers State University of New Jersey

summary: Assessing the dynamics of chromatin and transcription factor (TF) DNA binding in plants remains a significant challenge, primarily due to the lack of efficient tools for capturing these features on a larger scale. Here, we present PHILO (Plant HIgh-throughput LOw input) ChIP-seq, a high-throughput ChIP-seq platform that enables the cost-effective and extensive capture of TF DNA binding and the genome-wide distribution of histone modifications. Through the application of H3K9ac PHILO ChIP-seq to eight jasmonic acid pathway mutants, involving the parallel processing of over 100 samples, we not only substantially corroborate the previously described interplay among chromatin regulators at the target genes of the JA master TF MYC2 but also unveil previously unknown histone acetylation patterns in the regulatory regions of MYC2 target genes. Overall, our PHILO ChIP-seq platform proves to be a highly effective approach for simultaneously examining chromatin on an unprecedented scale opening the door to conducting large-scale epigenome studies in plants.

overall_design: ChIP-seq and PHILO ChIP-seq for histone modifications H3K9c, H3K4me3, H3K27me3, H3K36me3, transcription factor MYC2 and Pol II in Arabidopsis wildtype Col-0 seedlings and various jasmonic acid pathway mutants.

first: Aanchal

last: Choudhary

first: Moonia

last: Ammari

first: Hyuk

last: Yoon


title: Transcriptome analysis of rice seedlings treated with Z-3-hexenyl acetate

city: Ghent

first: Willem

last: Desmedt

address: Valentin Vaerwyckweg 1

department: Plants and Crops

institute: Ghent University

summary: mRNA-sequencing based transcriptome analysis was used to gain insights into the molecular basis of broad-spectrum induced resistance in rice seedlings upon exposure to the model green leaf volatile Z-3-hexenyl acetate

overall_design: One and seventeen hours after the start of exposure, seedlings exposed to air laden with Z-3-hexenyl acetate or to pure air (mock-treated controls) were harvested and their roots and shoots were seperately flash-frozen.

first: Kris

last: Audenaert


title: Long range regulation of transcription scales with genomic distance in a gene specific manner

city: Palo Also

first: Joanna

last: Wysocka

address: 265 Campus Drive, rm G3065

department: Chemical and Systems Biology

institute: Standford University

summary: While critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here we aim to bypass the need for sequence-specific transcription factors and recruit activators directly using catalytically dead Cas9 fused to the synthetic activator VPR. We perform a CRISPRa tiling screen in mouse ESCs that express doxycycline-inducible dCas9-VPR and were engineered with an endogenous T2A-EGFP reporter at the end of the Fgf5 gene. We used a library of over 13,000 guides spanning a 500kb window centered at the Prdm8-Fgf5 TAD. We demonstrate that this approach is unable to achieve detectable dCas9-VPR binding to arbitrary genomic sites making it impossible to address whether any genomic site to which activators are recruited can function as an enhancer. To overcome this limitation, we turned to CARGO-VPR, an approach for targeting dCas9-VPR using a multiplexed array of RNA guides. We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible when targeted with a single guide. We utilize CARGO-VPR across the Prdm8-Fgf5 locus in mESCs, where neither gene is expressed. We demonstrate that while activator recruitment to any tested region results in transcriptional induction of at least one gene, the expression level strongly depends on the genomic distance between the promoter and activator recruitment site. However, the expression-distance relationship for each gene scales distinctly in a manner not attributable to differences in 3D contact frequency, promoter DNA sequence or presence of the repressive chromatin marks at the locus.

overall_design: R1 mESCs expressing doxycycline-inducible dCas9-VPR and an endogenous T2A-EGFP reporter were transfected with an sgRNA library. Cells expressing high EGFP were isolated along with a roughly equal number of unsorted cells. Four paired replicates of EGFP positive and unsorted populations were sequenced to identify sgRNAs in each population.

first: Christina

last: Jensen

first: Tomek

last: Swigut

first: David

last: Yao

first: Mike

last: Bassik

last: Wysoka


title: High-throughput capture of transcription factor-driven chromatin dynamics using PHILO ChIP-seq (RNA-Seq)

overall_design: To identify genes that are responsive to jasmonic acid (JA) after 2 h of JA treatment, RNA-seq with Col-0 seedlings was performed.


title: Human-correlated genetic HCC models identify combination therapy for precision medicine

first: Holly

last: Hall

address: Garscube Estate, Switchback Road, Bearsden

institute: Cancer Research UK Scotland Institute

summary: Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is a leading cause of cancer related mortality worldwide. HCC occurs typically from a background of chronic liver disease, caused by a spectrum of predisposing conditions. Tumour development is driven by the expansion of clones that accumulated progressive driver mutations, with hepatocytes the most likely cell of origin. However, the landscape of driver mutations in HCC is independent of the underlying aetiologies. Despite an increasing range of systemic treatment options for advanced HCC outcomes remain heterogeneous and typically poor. Emerging data suggest that drug efficacies depend on disease aetiology and genetic alterations. Exploring subtypes in preclinical models with human relevance will therefore be essential to advance precision medicine in HCC. We generated over twenty-five new genetically-driven in vivo and in vitro HCC models. Our models represent multiple features of human HCC, including clonal origin, histopathological appearance, and metastasis to distant organs. We integrated transcriptomic data from the mouse models with human HCC data and identified four common human-mouse subtype clusters. The subtype clusters had distinct transcriptomic characteristics that aligned with histopathology. In a proof-of-principle analysis, we verified response to standard of care treatment and used a linked in vitro-in vivo pipeline to identify a promising therapeutic candidate, cladribine, that has not been linked to HCC treatment before. Cladribine acts in a highly effective subtype-specific manner in combination with standard of care therapy.

