title: The transcription factor HIF2a partakes in the differentiation blockade of acute myeloid leukaemia [ChIP-seq]

city: Milan

first: Rosa

last: Bernardi

address: via Olgettina 60

country: Italy

institute: San Raffaele Scientific Institute

summary: Acute myeloid leukemia (AML) is characterized by an accumulation of aberrant myeloid cells arrested at different stages of differentiation. Therapeutic approaches that prompt AML blasts to differentiate represent an attractive opportunity in the landscape of AML therapies, as they aim to induce terminal maturation and leukemia debulking without intensive cytotoxic treatments. In the present study, we investigate the involvement of HIF1a and HIF2a transcription factors in AML pathogenesis, and position HIF2a as a novel regulator of the AML differentiation block. We performed a comparative analysis of HIF1a and HIF2a function in AML cell lines via their inhibition with genetic or pharmacological strategies and found that both factors promote AML proliferation and clonogenicity. Importantly, specific inhibition of HIF2a provokes AML cell differentiation in cell lines and patient-derived xenograft (PDX) models.  Additionally, we demonstrate that HIF2a is positively regulated by the pro-differentiation agent all-trans retinoic acid (ATRA), and its inhibition cooperates with ATRA in triggering AML cell differentiation. In conclusion, we report evidence of a new role of HIF2a in the pathogenesis of AML, and we propose that HIF2a inhibition may open new therapeutic avenues for AML treatment by licensing AML differentiation and synergizing with ATRA towards leukemia exhaustion.

overall_design: ChIP-sequencing (ChIP-seq) analysis of H3K27me3 in Kasumi1 cells stably transfected with shRNA against HIF2a (shHIF2a) or a scramble shRNA as control (shCTRL). Each condition was conducted in experimental triplicate (6 samples).

first: Daniela

last: Magliulo

first: Fracassi

last: Cristina

first: Zapparoli

last: Ettore


title: ROCY1 binding sites in Toxoplasma gondii

status: Public on Aug 11 2023

city: Indianapolis

first: Michael

middle: James

last: Holmes

state: IN

address: 635 Barnhill Drive

department: Pharmacology & Toxicology

country: USA

institute: Indiana University School of Medicine

summary: Cyst formation is a key feature of the T. gondii life cycle but the genetic networks that drive this process are not yet fully characterized. To identify new components of this network, we compared T. gondii to its nearest extant relative Hammondia hammondi given the critical differences between these species in the timing and efficiency of cyst formation. Using transcriptional data from critical developmental and pH exposure time points from both species, we identified the gene TGVEG_311100, which we named Regulator of Cystogenesis 1 (ROCY1), as being both necessary and sufficient for cyst formation in T. gondii. Compared to WT parasites, TGVEG?ROCY1 parasites formed significantly fewer tissue cysts in response to alkaline pH stress in vitro and cysts were nearly undetectable in mouse brains for up to 9 weeks post-infection. Overexpression of tagged ROCY1 in WT parasites was sufficient to induce cyst formation in vitro in both WT and ROCY1-deficient parasites, demonstrating that ROCY1 is both necessary and sufficient for cyst formation. Moreover this induction of cyst formation required at least 1 of 3 predicted CCCH Zinc finger domains. Mice chronically infected with ?ROCY1 parasites had detectable tachyzoites in the brain for up to 37 days post-infection (while mice infected with WT parasites did not), and CNS transcriptional analyses at day 30 post-infection throughout the chronic phase of infection revealed inflammatory signatures consistent with acute infection in ?ROCY1 parasites compared to WT. Despite our inability to detect brain cysts in infected mice, both WT and ?ROCY1 knockout parasites reactivated after dexamethasone treatment with similar timing and magnitude for up to 5 months post infection, challenging the paradigm that long term parasite persistence in the CNS requires cyst formation. These data identify a new regulator of cyst formation in T. gondii that is both necessary and sufficient for cyst formation, and whose function relies on its conserved nucleic acid binding motif.

overall_design: FLAG-ROCY1 from 48h pH stressed parasites were collected and processed by eCLIP protocol to determine ROCY1 binding sites transcriptome-wide during Toxoplasma gondii bradyzoite formation

middle: J

first: William

last: Sullivan Jr


title: The transcription factor HIF2a partakes in the differentiation blockade of acute myeloid leukaemia

status: Public on Aug 10 2023

type: Expression profiling by high throughput sequencing

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: The transcription factor HIF2a partakes in the differentiation blockade of acute myeloid leukaemia [ATAC-seq]

summary: Acute myeloid leukemia (AML) is characterized by an accumulation of aberrant myeloid cells arrested at different stages of differentiation. Therapeutic approaches that prompt AML blasts to differentiate represent an attractive opportunity in the landscape of AML therapies, as they aim to induce terminal maturation and leukemia debulking without intensive cytotoxic treatments. In the present study, we investigate the involvement of HIF1 and HIF2 transcription factors in AML pathogenesis, and position HIF2 as a novel regulator of the AML differentiation block. We performed a comparative analysis of HIF1 and HIF2 function in AML cell lines via their inhibition with genetic or pharmacological strategies and found that both factors promote AML proliferation and clonogenicity. Importantly, specific inhibition of HIF2 provokes AML cell differentiation in cell lines and patient-derived xenograft (PDX) models. Mechanistically, we found that HIF2 and EZH2, the catalytic subunit of polycomb repressive complex 2, cooperate at favoring EZH2-mediated deposition of the repressive histone mark H3K27me3 on the regulatory regions of myeloid differentiation genes. Additionally, we demonstrate that HIF2 is positively regulated by the pro-differentiation agent all-trans retinoic acid (ATRA), and its inhibition cooperates with ATRA in triggering AML cell differentiation. In conclusion, we report evidence of a new role of HIF2 in the pathogenesis of AML, by promoting an undifferentiated state via EZH2-mediated epigenetic silencing of myeloid differentiation genes. We propose that HIF2 inhibition may open new therapeutic avenues for AML treatment by licensing AML differentiation and synergizing with ATRA towards leukemia exhaustion.

overall_design: ATAC-sequencing (ATAC-seq) analysis of 6 samples. Kasumi1 cells were stably transfected with shRNA against HIF2a or a scamble shRNA as control (shCTRL). Each condition was conduced in experimental triplicate.

first: Kety

last: Giannetti

first: Ettore

last: Zapparoli

first: Raffaella

last: Di Micco


title: Late fetal hematopoietic failure results from ZBTB11 deficiency despite abundant HSC specification

city: Parkville

first: Peter

last: Hickey

address: 1G Royal Parade

country: Australia

institute: WEHI

summary: We have used a conditional mouse model to investigate the role of Zbtb11 specifically in hematopoiesis. When Zbtb11 was deleted in the hematopoietic compartment, embryos died at embryonic day E18.5 with hematopoietic failure. Zbtb11 hematopoietic knockout (Zbtb11hKO) hematopoietic stem cells (HSCs) were overabundantly specified at E14.5 through E17.5 compared to controls. Overspecification was accompanied by loss of stemness, inability to differentiate into committed progenitors and mature lineages in fetal liver, failure to seed fetal bone marrow and total hematopoietic failure. Zbtb11hKO HSCs did not proliferate in vitro and were constrained in cell cycle progression, demonstrating a cell-intrinsic role for Zbtb11 in proliferation and cell cycle regulation in mammalian HSCs. scRNAseq analysis identified Zbtb11-deficient HSCs were underrepresented in an erythroid-primed subpopulation and showed downregulation of oxidative phosphorylation (OXPHOS) pathways and dysregulation of genes associated with the hematopoietic niche. We have identified a cell-intrinsic requirement for Zbtb11-mediated gene regulatory networks in sustaining a pool of maturation-capable hematopoietic stem and progenitor cells.

overall_design: We have compared Zbtb11-deleted LSK/SLAM HSCs with controls that are either heterozygous or WT for the same Zbtb11 deletion. A total of 186 single E14.5 fetal liver Zbtb11hKO and control HSCs were assayed on one 384-well plate. Cells were evenly split between WT and Zbtb11hKO (n=93 for each).

first: M

middle: C

last: Keightley

first: Huimin

last: Cao

first: Shalin

middle: H

last: Naik

last: Amann-Zalcenstein

middle: F

first: Agus

last: Salim

first: Benjamin

first: Susan

middle: K

last: Nilsson

first: Graham

last: Lieschke


title: RNA-seq analysis of Alyref and Gabpb1 knockout embryos

city: Fukuoka

first: Kei

last: Miyamoto

address: 744 Moto-oka, Nishi-ku

department: Faculty of Agriculture

country: Japan

institute: Kyushu University

summary: We have identified Alyref and Gabpb1 as developmentally important genes by siRNA screening.  Gene knockout (KO) of Alyref and Gabpb1 by the CRISPR/Cas9 system resulted in early developmental arrest in mice. To gain mechanistic insight into the developmental role of Alyref and Gabpb1, we performed RNA sequencing (RNA-seq) analysis of the KO embryos.