overall_design: Mouse liver tumour tissue for HCC. Primarily GEMM models, with some diet and chemical induction.

first: Tom

last: Bird

first: Miryam

last: Müller

first: Crispin

last: Miller

first: Robin

last: Shaw


title: High-throughput capture of transcription factor-driven chromatin dynamics using PHILO [ChIP-seq 3]

last: Zander

sample_organism:
Cannabis sativa
Arabidopsis thaliana
Solanum lycopersicum

overall_design: ChIP-seq and PHILO ChIP-seq for histone modifications H3K9c, H3K4me3, H3K27me3, H3K36me3, transcription factor MYC2 and Pol II in Arabidopsis wildtype Col-o seedlings and various jasmonic acid pathway mutants


title: Human-correlated genetic HCC organoid models identify combination therapy for precision medicine

overall_design: Organoids derived from 3 GEMM murine models of HCC. Early and late passages, some taken from different mice of the same GEMM background


title: Human-correlated genetic HCC and HCC-organoid models identify combination therapy for precision medicine


title: High-throughput capture of transcription factor-driven chromatin dynamics using PHILO ChIP-seq (ChIP-seq)

last: Choudhary


title: Multiplex generation and single cell analysis of structural variants in mammalian genomes [scRNA-seq]

status: Public on Dec 01 2024

city: Seattle

first: Sudarshan

last: Pinglay

address: 3720 15th Ave NE

institute: University of Washington

summary: Studying the functional consequences of structural variants (SVs) in mammalian genomes is challenging because: 1) SVs arise much less commonly than single nucleotide variants or small indels; and 2) methods to generate, map and characterize SVs in model systems are underdeveloped. To address these challenges, we developed Genome-Shuffle-seq, a method that enables the multiplex generation and mapping of thousands of SVs (deletions, inversions, translocations, extrachromosomal circles) throughout mammalian genomes. We also demonstrate the co-capture of SV identity with single-cell transcriptomes, facilitating the measurement of SVs’ impact on gene expression. We anticipate Genome-Shuffle-seq will be broadly useful for the systematic exploration of the functional consequences of SVs on gene expression, chromatin landscape, and 3D nuclear architecture, while also initiating a path towards a minimal mammalian genome.

overall_design: scRNA-seq libraries were constructed using the 10X Genomics 3' Gene expression with feature barcoding HT kit. Cells were fixed with methanol, subject to in situ transcription (IST) with T7 polymerase and then loaded on to the 10X Genomics chip. Gene expression libraries were constructed according to the manufacturer's protocol. A custom library protocol was employed to sequence T7 derived transcripts corresponding to integrated shuffle-cassettes.

first: Jean-Benoit

last: Lalanne

first: Riza

last: Daza

first: Sanjay

last: Kottapalli

first: Faaiz

last: Quaisar

first: Jonas

last: Koeppel

first: Riddhiman

last: Garge

first: Xiaoyi

last: Li

last: Lee

first: Jay

last: Shendure


title: Definitive Evidence for the Identification and Function of Renin-Expressing Cholinergic Neurons in the Nucleus Ambiguus

city: Wauwatosa

first: Ibrahim

last: Vazirabad

state: Wisconsin

address: 8915 W Connell Ave

department: Physiology

institute: Medical College of Wisconsin

summary: Background: The importance of the brain renin-angiotensin system (RAS) incardiovascular function is well accepted. However, not knowing the precise source ofrenin in the brain has been a limitation towards a complete understanding of how the brainRAS operates. Methods: Highly sensitive in situ hybridization techniques and conditionalknockout mice were used to address the location and function of renin in the brainstem.Results: We identified novel renin-expressing cholinergic neurons in the nucleusambiguus (NuAm), a major vagal cardioinhibitory center in the brainstem. The expressionof RAS genes was relatively abundant in the NuAm, implying that angiotensin II mightmediate an important regulatory role in this nucleus and other regions with neuralconnectivity to the NuAm. Then, we generated conditional knockout mice lacking theclassical renin isoform (Ren-aChAT-KO), specifically in cholinergic neurons. Ablation of Ren-a in cholinergic neurons abrogated renin expression in the NuAm. Moreover, studiesusing radiotelemetry, heart rate variability analyses, and pharmacological approachesrevealed that the parasympathetic nervous system is depressed in Ren-aChAT-KO maleswhile augmented in the Ren-aChAT-KO females. Subsequently, transcriptomic approacheswere used to infer putative genes and signaling pathways regulated by renin within theNuAm. Conclusions: This study revealed that renin in cholinergic neurons plays afundamental role in preserving autonomic balance and cardiovascular homeostasis in asex-dependent manner. These findings define the NuAm as an endogenous, local sourceof renin with biological function and serve as conclusive evidence for the presence andfunctionality of the brain RAS.

overall_design: Brains were collected from male and female WT and Ren-aChAT-KO mice. Brains were immediately harvested and frozen in 2-methyl butane on dry ice and stored in -80° C. Then, Palkovits technique was used to obtain bilateral NuAm brain punches samples as previously described.5  Briefly, the brains were embedded in Tissue-Tek O.C.T compound (Sakura) in a cryostat until AP: -7.2. Then, a 1.0 mm needle (Stoelting) was used to collect bilateral samples of the NuAm between AP: -7.2 to -6.5 mm coordinates caudal from bregma. The samples were maintained frozen until processing. Total RNA was extracted using Nucleospin RNA Plus XS kit (Takara). Total RNA was quantified using spectrophotometry (NanoDrop, Thermo Fisher Scientific) and confirmed using fluorometric methods (Qubit, Invitrogen). Samples were then submitted to Novogene (Beijing) for library preparation and RNA sequencing analysis. A total of 22 samples were submitted (WT male=6, Ren-aChAT-KO male=6, WT female=4, Ren-aChAT-KO female=6).

first: Eva

last: Fekete

first: Javier

last: Vargas

first: Mina

last: Ghobrial

first: Kathren

last: Kaminski

first: Patricia

last: Muskus

first: Carie

last: Boychuk

first: Ana

last: Guixa

first: Michelle

last: Xie

first: Azeez

last: Ganiyu

first: Daria

last: Golosova

first: Natalia

last: Mathieu

first: Yoko

last: Wang

first: Ko-Ting

last: Lu

first: Kelsey

last: Wackman

first: Daniel

last: Brozoski

first: Gary

last: Mouradian

first: Matthew

last: Hodges

first: Jeffrey

last: Segar

first: Justin

last: Grobe

first: Curt

last: Sigmund

first: Pablo

last: Nakagawa


title: Multiplex generation and single cell analysis of structural variants in mammalian genomes [Amplicon-seq]

first: Sudarshan

last: Pinglay

overall_design: Amplicon-seq libraries were constructed from genomic DNA extracted from mammalian cell lines (mESC, K562), in some cases at various time points after transfection with recombinase expressing plasmids and/or other treatments.

first: Jean-Benoit

last: Lalanne

first: Riza

last: Daza

first: Sanjay

last: Kottapalli

first: Faaiz

last: Quaisar

first: Jonas

last: Koeppel

first: Riddhiman

last: Garge

first: Xiaoyi

last: Li

first: David

last: Lee

first: Jay

last: Shendure


title: Investigating the Mechanism of Neurotoxic Effects of PFAS in Differentiated Neuronal Cells through Transcriptomics and Lipidomics Analysis

city: Buffalo

first: Charikleia

last: Karageorgiou

address: 109 Cooke Hall, Mary Talbert Way

department: Department of Biological Sciences

institute: University at Buffalo

summary: Per- and polyfluorinated alkyl substances (PFAS) are pervasive environmental contaminants that bioaccumulate in tissues and pose risks to human health. Increasing evidence links PFAS to neurodegenerative and behavioral disorders, yet the underlying mechanisms of their effects on neuronal function remain largely unexplored. In this study, we utilized SH-SY5Y neuroblastoma cells, differentiated into neuronal-like cells, to investigate the impact of six PFAS compounds perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), perfluorodecanoic acid (PFDA), perfluorodecanesulfonic acid (PFDS), 8:2 fluorotelomer sulfonate (8:2 FTS), and 8:2 fluorotelomer alcohol (8:2 FTOH) neuronal health. Following a 30 μM exposure for 24 h, PFAS accumulation ranged from 40−6500 ng/mg of protein. Transcriptomic analysis revealed 721 differentially expressed genes (DEGs) across treatments (padj < 0.05), with 11 DEGs shared among all PFAS exposures, indicating potential biomarkers for neuronal PFAS toxicity. PFOA-treated cells showed downregulation of genes involved in synaptic growth and neural function, while PFOS, PFDS, 8:2 FTS, and 8:2 FTOH exposures resulted in the upregulation of genes related to hypoxia response and amino acid metabolism. Lipidomic profiling further demonstrated significant increases in fatty acid levels with PFDA, PFDS, and 8:2 FTS and depletion of triacylglycerols with 8:2 FTOH treatments. These findings suggest that the neurotoxic effects of PFAS are structurally dependent, offering insights into the molecular processes that may drive PFAS-induced neuronal dysfunction.