overall_design: Sperm and oocytes were collected from hetero mice (Alyref+/- or Gabpb1+/-), and in vitro fertilization (IVF) was performed ([Alyref+/- x Alyref+/-] or [Gabpb1+/- x Gabpb1+/-]). As a control, IVF using wild type (WT) mice were also carried out. The resulting IVF embryos were cultured in potassium simplex optimized medium (KSOM) at 37°C under 5% CO2. At 96 hours post insemination (hpi), embryos at the morula and blastocyst stages were collected and subjected to single embryo RNA-seq. Single embryo RNA-seq enabled us to distinguish KO embryos (Alyref-/- or Gabpb1-/-) from hetero and WT embryos due to the lack of sequence reads at the deleted region. Then, transcriptomes of KO and other embryos were compared.

first: Shunya

last: Ihashi


title: Skeletal muscle tissue RNA-Seq and RRBS in T2D, Obese and Active subjects

city: Orlando

first: Lauren

last: Sparks

state: Florida

address: 301 E Princeton St, 301

institute: AdventHealth

type: Methylation profiling by high throughput sequencing

summary: We performed RNA-Seq on skeletal muscle tissue in Active individuals, indivduals with obesity (Obese) and individuals with obesity and T2D (T2D) (n =6 per group). PCA of top ranked contributing genes show clear separation of the Active group from Obese and T2D with considerable overlap between Obese and T2D. Over-representation analysis highlighted that Active had an upregulation of genes related to respiration/mitochondrial capacity in comparison to Obese and T2D. RRBS was performed on skeletal muscle tissue from a subset of participants from each group (n = 3). Data was analyzed to reveal (DMS) differentially methylated sites located at differentially expressed genes related to mitochondrial respiration/function. Out of a combined 488 DMS related to mitochondrial related differentially expressed genes, 11% were significantly positively correlated with gene expression and 12% were significantly negatively associated with gene expression. Significantly correlated DMS were mainly located in the promoter and 1st intron region

overall_design: Skeletal muscle tissue from M. Vastus Lateralis was obtained from lean Active individuals, individuals with Obesity and individuals with T2D and Obesity to perfrom transcriptomics by RNA-Seq and DNA methylation by RRBS.

first: L

middle: M


title: Drug repurposing unveils new combinatorial treatments to treat mutant KRAS lung cancer

city: Pamplona

first: Silvestre

last: Vicent

state: Outside the US or Canada

address: 55 PIO XII Ave

department: Oncology

country: Spain

institute: Center for Applied Medical Research (CIMA)

summary: We report data on a drug combination with synergistic antitumor effect in KRAS-driven lung cancer. We used RNAseq to characterize the transcriptional changes elicited by each drug in a mutant KRAS lung cancer cell lines (H1792)

overall_design: H1792 cell line treated with Trametinib (MEKi) or Lestaurtinib (multikinase FLT3i) for 16 hours

first: Silve

first: Marta

last: Román


title: Combination of an ACLY inhibitor with a GLP-1R agonist exerts additive benefits on NASH and hepatic fibrosis [NASH Custom panel]

status: Public on Aug 09 2023

city: Hamilton

first: Dongdong

last: Wang

state: Ontario

address: 1200 Main Street West

department: Department of Medicine

country: Canada

institute: McMaster University

summary: Increased liver de novo lipogenesis (DNL) is a hallmark of nonalcoholic steatohepatitis (NASH).  A key enzyme controlling DNL upregulated in NASH is ATP citrate lyase (ACLY). In mice, inhibition of ACLY reduces liver steatosis, ballooning and fibrosis and inhibits activation of hepatic stellate cells. Glucagon like peptide-1 receptor (GLP-1R) agonists lower body mass, insulin resistance and steatosis without improving fibrosis. Here, we find that combining an inhibitor of liver ACLY, bempedoic acid, and the GLP-1R agonist liraglutide reduces liver steatosis, hepatocellular ballooning, and hepatic fibrosis in a mouse model of NASH. Liver RNA analyses revealed additive downregulation of pathways that are predictive of NASH resolution, reductions in the expression of prognostically significant genes compared to clinical NASH samples, and a predicted gene signature profile that supports fibrosis resolution. These findings support further investigation of this combinatorial therapy to treat obesity, insulin resistance, hypercholesterolemia, steatohepatitis, and fibrosis in people with NASH.

overall_design: For NanoString analysis, 4-5 RNA samples per group were inspected by a BioAnalyzer quality control test. The McMaster Genomics Facility ran an nCounter Fibrosis v2 Panel (NanoString Technologies) containing 760 target genes as well as a CustomSet Panel consisting of 22 orthologous mus musculus genes that correspond to the 25-gene NASH severity signature.

first: Eric

last: Desjardins

first: Gregory

middle: R

last: Steinberg


title: Combination of an ACLY inhibitor with a GLP-1R agonist exerts additive benefits on NASH and hepatic fibrosis


title: Combination of an ACLY inhibitor with a GLP-1R agonist exerts additive benefits on NASH and hepatic fibrosis [Fibrosis panel]


title: MRTX1719 is an MTA-cooperative PRMT5 inhibitor that exhibits synthetic lethality in preclinical models and patients with MTAP deleted cancer

city: San Diego

first: Lars

last: Engstrom

state: CA

address: 3545 Cray Court

institute: Mirati Therapeutics

summary:
Previous studies implicated PRMT5 as a synthetic lethal target for MTAP deleted (MTAP del) cancers, however, the pharmacological characterization of small molecule inhibitors that recapitulate the synthetic lethal phenotype have not been described. MRTX1719 selectively inhibited PRMT5 in the presence of MTA, which is elevated in MTAP del cancers, and inhibited PRMT5-dependent activity and cell viability with >70-fold selectivity in HCT116 MTAP del compared to HCT116 MTAP WT cells. MRTX1719 demonstrated dose-dependent anti-tumor activity and inhibition of PRMT5-dependent SDMA modification in MTAP del tumors. In contrast, MRTX1719 demonstrated minimal effects on SDMA and viability in MTAP WT tumor xenografts, mouse or human hematopoietic cells. MRTX1719 demonstrated marked anti-tumor activity across a panel of xenograft models at well-tolerated doses. Early signs of clinical activity were observed including objective responses in patients with MTAP del melanoma, gallbladder adenocarcinoma, mesothelioma, non-small cell lung cancer, and MPNST from the Phase 1/2 study.
Significance:
PRMT5 was identified as a synthetic lethal target for MTAP del cancers, however, previous PRMT5 inhibitors do not selectively target this genotype. The differentiated binding mode of MRTX1719 leverages the elevated MTA in MTAP del cancers and represents a promising therapy for the ~10% of cancer patients with this biomarker.

overall_design: LU99 cells treated with DMSO or MRTX1719 for 3 or 5 days

middle: D

first: Peter

last: Olson


title: Discrimination of cell-intrinsic and environment-dependent effects of natural genetic variation on Kupffer cell epigenomes and transcriptomes [ChIP]

city: La Jolla

first: Hunter

last: Bennett

state: California

address: 9500 Gilman Drive, MC 0651

department: Dept. of Cellular & Molecular Medicine

institute: University of California, San Diego

summary: Noncoding genetic variation is a major driver of phenotypic diversity but determining the underlying mechanisms and the cell types in which it acts remain challenging problems. Here, we investigate the impact of natural genetic variation provided by phenotypically diverse inbred strains of mice on gene expression and epigenetic landscapes of Kupffer cells.  Analysis of gene expression in Kupffer cells and other liver cell types derived from C57BL/6J, BALB/cJ and A/J mice provided evidence for strain-specific differences in environmental factors influencing Kupffer cell phenotypes, including preferential Leptin signaling in BALB/cJ Kupffer cells. Systematic analysis of transcriptomic and epigenetic data from F1 hybrids of these mice, and transcriptomic data from strain-specific Kupffer cells engrafted into a common host enabled quantitative assessment of cis versus trans effects of genetic variation on gene expression and an estimate of cell autonomous versus non cell autonomous effects.  Under homeostatic conditions, trans effects of genetic variation were dominant, with the majority of trans regulation being non cell autonomous. In contrast, strain specific responses to acutely administered LPS were primarily associated with genetic variation acting in cis to modify response elements for lineage determining and signal dependent transcription factors.  Collectively, these findings reveal cell intrinsic and environmental effects of natural genetic variation on gene expression, demonstrate the use of enhancers as detectors of trans effects of genetic variation, and provide a new resource for understanding the impact of genetic variation on gene expression in Kupffer cells.