overall_design:
To investigate neurotoxic effects of PFAS in differentiated SH-SY5Y cells we performed transcriptomics analysis.
We conducted RNA-seq in neuroblastoma cells differentiated to neuronal-like cells after exposure to sub-toxic concentrations of PFOA, PFOS, PFDA, PFDS, 8:2 FTS and 8:2 FTOH

first: Logan

last: Running

first: Judith

last: Cristobal

first: Michelle

last: Camdzic

first: John Michael

last: Aguilar

first: Omer

last: Gokcumen

first: Diana

last: Aga

first: G. Ekin

last: Atilla-Gokcumen


title: Cell-type-specific requirement for TYK2 in murine immune cells under steady state and challenged conditions [RNA-Seq]

city: Vienna

first: Christian

last: Schoefer

address: Schwarzspanierstraße 17

department: Center for Anatomy and Cell Biology

country: Austria

institute: Medical University of Vienna

summary: Tyrosine kinase 2 (TYK2) deficiency and loss or inhibition of kinase activity in men and mice leads to similar immune compromised phenotypes, predominantly through impairment of interferon (IFN) and interleukin 12 family responses. Here we relate the transcriptome changes to phenotypical changes observed in TYK2-deficient (Tyk2-/-) and TYK2 kinase-inactive (Tyk2K923E) mice in naïve splenic immune cells and upon ex vivo IFN treatment or in vivo tumor transplant infiltration. The TYK2 activities under homeostatic and both challenged conditions are highly cell-type-specific with respect to quantity and quality of transcriptionally dependent genes. The major impact of loss of TYK2 protein or kinase activity in splenic homeostatic macrophages, NK and CD8+ T cells and tumor-derived cytolytic cells is on IFN responses. While reportedly TYK2 deficiency leads to partial impairment of IFN-I responses, we identified cell-type-specific IFN-I-repressed gene sets completely dependent on TYK2 kinase activity. Reported kinase-inactive functions of TYK2 relate to signaling crosstalk, metabolic functions and cell differentiation or maturation. None of these phenotypes relates to respective enriched gene sets in the TYK2 kinase-inactive cell types. Nonetheless, the scaffolding functions of TYK2 are capable to change transcriptional activities at single gene levels and chromatin accessibility at promoter-distal regions upon cytokine treatment most prominently in CD8+ T cells. The cell-type-specific transcriptomic and epigenetic effects of TYK2 shed new light on the biology of this JAK family member and are relevant for current and future treatment of autoimmune and inflammatory diseases with TYK2 inhibitors.

overall_design: Gene expression profiling by RNA-seq of 2 splenic cells types (NK and T CD8+ cells) in WT, Tyk2-/- (KO) and Tyk2K923E (KE, kinase-inactive) genotypes, with or without stimulation by IFN beta.

first: Anzhelika

last: Karjalainen

first: Agnieszka

last: Witalisz-Siepracka

first: Michaela

last: Prchal-Murphy

last: Martin

first: Felix

last: Sternberg

first: Milica

last: Krunic

first: Marlies

last: Dolezal

first: Nikolaus

last: Fortelny

first: Matthias

last: Farlik

first: Sabine

last: Macho-Maschler

first: Caroline

last: Lassnig

first: Katrin

last: Meissl

first: Lena

last: Amenitsch

first: Therese

last: Lederer

first: Elena

last: Pohl

first: Dagmar

last: Gotthard

first: Christoph

last: Bock

first: Thomas

last: Decker

first: Birgit

last: Strobl

first: Mathias

last: Müller


title: Multiplex generation and single cell analysis of structural variants in mammalian genomes [IVT-seq]

first: Sudarshan

last: Pinglay

overall_design: IVT-seq libraries were constructed with T7 In vitro transcription (IVT) derived RNA from genomic DNA extracted from mammalian cell lines (mESC, K562) in which shuffle cassettes were integrated. IVT-seq was largely performed to map integration sites of shuffle cassettes in parental populations but also to validate rearrangements induced by the recombinase.

first: Jean-Benoit

last: Lalanne

first: Riza

last: Daza

first: Sanjay

last: Kottapalli

first: Faaiz

last: Quaisar

first: Jonas

last: Koeppel

first: Riddhiman

last: Garge

first: Xiaoyi

last: Li

first: David

last: Lee

first: Jay

last: Shendure


title: Single cell gene expression profile of cells isolated from human aortic valve leaflets.

city: La Jolla

first: Talia

last: Baddour

state: California

address: 9500 Gilman Dr.

department: Bioengineering

institute: University of California San Diego

summary: Aortic valve cell  heterogeneity increases during Aortic Valve Stenosis (AVS) progression. We used single cell RNA sequencing (scRNA-seq) to characterize valve cell phenotype in AVS patients.

overall_design: Whole cells were isolated from aortic valve leaflets of age and disease-matched AVS patients. We chose one male and one female patient.