overall_design: RNA-seq of hepatic cells and BMDMs from three strains of mice (A/J, BALB/cJ, and C57BL/6J). RNA-seq of Kupffer cells in each strain after 30 minutes of AMLN control diet. RNA-seq from Kupffer cells derived from CB6F1/J hybrid mice and Nod-Scid mice transplanted with C57BL/6J or BALB/cJ bone marrow. RNA-seq and ATAC-seq from C57BL/6J, BALB/cJ, and CB6F1/J Kupffer cells and BMDMs treated with LPS. ATAC-seq and H3K27Ac ChIP-seq from Kupffer cells derived from A/J, BALB/cJ, C57BL/6J, and CB6F1/J mice.

first: Christopher

last: Glass


title: Effect of markerless deletion of ptsP from P. aeruginosa PA14 biofilm colonies on M6301-glycerol agar for 3 d at 25C

city: Stillwater

first: Matthew

last: Cabeen

state: Oklahoma

address: 307 Life Science East

department: Microbiology and Molecular Genetics

institute: Oklahoma State University

summary: To determine the transcriptomic effect when ptsP, which encodes enzyme I of the nitrogen-related phosphotransfer system (Nitro-PTS), is absent from cells. Deletion of this gene results in lower exopolysaccharide levels (a biofilm defect). Here, we compared PA14 deleted for amrZ (which has a moderately hyper-biofilm phenotype) and cells deleted for both amrZ and ptsP. We also analyzed wild-type PA14 cells (see GSE226104).

overall_design:
Total RNA was extracted from mechanically dispersed colonies grown on M6301-1% agar for 3 d at 25C. Transcriptomes were compared among PA14, PA14 delta-amrZ (raw data for these samples are deposited under GSE226104), and PA14 delta-amrZ delta-ptsP.
GSE226104/PRJNA938508 re-analyzed samples:
GSM7063834  PA14 delta-amrZ cells, biol rep 1  SRX19499901  SAMN33439943
GSM7063835  PA14 delta-amrZ cells, biol rep 2  SRX19499902  SAMN33439942
GSM7063836  PA14 delta-amrZ cells, biol rep 3  SRX19499903  SAMN33439941
GSM7063837  PA14 delta-amrZ cells, biol rep 4  SRX19499904  SAMN33439940

first: Somalisa

last: Pan

middle: T


title: Discrimination of cell-intrinsic and environment-dependent effects of natural genetic variation on Kupffer cell epigenomes and transcriptomes [RNA-Seq]


title: Discrimination of cell-intrinsic and environment-dependent effects of natural genetic variation on Kupffer cell epigenomes and transcriptomes [ATAC-Seq]


title: The Trophoblast Compartment Helps Maintain Embryonic Pluripotency and Delays Differentiation towards Cardiomyocytes

city: Calgary

first: Myriam

last: Hemberger

state: Alberta

address: 3330 Hospital Dr NW

institute: University of Calgary

summary: Normal developmental progression relies on a close crosstalk between the embryonic and extraembryonic lineages in the pre- and peri-gastrulation conceptus. This has been demonstrated in the mouse where epiblast-derived FGF and NODAL signals are required to maintain a stem-like state in trophoblast cells of the extraembryonic ectoderm, while visceral endoderm signals are pivotal to pattern the anterior region of the epiblast. These developmental stages also coincide with the specification of the first heart precursors. Here, we established a robust differentiation protocol of mouse embryonic stem cells (ESCs) into cardiomyocytes that we used to test the impact of trophoblast on this key developmental process. Using trophoblast stem cells (TSCs) to produce trophoblast-conditioned medium (TCM), we show that TCM profoundly slows down the cardiomyocyte differentiation dynamics and specifically delays the emergence of cardiac mesoderm progenitors. TCM also strongly promotes the retention of pluripotency transcription factors. By applying TCM from various mutant TSCs, we further show that specifically those mutations that cause a trophoblast-mediated effect on early heart development alter the normal cardiomyocyte differentiation trajectory. Our approaches provide a meaningful deconstruction of the intricate crosstalk between the embryonic and the extraembryonic compartments. They demonstrate that trophoblast helps prolong a pluripotent state in embryonic cells by delaying early differentiative processes, likely through secretion of leukemia inhibitory factor (LIF). These data expand our knowledge of the multifaceted signalling interactions between distinct compartments of the early conceptus that ensure normal embryogenesis, insights that will also be of great importance for the field of synthetic embryo research.

overall_design: RNA-seq of mouse EBs differnitated in standard base media or media conditioned by mTSCs for 4 or 8 days (N=3 for each condition at each timepoint). Every sample was sequenced in two lanes.

first: Xiang

last: Zhao

first: Bethany

middle: N

last: Radford


title: Transcriptome changes reveal the toxic mechanism of cadmium and lead combined exposure on silk production and web-weaving behavior of spider A. ventricosus

status: Public on Aug 15 2023

city: changsha

first: Huilin

middle: huilin

last: Yang

state: huannan

address: Donghu Street

country: China

institute: Hunan Agricultural University

summary: The goals of this study are to compare NGS-derived transcriptome of ampullate gland and cerebral ganglia under single and combined exposure to cadmium (Cd) and lead (Pb). The upregulated ampullate spidroin genes, downregulated protein anabolic genes, upregulated Far genes, and downregulated TUBA genes exhibit promising potential as biomarkers for silk protein synthesis and cellular degeneration, respectively. Moreover, the overexpression of AChE, Glu, and Unc13a genes may induce crucial neurotransmitters. This study offers an enormous capability for comprehensively understanding of the eco-toxicological effects and mechanisms of multi-heavy metals pollution.

overall_design: The mRNA profiles of ampullate gland and cerebral ganglia under single and combined exposure of Cd and Pb for 30-days were generated by deep sequencing, in triplicate, using Illumina NovaSeq.


title: DUX4 expression in cancer induces a metastable early embryonic totipotent program

city: Seattle

first: Stephen

last: Tapscott

state: Washington

address: 1100 Fairview Ave N, Room C3-123

institute: Fred Hutchinson Cancer Research Center

summary: The transcription factor DUX4 regulates a portion of the zygotic gene activation (ZGA) program in the early embryo. Many cancers express DUX4 but it is unknown whether this generates cells similar to early embryonic stem cells. Here we identify cancer cell lines that express DUX4 and show that DUX4 is transiently expressed in a small subset of the cells. DUX4 expression activates the ZGA transcriptional program, the subsequent 8C-like program, and markers of early embryonic lineages; while suppressing steady-state and interferon-induced MHC class I expression. Although DUX4 was expressed in a small number of cells under standard culture conditions, DNA damage or changes in growth conditions dramatically increased the fraction of cells expressing DUX4 and its downstream programs. Demonstrating that the transient expression of endogenous DUX4 in cancer cells induces a metastable early embryonic stem cell program and suppresses antigen presentation has implications for cancer growth, progression, and immune evasion.

overall_design: 10X single cell RNA-seq of KLE and SuSa cancer cell lines at log phase and 24, 48, and 72hrs after starvation release.

first: Sean

first: Andrew

middle: A

last: Smith


title: Mass production of lumenogenic human embryoid bodies and functional cardiospheres using in-air-generated microcapsules

city: Nijmegen

first: Rebecca

middle: Regina

last: Snabel

state: Gelderland

address: Geert Grooteplein 26-28

department: Molecular Developmental Biology

country: Netherlands

institute: RIMLS

summary: Organoids are engineered 3D miniature tissues that are defined by their organ-like structures, which drive novel fundamental understanding of human development and can revolutionize our healthcare system. However, current organoid generation methods associate with low production throughputs and poor control over size and function due to merging, which limits their clinical and industrial translation. Here, we present an innovative microfluidic platform for mass production of functional organoids. Specifically, triple-jet In Air microfluidics (IAMF) enables ultra-high throughput generation of hollow, thin-shelled, hydrogel microcapsules that can act as organoid forming bioreactors in a cytocompatible, oil-free, and size-controlled manner. IAMF microencapsulation of human pluripotent stem cells (hPSCs) allowed for long-term stable 3D suspension culturing and self-organizing hPSC spheroids with maintained pluripotency. Uniquely, IAMF microcapsules provided a lumenogenic microenvironment with near 100% efficient cavitation of hPSC spheroids resembling embryoid body’s rosette-stage; autonomous self-organization with polarized cytoarchitecture was achieved without added growth factors. Upon chemical stimulation, hPSC-derived spheroids underwent cardiomyogenic differentiation effectively resulting in mass production of a homogeneous and functional (i.e., contracting) cardiospheres that were responsive to external electrical stimulation. In short, we report on an innovative method that enables efficient production of high-quality organized spheroids, embryoid bodies, and organoids in a cytocompatible, ultra-high-throughput, and clean manner, thereby driving clinical and industrial adaption of stem cell technology in application areas such as tissue engineering, drug testing, and cultured meat.

overall_design: Comparative scRNA-seq analysis of encapsulated embryoid bodies and cardiospheres tissues and control (non-encapsulated) embryoid body tissues.

first: Bas

last: van Loo

first: Simone

last: ten Den

first: Nuno

last: Araújo-Gomes

first: Vincent

last: de Jong

first: Maik

last: Schot

first: José

last: Rivera-Arbeláez

first: GertJan

last: Veenstra

first: Robert

last: Passier

first: Tom

last: Kamperman

first: Jeroen

last: Leijten


title: LKB1 inhibition in alveolar epithelial cells induces fibroblast activation in pulmonary fibrosis

city: Southampton

first: Yihua

last: Wang

address: University Road

country: United Kingdom

institute: University of Southampton


title: LKB1 inhibition in alveolar epithelial cells induces fibroblast activation in pulmonary fibrosis [RNA-seq in 3D co-cultured ATII+MRC5]

summary: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, irreversible, and lethal lung disease. The initiation of IPF involves microinjuries to and/or dysfunction of the alveolar epithelium, but factors that determine fibrosis progression or normal tissue repair are largely unknown. We previously demonstrated that autophagy inhibition-mediated epithelial-mesenchymal transition (EMT) in human alveolar epithelial type II (ATII) cells augments local myofibroblast differentiation in pulmonary fibrosis by paracrine signalling. Here, we report that liver kinase B1 (LKB1) inactivation in ATII cells induces autophagy inhibition and EMT as a consequence. In IPF lungs, this is caused by a downregulation of CAB39L, a key subunit within the LKB1 complex. 3D co-cultures of ATII cells and lung fibroblast MRC5 coupled with RNA sequencing (RNA-seq) confirmed that paracrine signalling between LKB1-depleted ATII cells and fibroblasts augmented myofibroblast differentiation. Together these data suggest that reduced autophagy caused by LKB1 inhibition can induce EMT in ATII cells and contribute to fibrosis via aberrant epithelial–fibroblast crosstalk.

overall_design: 3D co-cultures of LKB1-depleted or control ATII cells with lung fibroblast MRC5

first: Elizabeth

last: Davies

first: Zijian

last: Xu

first: Yilu

last: Zhou


title: A simplified platform to generate and purify human primordial germ cells in vitro provides new insights into germline specification [scRNA-Seq]

city: Stanford

first: Sivakamasundari

last: Vijayakumar

address: 265 Campus Drive, Lokey Stem Cell Building, Rm G3105

department: Institute for Stem Cell Biology and Regenerative Medicine

institute: Stanford University

summary: Generating primordial germ cells (PGCs) from human pluripotent stem cells (hPSCs) advances studies of human reproduction and development of infertility treatments, but currently entails complex 3D aggregates. Here we develop a simplified, monolayer method to differentiate hPSCs into PGCs within 3.5 days, with higher efficiencies and improved consistency across multiple hPSC lines. We used our simplified differentiation platform and single-cell RNA-sequencing to uncover new insights into PGC specification. Transient WNT activation for 12 hours followed by WNT inhibition specified PGCs; by contrast, sustained WNT instead induced primitive streak. Thus, somatic (primitive streak) and PGCs are related—yet distinct—lineages segregated by temporally-dynamic signaling. Pluripotency factors are continuously expressed during the transition from pluripotency to posterior epiblast to PGCs, thus bridging pluripotent and germline states. Finally, hPSC-derived PGCs can be easily purified by virtue of their CXCR4+PDGFRA-GARP- surface-marker profile and single-cell RNA-sequencing reveals that they harbor strong transcriptional similarities with fetal PGCs. We report single-cell RNA-sequencing data of in vitro differentiated hPSCs (H9) cells into human Primordial germ cell-like cells (PGCLCs), profiled at different time-points of differentiation - Day 0 (H9), D0.5, D3.5 (sorted) and D3.5 unsorted population and a negative control of hPSCs differentiated into definitive endoderm (DE) (using the protocol described in (Loh et al., 2014, Cell Stem Cell 14, 237-252)). The data comprises of 5 single-cell RNA-sequencing libraries (H9, D0.5, D3.5S, D3.5U and DE) generated using 10xChromium Single cell RNA-expression platform and 10xGenomics Chromium version 2 chemistry with a targeted capture of ~6000 cells each. Single cell libraries were sequenced on Illumina HiSeq4000 platform as paired-end 150bp reads.

overall_design: single-cell RNA-sequencing of hPSCs (H9) cells differentiated into human Primordial germ cell-like cells (PGCLCs) using our simplified monolayer serum-free differentiation protocol was performed for the following time-points: Day 0 (H9), Day 0.5 (D05), Day 3.5 unsorted (D35U), Day 3.5 sorted for CXCR4+ PDGFRα- GARP-  (D35S). As a negative control, hPSCs were differentiated into Definitive endoderm (day 2) (DE) were profiled as well.

first: Gugene

last: Kang

first: Roberta

last: Sala

first: Angela

last: Chen

first: Abidemi

middle: I

last: Adebayo

first: Andrea

last: Cipriano

first: Jonas

middle: L

last: Fowler

first: Lay

last: Ang

first: Kyle

last: Loh

first: Vittorio

last: Sebastiano


title: The small RNA component of Arabidopsis phloem sap and its response to iron deficiency.

status: Public on Aug 08 2023

city: Amherst

first: Elsbeth

last: Walker

state: MA

address: 611 N Pleasant St Morrill Science Center IV South

department: Biology

institute: University of Massachusetts Amherst

type: Non-coding RNA profiling by high throughput sequencing

summary: We performed small RNA-seq (sRNA-seq) study of Arabidopsis phloem sap under iron-sufficient (+Fe; control) and iron deficient (-Fe)  conditions to investigate and identify sRNAs whose expression is regulated by iron deficiency in the phloem sap.

overall_design: Comparative small RNA expression profiling analysis of sRNA-seq data for Col-0 Arabidopsis phloem sap under Fe deficiency and control conditions

first: Ahmet

last: Bakirbas

first: Rosario

last: Castro-Rodriguez


title: Gene Expression Changes in Sickle Cell Reticulocytes and their Clinical Associations

city: Chicago

first: Xu

last: Zhang

state: Illinois

address: 820 South Wood St

institute: University of Illinois at Chicago

summary: Transcriptional changes in compensatory erythropoiesis in sickle cell anemia (SCA) and their disease modulation are unclear. We detected 1226 differentially expressed genes in hemoglobin SS reticulocytes compared to non-anemic hemoglobin AA controls. Assessing developmental expression changes in hemoglobin AA erythroblasts for these genes suggests heightened terminal differentiation in early erythroblasts in SCA that diminishes toward the polychromatic to orthochromatic stage transition. Comparison of reticulocyte gene expression changes in SCA with that in Chuvash erythrocytosis, a non-anemic disorder of increased erythropoiesis due to constitutive activation of hypoxia inducible factors, identified 453 SCA-specific changes attributable to compensatory erythropoiesis. Peripheral blood mononuclear cells (PBMCs) in SCA contain elevated proportions of erythroid progenitors due to heightened erythropoiesis. Deconvolution analysis in PBMCs from 131 SCA patients detected 54 genes whose erythroid expression correlated with erythropoiesis efficiency, which were enriched with SCA-specific changes (OR=2.9, P=0.00063) and annotation keywords of “ubiquitin-dependent protein catabolic process” and “protein ubiquitination” (OR=4.1, P=9.6×10-5). An erythroid expression quantitative trait locus of one of these genes, LNX2 encoding an E3 ubiquitin ligase, associated with severe pain episodes in 774 SCA patients (OR=1.7, P=3.9×10-5). Thus, erythroid gene transcription responds to unique conditions within SCA erythroblasts and these changes potentially correspond to vaso-occlusive manifestations.