first: T

first: B

last: Aguado


title: High-throughput capture of transcription factor-driven chromatin dynamics using PHILO ChIP-seq

first: Mark

last: Zander

platform_organism:
Cannabis sativa
Arabidopsis thaliana
Solanum lycopersicum

overall_design: Refer to individual Series


title: Olaparib and radiotherapy induce type I interferon and CD8+ T cell-dependent sensitization to immunotherapy in pancreatic cancer

city: Ann Arbor

first: Meredith

last: Morgan

state: Michigan

address: 1301 Catherin St

department: Radiation Oncology

institute: University of Michigan

summary: PARP inhibitors sensitize pancreatic ductal adenocarcinoma (PDAC) to radiation by inducing DNA damage and replication stress. These mechanisms also have the potential to enhance radiation-induced type I interferon (T1IFN) mediated anti-tumoral immune responses. We hypothesized that the PARP inhibitor olaparib would also potentiate radiation-induced T1IFN to promote anti-tumor immune responses and sensitization of otherwise resistant PDAC to immunotherapy. To test this hypothesis, we assessed the effects of olaparib and radiation on T1IFN production and sensitivity to αPD-L1 immunotherapy, as well as consequent effects on the tumor microenvironment by single-cell RNA sequencing (scRNA-seq). We found that olaparib enhanced T1IFN production following radiation and had superior therapeutic efficacy in immune competent models. Treatment with olaparib and radiation sensitized PDAC tumors to αPD-L1 resulting in decreased tumor burden and a 33% complete response rate. Combination treatment provided durable immune responses as shown by tumor rejection upon tumor rechallenge of previously cured mice. Furthermore, scRNA-seq analysis revealed that combination treatment induced an immunogenic tumor microenvironment, characterized by interferon responses in both PDAC and myeloid cell populations, macrophage polarization, and increased CD8+ terminal effector T cell frequency and activity. Furthermore, CD8+ T cells and T1IFN signaling were required for therapeutic efficacy as host depletion of CD8+ T cells or the T1IFN receptor diminished treatment responses. Taken together, our results indicate that olaparib enhances radiation-induced T1IFN-mediated immune signaling and subsequently an adaptive immune response thus sensitizing pancreatic cancer to αPD-L1 therapy, supporting an ongoing clinical trial of this therapy in patients with PDAC.

overall_design: 6-8 week old C57/Bl6 mice with subcutaneous mT4 tumors were treated with AZD1390 (20mg/kg) for 5 days starting on day 0, 1 dose of radiation (8Gy) on day 0, and 3 doses of anti-PD-L1 blocking antibody (100µg) once every three days starting on day -1. 6-8 tumors were harvested for each of the following treatment groups: IgG/control, AZD1390, RT+anti-PD-L1, and AZD1390+RT+anti-PD-L1. Tumors were harvested on day 5 and digested in a collagenase digest buffer, and filtered through 40µm mesh filter. Samples were depleted of dying cells twice using Dead Cell Removal Kit from Miltenyi. Cells were then seperated based on CD45 status, and mixed back in equal parts CD45+ and CD45- to enrich immune cells present in the samples. Samples were diluted to a final concentraiotn of 700-1000 cells/ul. Single cell sequencing was performed by the University of Michigan Advanced Genomics Core Research Facility. Library generation was performed on the 10x Genomica Chromium Controller following manufacturers protocol for the 3'v3.1 chemistry with NextGEM Chip G reagents. Library quality was assess by LabChip GXII HT and quantified by Qubit. Pooled libraries were subjected to 150 base pair, paired-end sequencing according to manufacturer protocol (Illumina NovaSeq 6000). Bcl2fastq2 Conversion Software was used to de-multiplex fastq files and CellRanger v7 Pipeline was used to align reads and generate count matrices.

first: Victoria

last: Valvo

first: Qiang

last: Zhang

first: Long

last: Jiang

first: Anna

last: Edmunds

first: Akshay

last: Tate

first: Amanda

last: Huber

first: Erin

last: Holcomb

first: Ashley

middle: N

last: Pearson

first: Zhuwen

last: Wang

first: Yupei

last: Guo

last: Karnak

first: Leslie

last: Parsels

first: Joshua

first: Yu

last: Lei

first: Alnawaz

last: Rehemtulla

first: Eileen

last: Carpenter

first: Daniel

last: Wahl

first: Vaibhav

last: Sahai

first: Theodore

last: Lawrence

first: Michael

last: Green


title: Cell-type-specific requirement for TYK2 in murine immune cells under steady state and challenged conditions

first: Christian

last: Schoefer

summary:
Tyrosine kinase 2 (TYK2) deficiency and loss or inhibition of kinase activity in men and mice leads to similar immune compromised phenotypes, predominantly through impairment of interferon (IFN) and interleukin 12 family responses.
Here we relate the transcriptome changes to phenotypical changes observed in TYK2-deficient (Tyk2-/-) and TYK2 kinase-inactive (Tyk2K923E) mice in naïve splenic immune cells and upon ex vivo IFN treatment or in vivo tumor transplant infiltration.
The TYK2 activities under homeostatic and both challenged conditions are highly cell-type-specific with respect to quantity and quality of transcriptionally dependent genes.
The major impact of loss of TYK2 protein or kinase activity in splenic homeostatic macrophages, NK and CD8+ T cells and tumor-derived cytolytic cells is on IFN responses.
While reportedly TYK2 deficiency leads to partial impairment of IFN-I responses, we identified cell-type-specific IFN-I-repressed gene sets completely dependent on TYK2 kinase activity.
Reported kinase-inactive functions of TYK2 relate to signaling crosstalk, metabolic functions and cell differentiation or maturation.
None of these phenotypes relates to respective enriched gene sets in the TYK2 kinase-inactive cell types.
Nonetheless, the scaffolding functions of TYK2 are capable to change transcriptional activities at single gene levels and chromatin accessibility at promoter-distal regions upon cytokine treatment most prominently in CD8+ T cells.
The cell-type-specific transcriptomic and epigenetic effects of TYK2 shed new light on the biology of this JAK family member and are relevant for current and future treatment of autoimmune and inflammatory diseases with TYK2 inhibitors.

overall_design: The study is based on RNA-Seq profiling and functional analyses of TYK2-STAT signaling in splenic homeostatic and interferon beta-situmulated macrophages, NK cells and CD8+ T cells and in NK cells and CD8+ T cells FACS-purified from tumor transplants. In addition we performed ATAC-Seq to correlated the RNA expression profile with the chromatin accessibility of interferon treated T cells. We systematically compared wild type mice and mice lacking TYK2 protein or carrying kinase-inactive TYK2, thereby mimicking pharmaceutical TYK2 inhibition.

first: Anzhelika

last: Karjalainen

first: Agnieszka

last: Witalisz-Siepracka

first: Michaela

last: Prchal-Murphy

first: David

last: Martin

first: Felix

last: Sternberg

first: Milica

last: Krunic

first: Marlies

last: Dolezal

first: Nikolaus

last: Fortelny

first: Matthias

last: Farlik

first: Sabine

last: Macho-Maschler

first: Caroline

last: Lassnig

first: Katrin

last: Meissl

first: Lena

last: Amenitsch

first: Therese

last: Lederer

first: Elena

last: Pohl

first: Dagmar

last: Gotthardt

first: Christoph

last: Bock

first: Thomas

last: Decker

first: Birgit

last: Strobl

first: Mathias

last: Müller


title: Locus-specific and genome-wide analysis of adaptations to hypoxia in iPSC-derived endothelium from a population adapted to high altitude [scRNA-Seq]

city: Chicago

first: Anna

last: Di Rienzo

state: IL

department: Human Genetics

institute: University of Chicago

summary: Tibetan adaptation to high-altitude hypoxia remains a classic example of Darwinian selection in humans. Amongst Tibetans, alleles in the EPAS1 gene - whose protein product, Hif-2α, is a central regulator of the hypoxia response - have repeatedly been shown to carry some of the strongest signals of positive selection in humans and to influence several adaptive phenotypes. We recently showed that the selected haplotype at this locus spans a hypoxia-dependent enhancer (ENH5) that contributes to the regulation of EPAS1 in a variety of cell types. However, selective sweep signals alone may account for only part of the phenotypes that differentiate Tibetans from closely related lowlanders. Therefore, there is a pressing need to functionally probe adaptive alleles and their impact at the genome-wide level and across cell types to uncover the full range of beneficial traits. To cast a wider net, we established a library of induced pluripotent stem cells (iPSCs) derived from Tibetan and Han Chinese individuals, a robust model system allowing precise exploration of both locus-specific and genome-wide effects on transcriptional responses. We harness this system by differentiating the iPSC library into vascular endothelium and investigating the locus-specific effects of the ENH5 enhancer in this cellular context. In addition, we use it to explore Tibetan-specific transcriptome-wide responses and find evidence that energy metabolism and immune pathways have been shaped by natural selection in Tibetans. Finally, to aid with the interpretation of the transcriptional differences between populations, we test for polygenic adaptations as a complementary in silico approach for the identification of beneficial Tibetan phenotypes.

overall_design:
We developed a panel of sex-matched Tibetan and Han Chinese iPSCs using lymphoblastoid cell lines (LCLs) from 10 unrelated individuals of Tibetan origin and 10 Han Chinese (CHB) from the 1000 Genome Project.These 20 LCLs were reprogrammed into iPSCs in batches, balanced by population and sex and then successfully differentiated into vascular endothelium.
We used CRISPR-CAS9 editing to delete the region (chr2: 46578867 to 46579857; hg19) spanning the ENH5 enhancer region  of the iPSC line derived from Han individual CHB633  in order to create knockout cells carrying a low-altitude haplotype across the entire EPAS1 locus, but with complete loss of ENH5 activity. These DENH5 iPS cells (subsequently referred to as knockout, KO) and the original CHB633 (wild type, WT) iPS cells were differentiated into vascular endothelium in five independent replicates and cultured in hypoxic conditions (1% O2) for 48 hours.
In order to assess the impact of the ENH5 deletion in vascular endothelial cells, we performed single cell RNA-sequencing in a targeted 10,000 cells per KO and WT replicate. We assessed the expression of 25 endothelial markers (in the 10 UMAP clusters identified by Seurat) and identified two cell clusters as being robustly enriched in all endothelial markers. Transcript data from the cells in these two clusters were pooled to compare expression differences between KO and WT in a cell-type specific manner.
We utilized our complete panel of 20 cell lines to compare transcript levels between TAC and CHB iPSC-derived vascular endothelium under both normoxic and hypoxic conditions. Given the larger number of cell lines and treatment conditions, we performed bulk RNA-sequencing rather than single cell RNA-sequencing.