overall_design: Five HbSS patients and Five age- and gender-matched healthy HbAA African American control individuals

first: Binal

last: Shah

first: Taif

last: Hassan

first: Victor

last: Gordeuk


title: Global transcriptional landscape of CtBP2 in pancreatic beta cells

city: Tsukuba

first: Motohiro

last: Sekiya

address: 1-1-1 Tennodai

institute: University of Tsukuba

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

overall_design: Refer to individual Series


title: N4-acetylcytidine-dependent GLMP mRNA stabilization by NAT10 promotes head and neck squamous cell carcinoma metastasis and remodels tumor microenvironment through MAPK/ERK signaling pathway

city: Shanghai

first: Pin

last: Dong

address: No. 650, New Songjiang Road, Shanghai

institute: Shanghai General Hospital, Shangahi Jiao Tong University School of Medicine

summary: N4-acetylcytidine (ac4C) is a posttransciptional RNA modification regulating in various important bioprocess. However, the role in human cancer, especially lymph node metastasis, remains largely unknown. Here, we showed NAT10, as the only “writer” of ac4C mRNA modification, was highly expressed in HNSCC patients with lymph node metastasis. High NAT10 levels in lymph node of HNSCC patients predicted poor overall survival. Moreover, we found the high expression of NAT10 was positively upregulated by NRF1 transcription factor. Gain and loss-of-function displayed that NAT10 promoted cell metastasis in mice. Mechanistically, NAT10 induced ac4C modification of GLMP and stabilized its mRNA, which triggered the activation of MAPK/ERK signaling pathway. Lastly, the NAT10’s specific inhibitor Remodelin could inhibit HNSCC tumorigenesis via 4-NQO-induced murine tumor model and remodel the tumor microenvironment, including angiogenesis, CD8+ T cell and Treg recruitment. These results demonstrated that NAT10 promoted lymph node metastasis of HNSCC via ac4C-dependent stabilization of GLMP transcript, providing a potential epitranscriptomic-targeted therapeutic strategy for HNSCC.

overall_design: The expression and overall survival (OS) probability of NAT10 in patients with HNSCC metastases were analyzed with the GEPIA website tool and our 43 clinical samples, respectively.  Gain-of-function and loss-of-function methods were envolved and a popliteal lymphatic metastasis model was constructed to investigate the functional roles of NAT10 on HNSCC cell metastasis in vitro and in vivo. The transcription factors regulated upstream of NAT10 were explored via three public website online tools. RIP, acRIP, luciferase reporter and mRNA stabilization experiments were performed to explore the epigenetic mechanisms of NAT10 in HNSCC. A 4-NQO-induced murine tumor model was further constructed to validate the effect of the inhibitor Remodelin on tumors and the tumor microenvironment.

first: Yuanyuan

last: Liu

first: Xing

last: Wang

first: Yuying

first: Jianqiang

last: Yang

first: Wei

last: Mao

first: Chen

last: Feng

first: Xiaoliang

last: Wu

first: Xinwei

first: Lixiao


title: Dasatinib Resistant K562 cell lines

city: Adelaide

first: Benjamin

middle: Chia Sing

last: Leow

state: South Australia

address: SAHMRI North Terrace

department: Cancer Theme

institute: SAHMRI

summary: A project to explore the acquisition of drug resistance mechanisms in a K562 erythroblastic leukemia cell line, with induced resistance to the tyrosine kinase inhibitor, dasatinib.

overall_design: Cells were exposed to increasing concentrations of dasatinib over ~11 months, up to a maximum dose of 200 nM dasatinib. Sequencing was performed in a selection of dasatinib dose escalation intermediates, ranging from 0 nM to 200 nM dsasatinib. Sequencing libraries were prepared using the NEXTFlex Rapid Directional RNA Seq Library Prep Kit (Perkin Elmer, Inc., Waltham, MA), depleting ribosomal and non-coding RNA. Quality control of RNA was performed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), using the Bioanalyzer RNA 6000 Nano assay. Libraries were sequenced on the Illumina MiSeq instrument (Illumina Inc., San Diego, CA), using a 150 cycle to produce 2x75 bp paired end reads. Sequencing data was uploaded to the Illumina BaseSpace Sequencing Hub (Illumina Inc.), which was used for FastQC quality control. Results were used for differential gene expression analysis (EdgeR).

first: Chung

last: Kok


title: p53-dependent R-loop formation and HPV pathogenesis

first: Conor

last: Templeton

address: 300 E Superior Street

institute: Northwestern University

summary: R-loops are trimeric RNA: DNA hybrids that are important physiological regulators of transcription; however, their aberrant formation or turnover leads to genomic instability and DNA breaks. High-risk human papillomaviruses are the causative agents of genital as well as oropharyngeal cancers and exhibit enhanced amounts of DNA breaks. The levels of R-loops were found to be increased up to 50-fold in cells that maintain high-risk HPV genomes and were readily detected in squamous cell cervical carcinomas in vivo but not in normal cells. The high levels of R-loops in HPV positive cells were present on both viral and cellular sites together with RNase H1, an enzyme that controls their resolution. Depletion of RNase H1 in HPV positive cells further increased R-loop levels, resulting in impaired viral transcription and replication and reduced expression of DNA repair genes such as FANCD2 and ATR, which are necessary for viral functions.  Overexpression of RNase H1 decreased total R-loop levels, reducing DNA breaks by over 50%. Furthermore, increased RNase H1 expression leads to reduced R-loop levels, blocking viral transcription and replication while enhancing the expression of factors in the innate immune regulatory pathway. This suggests that maintaining elevated R-loop levels is important for the HPV life cycle. The E6 viral oncoprotein was found to be responsible for inducing high levels of R-loops by inhibiting p53’s transcriptional activity. Our studies indicate that high R-loop levels are critical for HPV pathogenesis and that this depends on suppressing the p53 pathway.

overall_design: CIN 612 cells (keratinocytes isolated from a cervical biopsy that stably maintain the HPV 31 genome) were transduced with lentivirus to express either a scrambled sequence (Samples 1/2), shRNA targeting Rnase H1 (Samples 3/4), or a cassette expressing a GFP-tagged RNase H1 (Samples 5/6). RNA sequencing was performed on each of these samples and the scramble control CIN 612 cells were compared to CIN 612 cells either depleted of RNase H1 or overexpressing RNase H1.

first: Laimonis

last: Laimins


title: Guanosine-7 tRNA methylation steers tRNA-derived fragment biogenesis and translational control in prostate cancer

city: St Louis

first: Sabine

last: Dietmann

state: MO

address: 4565 McKinley Avenue

institute: Washington University School of Medicine

summary:
Abstract
Transfer RNAs (tRNAs) are exceptionally subject to modifications, including methylation. While mRNA methylation is emerging as an important regulator of biological and pathological processes in cancer, how post-transcriptional methylation of tRNAs contributes to cancer is largely unknown. Here we show that the RNA N7-methylguanosine (m7G) methyltransferase METTL1 is highly differentially expressed in prostate cancer compared to non-tumour prostate tissues. METTL1 expression regulation is mediated by the oncogenic regulator PI3K, which is altered in most advanced prostate tumours. Knockdown of METTL1 dramatically inhibits prostate cancer cell growth and tumour progression in vivo. In contrast, overexpression of the wild type but not the catalytically inactive METTL1 potentiates cell growth. Thus, METTL1-mediated methylation is important for prostate tumorigenesis. Mechanistically we find that METTL1 depletion causes loss of m7G tRNA methylation and increases endonucleolytic cleavage of Cysteine tRNA leading to an accumulation of 5′ tRNA-derived small RNA fragments. 5′ tRNA-derived fragments steer translation control to favour synthesis of key regulators of tumour growth suppression and immune rejection. In summary, our findings uncover a critical function of m7G tRNA methylation in directing translation control in cancer cells with important implications for tumour growth and unveil METTL1 inhibition as a promising anti-cancer therapeutic strategy.
induction and maintenance of naïve human pluripotency are governed by distinct signaling requirements.
tRNAs from WT and METTL1 KO cells were subjected to NaBH4-Aniline treatment followed by RNA-seq to unveil methylation of guanosine-7 in tRNA with nucleotie resolution. RNA-seq libraries were also analysed to unveil tRNA stability or processing into tRNA-derived fragments in METTL1 KO cells.

overall_design: Samll RNA from prostate cancer cells PC3 were extrated. Cells were either control CRIPSR (WT) or METTL1KO (using CRISPR). RNA were either non chemically treated (non-treated NT) or treated wih NaBH4 and aniline. Three replicates were used per condition. A total of 12 samples were sequenced.