first: O

last: Gray

first: D

last: Witonsky

first: J

last: Jousma

last: Sobreira

first: X

last: Van Alstyne

first: R

middle: T

last: Huang

first: Y

last: Fang

first: A


title: Cell-type-specific requirement for TYK2 in murine immune cells under steady state and challenged conditions [ATAC-Seq]

first: Christian

last: Schoefer

overall_design: ATAC-seq samples of spleen-derived immune cells with mutations of JAK-STAT signaling components

first: Anzhelika

last: Karjalainen

first: Agnieszka

last: Witalisz-Siepracka

first: Michaela

last: Prchal-Murphy

first: David

last: Martin

first: Felix

last: Sternberg

first: Milica

last: Krunic

first: Marlies

last: Dolezal

first: Nikolaus

last: Fortelny

first: Matthias

last: Farlik

first: Sabine

last: Macho-Maschler

first: Caroline

last: Lassnig

first: Katrin

last: Meissl

last: Gotthard


title: Locus-specific and genome-wide analysis of adaptations to hypoxia in iPSC-derived endothelium from a population adapted to high altitude [RNA-Seq]

first: Anna

last: Di Rienzo

first: O

last: Gray

first: D

last: Witonsky

first: J

last: Jousma

last: Sobreira

first: X

last: Van Alstyne


title: RORc expressing immune cells negatively regulate tertiary lymphoid structure formation and support their pro-tumorigenic functions

city: Jerusalem

first: Oren

last: Parnas

address: Faculty of Medicine, Ein-Kerem medical center

department: Immunology

country: Israel

institute: The Hebrew University of Jerusalem

summary: Tertiary lymphoid structures (TLSs) are formed in many cancer types and have been correlated with better prognosis and response to immunotherapy. In liver cancer, TLSs have been reported to be pro-tumorigenic as they harbor tumor progenitor cells and nurture their growth. The processes involved in TLS development and acquisition of pro- or anti-tumorigenic phenotype in cancer are largely unknown. RORc expressing immune cells have been previously implicated in TLS formation, however we find that they are not necessary for TLS neogenesis in the context of liver inflammation. On the contrary, RORc expressing cells negatively regulate TLS formation, since in their absence TLSs form in excess. CD4 cells are essential for liver TLS formation whereas B cells are required for TLS formation specifically in the absence of RORc expressing cells. Importantly, in chronically inflamed livers lacking RORc expressing cells, TLSs become anti-tumorigenic, resulting in reduced tumor load. Comparing liver pro- and anti-tumorigenic TLSs by transcriptional, proteomic and immunohistochemical analyses, revealed enrichment of exhausted CD8 cells that retained effector functions as well as germinal center B cells and plasma cells in anti-tumorigenic TLSs. Cell depletion experiments revealed a role mainly for B cells in limiting tumor development, possibly via tumor directed antibodies. Thus, RORc expressing cells negatively regulate B cell responses, and facilitate the pro-tumorigenic functions of hepatic TLSs.

overall_design: The goal of this experiment was to analyze the composition and phenotype of immune cells from hepatic tertiary lymphoid structures (TLSs). Whole body RORc knock out mice with chronically inflammed livers were compared to controls.  The genotypes compared were: Alb-IKKβ(EE)Hep;RORctm2Litt +/- (termed: IKK RORc Het or control) and Alb-IKKβ(EE)Hep;RORctm2Litt -/- (termed: IKK RORc KO or Experiment). CD45+ immune cells were isolated from livers of 6 months old DEN-injected mice. Immune cells were isolated from 3 IKK RORc Het mice (cells were pooled after isolation), and from 2 IKK RORc KO mice.

first: Einat

last: Cinnamon

first: Ilan

last: Stein

first: Elvira

last: Zino

first: Pikarsky

last: Eli

first: Yehuda

last: Shovman


title: Effect of over expression of MED13 IDR in human osteosarcoma U2OS cell line

first: Mo-Fan

last: Huang

address: 6431 Fannin Street

department: Integrative Biology and Pharmacology

institute: University of Texas Health science center

summary: The eukaryotic transcriptional Mediator comprises a large Core (cMED) and a dissociable CDK8 kinase module (CKM). cMED recruits RNA polymerase II (Pol II) and promotes pre-initiation complex formation in a manner repressed by the CKM through mechanisms presently unknown. Herein we report cryo-electron microscopy structures of the complete human Mediator and its CKM. Combined with biochemical and functional analyses, these structures provide a mechanistic framework to explain the basis for CKM-mediated repression of cMED function. The CKM binds to cMED at multiple sites through both MED12 and a large intrinsically disordered region (IDR) in MED13. The MED13 IDR blocks Pol II/MED26 recruitment onto cMED by direct occlusion of their corresponding binding sites, leading to functional repression of cMED-dependent transcription. Notably, the CKM is anchored to the cMED Hook, positioning CDK8 downstream and proximal to the transcription start site, which sheds new light on its stimulatory function in post-initiation events.

overall_design: To investigate the function of MED13 IDR in transcription initiation, we over-expressed MED13 IDR using lentivirus based system in U2OS cell line. We then performed gene expression profiling analysis using data obtained from RNA-seq. Comparative gene expression profiling analysis of RNA-seq data for triplicate of MED13 IDR and control cells.

first: Ti-chun

last: chao

first: An

last: Xu

first: Dung-Fang

last: Lee

first: Kuang-lei

last: Tsai


title: Differential gene expression patterns with ontogeny following an in situ depth translocation of coral planulae

city: Haifa

first: Jessica

last: Bellworthy

address: 199 Aba Khoushy Ave

institute: Haifa University

summary: Mesophotic coral reefs have been proposed as refugia for corals, providing shelter and larval propagules for shallow-water reefs that are disproportionately challenged by global climate change and local anthropogenic stressors. Yet, knowledge of the capacity of coral larvae to adjust to different depth environments is still limited. In this study, planulae of the reef-building coral Stylophora pistillata from 5-8 and 40-44 m depth in the Gulf of Aqaba were tested in a long-term in situ translocation experiment for their ability to settle and acclimate to reciprocal depth conditions. We assessed survival rates, photochemical, physiological and morphological characteristics, as well as gene expression variations in juveniles grown at different depths, comparing them to non-translocated adults, juveniles and planulae. We found high mortality rates among mesophotic-origin planulae, irrespective of translocation depth. Gene expression patterns suggested that deep planulae lacked settlement competency and experienced increased developmental stress upon release. Symbiont photochemical acclimation to depth occurred rapidly within 8 days, with symbiont populations showing changes in photochemical traits but no symbiont species shuffling between deep and shallow juveniles. In contrast, coral host physiological and morphological acclimation were less evident. We observed minimal overlap in gene expression patterns between different life stages and depths, indicating that gene expression significantly depends on life stage. The study also identified a set of DEGs associated with initial stress responses following translocation, lingering stress response, and environmental effects of depth. In conclusion, though our data reveal rapid symbiont acclimation, host acclimation to match deep coral phenotypes was incomplete within 60 days for planulae translocated to different depths. These results have implications for understanding the ecological significance of mesophotic reefs as potential larval sources in the face of environmental stressors.

overall_design: We sampled coral planulae from shallow (5m) and mesophotic reefs (45m) in Eilat, Israel. Gene expression patterns of planulae and their parents from each depth were assessed with RNAseq (Illumina HTS). Planulae were placed in situ in a fully corssed reciprocal depth transplant design. Samples for RNAseq were again taken at 8 and 60 days old.

first: Federica

last: Scucchia

first: Gretchen

last: Goodbody Gringley

first: Tali

last: Mass


title: Ngn family transcription factors reprogram mouse embryonic fibroblasts into neuron-like cells

city: Guangzhou

first: SHUYI

last: CHEN

state: Guangdong

address: 54 S. Xianlie Rd

department: State Key Laboratory of Ophthalmology

institute: Zhongshan Ophthalmic Center, SYSU

summary: Ngn family transcription factors (TFs) are important neurogenic regulators during development. The aim of this study is to examine the ability of two Ngn family members, Ngn1 and Ngn3, to induce neuron fates from mouse embryonic fibroblasts (MEFs) through direct cell fate reprogramming.

overall_design: MEFs were infected with Ngn1/Ngn3-expressing lentiviruses; Cells were collected 24hours and 13 days after Ngn1/Ngn3 overexpression, and processed for RNA-Seqs.

first: Shuyi

last: Chen


title: The role of ATAD2 in ovarian cancer growth and progression

city: Birmimgham

first: Romi

last: Gupta

state: Alabama

address: 720, 20th street south

institute: University of Alabama at Birmingham

summary: ATPase Family AAA domain containing 2 (ATAD2) expression is elevated in many cancer types including ovarian cancer. Therefore, we analyzed the role of ATAD2 in ovarian cancer. Pharmacological inhibition of ATAD2 expression in ovarian cancer cell lines resulted in decreased proliferation, and increased cell death.

overall_design: PA-1 and SK-OV-3 cells were treated with  ATAD2 inhibitor (5 micomolar) BAY-850 for 48 hours, samples were harvested and were used to prepare the total RNA, followed by RiboRNA depletion with random priming and RNASeq Illumina platform, with three biological replicates for each sample.


title: Effect of Calcipotriol on gene expression in gastrocnemius muscle of SKO mice.

city: Shenzhen

first: Aolin

last: Yang

address: 66 Gongchang Road

institute: Sun Yat-sen University

summary: Further investigation is needed to understand the role of vitamin D in muscle function. In this study, we utilized SOD1 gene knockout mice as a model of muscular atrophy and intervened with the VDR ligand calcipotriol to observe its effects on muscle function. RNAseq analysis was employed to examine the impact of calcipotriol on gene expression in the gastrocnemius muscle, aiming to elucidate its underlying mechanisms. The results revealed that calcipotriol primarily exerted its effects through pathways related to protein synthesis and mitochondrial function.

overall_design: We utilized SOD1 gene knockout mice and their wild-type littermates, and intervened with either calcipotriol or a vehicle control.

first: Qingqing

last: Lv