first: Sandra

last: Blanco

first: Raquel

last: García-Vílchez


title: The global anaerobic metabolism regulator fnr is necessary for the degradation of food dyes and drugs by Escherichia coli

city: San Francisco

first: Peter

last: Turnbaugh

address: 513 Parnassus Ave, HSW 1501

institute: University of California, San Francisco

summary: The microbiome is an underappreciated contributor to intestinal drug metabolism with broad implications for drug efficacy and toxicity. While considerable progress has been made towards identifying the gut bacterial genes and enzymes involved, the role of environmental factors in shaping their activity remains poorly understood. Here, we focus on the gut bacterial reduction of azo bonds (R-N=N-R’), found in diverse chemicals in both food and drugs. Surprisingly, the canonical azoR gene in Escherichia coli was dispensable for azo bond reduction. Instead, azo reductase activity was controlled by the fumarate and nitrate reduction (fnr) regulator, consistent with a requirement for the anoxic conditions found within the gastrointestinal tract. Paired transcriptomic and proteomic analysis of the fnr regulon revealed that in addition to altering the expression of multiple reductases, FNR is necessary for the metabolism of L-Cysteine to hydrogen sulfide, enabling the degradation of azo bonds. Furthermore, we found that FNR indirectly regulates this process though the small non-coding regulatory RNA fnrS. Taken together, these results show how gut bacteria sense and respond to their intestinal environment to enable the metabolism of chemical groups found in both dietary and pharmaceutical compounds.

overall_design: We sought to investigate the transcriptomic response of E. coli to the presence of FD&C Red No. 40. We grew wt and Δfnr E. coli BW25113 in LB media with 0.05% L-Cysteine and inoculated the cultures with 250 µM FD&C Red No. 40 or DMSO vehicle at either mid-exponential or stationary growth phases . After a 40 minute incubation period, samples were collected and split for paired transcriptomics (RNA-seq) and proteomics.

first: Lindsey

last: Pieper

last: Spanogiannopoulos

first: Regan

last: Volk

first: Carson

last: Miller

first: Aaron

last: Wright


title: Using combined single-cell gene expression, TCR sequencing and cell surface protein barcoding to characterize and track CD4 T cell clones from murine tissues

city: Mainz

first: Michael

last: Delacher

state: Rhineland-Palatinate

address: Langenbeckstrasse 1

department: Institute for Immunology

country: Germany

institute: University Medical Center of the Johannes Gutenberg-University Mainz

summary: Single-cell gene expression analysis using sequencing (scRNA-seq) has gained increased attention in the past decades for studying cellular transcriptional programs and their heterogeneity in an unbiased manner, and novel protocols allow the simultaneous measurement of gene expression, T-cell receptor clonality and cell surface protein expression. In this article, we describe the methods to isolate scRNA/TCR-seq-compatible CD4+ T cells from murine tissues, such as skin, spleen, and lymph nodes. We describe the processing of cells and quality control parameters during library preparation, protocols for multiplexing of samples, and strategies for sequencing. Moreover, we describe a step-by-step bioinformatic analysis pipeline from sequencing data generated using these protocols. This includes quality control, preprocessing of sequencing data and demultiplexing of individual samples. We perform quantification of gene expression and extraction of T-cell receptor alpha and beta chain sequences, followed by quality control and doublet detection, and methods for harmonization and integration of datasets. Next, we describe the identification of highly variable genes and dimensionality reduction, clustering and pseudotemporal ordering of data, and we demonstrate how to visualize the results with interactive and reproducible dashboards. We will combine different analytic R-based frameworks such as Bioconductor and Seurat, illustrating how these can be interoperable to optimally analyze scRNA/TCR-seq data of CD4+ T cells from murine tissues.

overall_design: We performed combined single-cell RNA/TCR-sequencing of CD4 T cells from tissues of an individual mouse.

first: Annekathrin

middle: S

last: Nedwed

first: Sara

last: Helbich

first: Kathrin

last: Braband

first: Federico

last: Marini

last: Volkmar


title: LKB1 inhibition in alveolar epithelial cells induces fibroblast activation in pulmonary fibrosis [RNA-seq in 2D-cultured ATII]

first: Yihua

last: Wang

overall_design: Alveolar epithelial type II (ATII) cells were transfected with siRNA_LKB1 (STK11)  or siRNA control for 72 hours.

first: Zijian

last: Xu

first: Liudi

last: Yao

first: Yilu

last: Zhou


title: Dynamic changes of mRNA expression during development of human embryonic stem cells derived cardiomyocytes

status: Public on Aug 07 2023

city: Hong Kong

first: Ellen

middle: Ngar-Yun

last: Poon

state: Hong Kong

address: Shatin

department: School of Biomedical Sciences

institute: The Chinese University of Hong Kong

summary: To dissect complex regulatory mechanisms on cardiac development, we performed the time-series gene expression experiments of human embryonic stem cells (ESCs) differentiated to cardiomyocytes (CMs). The RNA-sequencing at 4 stages of human to CM maturation, representing timepoints with days 15, 30, 45 and 60 of ESC-CMs. The early CMs from differentiation days (D) 0-15 are commonly considered to be immature and embryonic-like. We define late CMs after D15. During this phase, the cells stop dividing and undergo some degree of maturation. This study established distinct dynamic expression patterns of mRNAs/genes and identified several subsets of up- or down-regulated genes with similar expression patterns during CM differentiation and maturation.

overall_design: The RNA-sequencing of mRNA levels were conducted at 4 stages (15, 30, 45 and 60 days, +/- 2 days) of human H7 ESC-directed CM differentiation.  Each stage included 3 biological replicates.

first: Ellen Ngar-Yun

first: Kenneth

last: Boheler


title: RNA-seq analysis of sorted colon epithelial cells and colon lamia propria macrophages from mice where Tsc2 is deleted in Lyz2-expressing cells

city: Vienna

first: Thomas

last: Weichhart

address: Waehringerstraße 10

department: Center for Pathobiochemistry and Genetics

country: Austria

institute: Medical University of Vienna

summary: The intestinal epithelium has a high turnover and constantly renews itself through proliferation of intestinal crypt cells, which depends on insufficiently characterized signals from the microenvironment. Here we show that colonic macrophages were located directly adjacent to epithelial crypt cells in mice, where they metabolically supported epithelial cell proliferation in an mTORC1-dependent manner. Specifically, mTORC1 activation in macrophages protected against colitis-induced intestinal damage and induced the synthesis of the polyamines spermidine and spermine. Epithelial cells ingested these polyamines and rewired their cellular metabolism for optimizing proliferation and defense. Notably, spermine directly stimulated proliferation of colon epithelial cells and colon organoids. Genetic interference with polyamine production in macrophages altered global polyamine levels in the colon and modified epithelial cell proliferation. Our results suggest that macrophages act as “commensals” that provide metabolic support to promote efficient self-renewal of the colon epithelium.

overall_design: Colon lamina propria macrophages (Cd45+, Ly6G-, SiglecF-, Cd24-, Cd11c+/-, Cd103-, Cd11b+, Ly6C-, Cd64+, MHCII+) and Cd44+ epithelial cells (Cd45-, EpCAM+, Cd44+) from 3 Tsc2fl/fl and 4 Tsc2Lyz2-cre mice were isolated as per manufacturer’s instructions (MACS Miltenyi Biotec, 130-097-410) and FACS sorted at the FACS Core Unit of the Children’s Cancer Research Institute (CCRI) of Vienna. Freshly sorted cells were harvested in RLT-Buffer (RNeasy RNA isolation kits, QIAGEN) containing ß-mercaptoethanol (Sigma) and stored at -80 °C. RNA was isolated using an RNeasy Micro Kit (QIAGEN, Cat No.ID: 74004).

first: Stephanie

last: Fritsch

first: Nyamdelger

last: Sukhbaatar

last: Schuster


title: single nucleus RNA-seq of early development S. purpuratus

city: PITTSBURGH

first: Hinman

last: Veronica

state: PA

address: 587 S Negley Ave, Apt 17

department: Biological Sciences

institute: Carnegie Mellon University

summary: To explore the evolution of different cell types across species, we compared transcriptomes between different cell types between two echinoderm species, P. miniata and S. purpuratus. snRNA-seq was used to analyze to characterize diffente cell states. For each species, the time points were integrated into a common atlas and annotated. The fully processed and annotated P. miniata and S. purpuratus atlases are available as supplementary files.

overall_design: Fresh nuclei were isolated from embryonic cultures and analyzed using snRNA-seq. Two datasets were produced- an exploratory S. purpuratus dataset that was used to validate snRNA-seq methods, and a P. miniata dataset

first: Anne

last: Meyer

first: Carolyn

last: Ku

first: Cheryl

last: Telmer

first: William

last: Hatleberg

first: Veronica

last: Hinman


title: single nucleus RNA-seq of early development in P. miniata


title: The IGF2BP3-COPS7B axis facilitates mRNA translation to drive colorectal cancer progression

city: Harbin

first: Jing

last: Tang

state: Heilongjiang

address: No. 157 Baojian Road, Nangang District

institute: Harbin medical university

summary: Many studies have provided valuable information about genomic and transcriptomic changes that occur in colorectal cancer (CRC). However, protein abundance cannot be reliably predicted by DNA alteration or mRNA expression, which can be partially attributed to post-transcriptional and/or translational regulation of gene expression. In this study, we identified increased translational efficiency (TE) as a hallmark of CRC by evaluating the transcriptomic and proteomic features of CRC patients, along with comparative transcriptomic and ribosome-protected mRNA analysis in colon epithelial cells and colon cancer cells. COPS7B was among the key genes that consistently showed both significant TE increase and protein elevation without transcriptional alteration in CRC. IGF2BP3 enhanced the TE of COPS7B mRNA to promote CRC growth and metastasis. COPS7B was found to be a component of the ribo-interactome that interacted with ribosomes to facilitate ribosome biogenesis and mRNA translation initiation. Collectively, this study revealed the proteomic features of CRC and highlighted elevated mRNA translation as a hallmark of CRC. The identification of the  IGF2BP3-COPS7B axis underlying the increased protein synthesis rate in CRC provided a  promising therapeutic target to treat this aggressive disease.

overall_design: To investigate the TE changes of global mRNAs in three colon cancer cell lines (HCT116, HCT15 and SW620) compared with colon epithelial cells (841-CON) respectively through Ribo-seq

first: Shuoshuo

first: Rui

last: Su

first: Xiaobo

last: Li


title: Glutamine metabolism controls amphiregulin-facilitated chemoresistance to cisplatin in human chondrosarcoma

city: Taichung

first: Chih-Hsin

address: 91 Xueshi

country: Taiwan

institute: China Medical University

summary: Chondrosarcoma is the second most common type of bone cancer. At present, the most effective clinical course of action is surgical resection. Cisplatin is the chemotherapeutic medication most widely used for the treatment of chondrosarcoma; however, its effectiveness is severely hampered by drug resistance. In the current study, we compared cisplatin-resistant chondrosarcoma SW1353 cells with their parental cells via RNA sequencing. Our analysis revealed that glutamine metabolism is highly activated in resistant cells but glucose metabolism is not. Amphiregulin (AR), a ligand of the epidermal growth factor receptor, enhances glutamine metabolism and supports cisplatin resistance in human chondrosarcoma by promoting NADPH production and inhibiting ROS accumulation. The MEK, ERK, and NrF2 signaling pathways were shown to regulate AR-mediated SLC1A5 and GLS expression as well as glutamine metabolism in cisplatin-resistant chondrosarcoma. The knockdown of AR expression in cisplatin-resistant chondrosarcoma cells was shown to reduce the expression of SLC1A5 and GLS in vivo. These results indicate that AR and glutamine metabolism are worth pursuing as therapeutic targets in dealing with cisplatin-resistant human chondrosarcoma.

overall_design: To identify the metabolic processes underlying chondrosarcoma cisplatin resistance. We established the chondrosarcoma cisplatin resistance cell line and performed RNA seq.


title: Astrocyte-targeting therapy rescues cognitive impairment caused by neuroinflammation via the Nrf2 pathway

city: Kyoto

first: Andres

last: Canela

state: Kyoto

address: Yoshida-Konoe-cho, Sakyo-ku, Kyoto, Kyoto University

department: The Hakubi Center for Advanced Research / Radiation Biology Center

institute: Kyoto University


title: MIN6 beta cell CtBP2 ChIP-seq

first: Motohiro

last: Sekiya

summary: CtBP2 ChIP-seq analysis was performed in a murine pancreatic beta cell line MIN6.

overall_design: ChIP-seq with anti-CtBP2 antibody (Active Motif 61261) was performed using MIN6 cells in a regular culture condition.


title: Astrocyte-targeting therapy rescues cognitive impairment caused by neuroinflammation via the Nrf2 pathway ( nascent RNA-Seq )

summary: Neuroinflammation is a common feature of neurodegenerative disorders such as Alzheimer's disease (AD). Neuroinflammation is induced by dysregulated glial activation, and astrocytes, the most abundant glial cells, become reactive upon neuroinflammatory cytokines released from microglia, and actively contribute to neuronal loss. Therefore, blocking reactive astrocyte functions is a viable strategy to manage neurodegenerative disorders. However, factors or therapeutics directly regulating astrocyte subtype remain unexplored. Here, we identified transcription factor NF-E2-related factor 2 (Nrf2) as a therapeutic target in neurotoxic reactive astrocytes upon neuroinflammation. We found that the absence of Nrf2 promoted the activation of reactive astrocytes in the brain tissue samples obtained from AD model 5xFAD mice, whereas enhanced Nrf2 expression blocked the induction of reactive astrocyte gene expression by counteracting NF-kB subunit p65 recruitment. Neuroinflammatory astrocytes robustly upregulated genes associated with type I interferon and the antigen-presenting pathway, which were suppressed by Nrf2 pathway activation. Moreover, impaired cognitive behaviors observed in AD mice were rescued upon ALGERNON2 treatment, which potentiated Nrf2 pathway and reduced the induction of neurotoxic reactive astrocytes. Thus, we highlight the potential of astrocyte-targeting therapy by promoting the Nrf2 pathway signaling for neuroinflammation-triggered neurodegeneration.

overall_design: Nascent RNA sequencing in primary astrocytes cultures treated with Sulforaphane (SLF) or DMSO, and then stimulated or not with a cytokine cocktail.

first: Akiko

last: Nakano-Kobayashi

first: Toru

last: Yoshihara

first: Masatoshi

last: Hagiwara


title: Astrocyte-targeting therapy rescues cognitive impairment caused by neuroinflammation via the Nrf2 pathway (ChIP-Seq)

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for p65 and NRF2 in primary astrocytes cultures treated with Sulforaphane (SLF) or DMSO, and then stimulated or not with a cytokine cocktail.


title: Single-cell RNA sequencing of  microglia under awake and anesthesia-state

city: Hangzhou

first: Kelei

last: Cao

address: 866 Yuhangtang Road

institute: Zhejiang University

summary: To probe potential  differences between awake and anesthesia microglia, we performed the single-cell RNA sequencing (scRNA-seq) . Microglia were isolated by FACS as CD11b high CD45 low cells from awake and anesthesia mouse brains.

first: Zhihua

last: Gao

first: Yaling

last: Hu

first: Weiyin

last: Wu


title: Evaluation of renal mononuclears cells in acute kidney injury after inhibiting retinoic acid signaling in proximal tubular epithelial cells

city: Nashville

first: Mark

last: de Caestecker

state: TN

address: 1161 21st Avenue South

institute: Vanderbilt University Medical Center

summary: Retinoic acid (RA) signaling is activted in proximal tubular epithelial cells (PTECs) after acute kidney injury (AKI). In these studies we evaluated the functional role of this by inducing ischemia reperfusion-induced AKI (IRI-AKI) in mice after inhibiting RA signaling in PTECs genetically. Here we evaluated the effects of this on the activation of renal mononuclear cells (MNCs) by evaluating RNA expression in CD11B+ cells isolated from kidneys after IRI-AKI. Compared with control mice, there was a reducion in expression of inflammatory marker gene expression 3 days after IRI-AKI in mice with inhibition of RAR signaling in PTECs. These findings suggest a mechanism by which inhibition of RAR signaling in PTECs is protective against AKI.

overall_design: PTEC DN-RAR mice (PEPCK CRE+; Rosa26-LSL-RARaT403X), and CRE- controls (PEPCK CRE-; Rosa26-LSL-RARaT403X) underwent bilateral IRI-AKI, and bulk RNA seq performed on renal CD11B+ cells isolated using CD11B antibody coated magnetic beads 3 days after injury.

last: de Castecker

first: Rachel

last: Delgado

first: Thitannee

last: Vanichapol

first: Alan

last: Davidson


title: Transcriptomics Analysis of Diploid and Tetraploid Worms Exposed to DNA-Damaging Chemotherapeutics

city: Charleston

first: Jessica

last: Hartman

state: South Carolina

address: 70 President St., Drug Discovery Bldg/Rm DD220

department: Biochemistry and Molecular Biology

institute: Medical University of South Carolina

summary: To establish the consequences of whole-animal tetraploidy, we generated stable C. elegans worm strains that are tetraploid in every cell in their body and exposed them to DNA damaging chemotherapeutics doxorubicin and cisplatin at doses that do not cause growth delay. Exposures were carried out for 48 h beginning at L1 stage.

overall_design: Samples include diploid and tetraploid ploidy groups, each exposed to vehicle (CTRL), 5 micromolar cisplatin (CIS), or 5 micromolar doxorubicin (DOX), in three biological replicates (1,2, and 3)

first: Kelly

last: Misare

first: Elizabeth

last: Ampolini

first: Hyland

middle: C

last: Gonzalez

first: Kaitlan

last: Sullivan

first: Xin

first: Camille

last: Miller

first: Bintou

last: Sosseh

first: Jaclyn

middle: B

last: Dunne

first: Christina

last: Voelkel-Johnson

first: Kacy

last: Gordon


title: CtBP2 beta cell KO islet RNA-seq

first: Motohiro

last: Sekiya

summary: RNA-seq analysis was performed using pancreatic islet samples from flox mice and beta-cell specific CtBP2 KO mice.

overall_design: Pancreatic beta cell specific CtBP2 KO mice were generated by crossing CtBP2 flox mice into Ins1-Cre transgenic mice. Pancreatic islets were isolated from flox and KO mice and the isolated RNAs were subjected to the RNA-seq analysis.


title: The EoE esophageal epithelial transcriptomic landscape

first: Marie-Pier

last: Tetreault

state: IL

address: 300 East Superior Street

department: Medicine/GI&Hep division

summary: We report alterations in the esophageal transcriptome of adult eosinophilic esophagitis (EoE) patients at a single-cell transcriptomic level.

overall_design: Examinationof 6 EoE patients and 6 healthy donors by scRNA-seq, with proximal and distal esophageal biopsies from each.

first: Tetreault

last: Marie-Pier

first: Clevenger

last: Margarette


title: snRNAseq in human fetal neocortex

city: Piscataway

first: Ronald

middle: P.

last: Hart

state: NJ

address: 604 Allison Rd Rm B430

department: Cell Biology & Neuroscience

institute: Rutgers University

summary: Abnormalities in neocortical and synaptic development have been associated with neurodevelopmental disorders. However, the molecular and cellular mechanisms regulating the formation of the initial synapses in an evolutionary advanced neocortical layer, the subplate (SP), are poorly understood. Our snRNAseq screen of human prefrontal neocortices from early (11/12 PCW), mid (14/15 PCW) to late (17/18 PCW) fetal developmental stages revealed the bipartite-to-tripartite differentiation of SP neuronal subclasses. Using polysome profiling with RNAseq, we report for the first time a set of mRNAs undergoing translational control in cellular subclasses of developing human prefrontal neocortices, including SP neurons. By examining both mouse and human neocortex, we further found that an autism spectrum disorder (ASD)-risk gene and RNA binding protein CUGBP Elav-Like Family Member 4 (CELF4) is selectively expressed in the neurons of two synapse-enriched compartments, the SP and the marginal zone. Furthermore, CELF4 binds mRNA targets that are encoded by the synaptic genes associated with ASD and adverse neurodevelopmental outcomes; albeit in an evolutionarily advanced fashion between mouse and human synaptic mRNAs. The selective forebrain Celf4 deletion from developing mouse cortical neurons disrupts the balance of SP synapses in a gender-specific fashion. Taken together, our results underscore the importance of RNA binding proteins and mRNA translation in evolutionarily advanced synaptic development, as well as their possible contribution to gender specific protein synthesis and vulnerability.

overall_design: Samples were prepared from fresh frozen human fetal neocortex at 11 to 18 PCW (post conception weeks).  Single nuclear RNA sequencing libraries were prepared using 10X Genomics Chromium technology and sequenced. The goal was to investigate regulation of translation by Celf4.

first: Iva

last: Salamon

first: Zeljka

last: Krsnik

first: Ivica

last: Kostovic

middle: P

first: Mladen-Roko

last: Rasin


title: Mouse Celf4 cKO fetal neocortex polysome fractions

overall_design: Samples prepared from neocortex of mouse wild type or Celf4 conditional knockout (cKO) collected at P0 were fractionated on sucrose gradients to yield polysomes, monosomes and unfractionated input.  The goal was to investigate regulation of translation by Celf4.


title: The role of mRNA translation in the synaptic development of prenatal mammalian neocortex

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Human fetal neocortex polysome fractions over development

overall_design: Samples prepared from neocortex of human 11-20 PCW (post conception weeks) were fractionated on sucrose gradients to yield polysomes, monosomes and unfractionated input.  The goal was to investigate regulation of translation over development.


title: Human CELF4 RNA immunoprecipitation (RIP)

overall_design: Samples were prepared from fresh frozen human fetal prefrontal lobe at 11 and 12 PCW (post conception weeks).  RNA-protein complexes were immunoprecipitated using either CELF4 antibody or control IgG. The goal was to investigate regulation of translation by Celf4.

first: Yongkyu

last: Park


title: Exercise Training Reduces the Inflammatory Response and Promotes Intestinal Mucosa-associated Immunity in Lynch Syndrome patients [GeoMx Digital Spatial Profiling (DSP)]

city: Houston

first: Eduardo

last: Vilar-Sanchez

state: TX

address: 1515 Holcombe Blvd

summary: This CYCling Lynch patients for Exercise and Prevention (CYCLE-P) trial was a prospective, non-randomized controlled trial conducted at the Familial High-Risk Gastrointestinal and Cancer Prevention Clinic of The University of Texas MD Anderson Cancer Center. Twenty-one LS participants with a confirmed pathogenic mutation in one of four DNA mismatch repair (MMR) genes were enrolled between April 2018 and January 2019. Follow-up was conducted 1-year from the start of the intervention. Statistical analysis by both per-protocol and intention-to-treat was performed from March to May 2021.

overall_design: CYCLE-P was a prospective, non-randomized controlled trial that enrolled 21 consecutive participants diagnosed with LS in a 12-month aerobic exercise intervention involving cycling classes in a local community (n=5) followed by enrollment of patients to usual care (n=3) between April 2018 to January 2019. All eligible participants provided written informed consent before enrollment, and the conduction of the trial complied with the Declaration of Helsinki. The trial protocol and all amendments were approved by the MD Anderson Cancer Center institutional review board. We followed the Transparent Reporting of Evaluations with Nonrandomized Designs (TREND) reporting guidelines.

last: Deng


title: Downregulation of TAB182 promotes cancer stem-like cell properties and therapeutic resistance in triple-negative breast cancer cells

city: Changchun

first: HUAN

last: HE

state: Jilin Province

address: No.1163 Xinmin Street

department: School of Public Health

institute: Jilin University

summary: TAB182 participates in DNA damage repair and radio-/chemosensitivity regulation in various tumors, but its role in tumorigenesis and therapeutic resistance in breast cancer remains unclear. In the current paper, we observed that triple-negative Breast Cancer (TNBC), a highly aggressive type of breast cancer, exhibits a lower expression of TAB182. TAB182 knockdown stimulates the proliferation, migration, and invasion of TNBC cells. Our study first obtained RNA-seq data to explore the cellular functions mediated by TAB182 at the genome level in TNBC cells. A transcriptome analysis and in vitro experiments enabled us to identify that TAB182 downregulation drives the enhanced properties of cancer stem-like cells (CSCs) in TNBC cells. Furthermore, TAB182 deletion contributes to the resistance of cells to olaparib or cisplatin, which can be rescued by silencing GLI2, a gene downstream of cancer stemness-related signaling pathways. Our results reveal a novel function of TAB182 as a potential negative regulator of cancer stem-like properties and drug sensitivity in TNBC cells, suggesting that TAB182 may be a tumor suppressor gene and is associated with increased therapeutic benefits for TNBC patients.

overall_design: Examination of genes regulated by knockdown of TAB182 in MDA-MB-231 cells.

first: Huan

last: He

first: Shaozheng

last: Wang

first: Wen

last: Zhang

first: Shanshan

last: Gao

first: Hua

last: Guan

first: Pingkun

last: Zhou


title: The transcription factor HIF2a partakes in the differentiation blockade of acute myeloid leukaemia [RNA-Seq]

city: Milan

first: Rosa

last: Bernardi

address: via Olgettina 60

institute: San Raffaele Scientific Institute

summary: Acute myeloid leukemia (AML) is characterized by an accumulation of aberrant myeloid cells arrested at different stages of differentiation. Therapeutic approaches that prompt AML blasts to differentiate represent an attractive opportunity in the landscape of AML therapies, as they aim to induce terminal maturation and leukemia debulking without intensive cytotoxic treatments. In the present study, we investigate the involvement of HIF1 and HIF2 transcription factors in AML pathogenesis, and position HIF2 as a novel regulator of the AML differentiation block. We performed a comparative analysis of HIF1 and HIF2 function in AML cell lines via their inhibition with genetic or pharmacological strategies and found that both factors promote AML proliferation and clonogenicity. Importantly, specific inhibition of HIF2 provokes AML cell differentiation in cell lines and patient-derived xenograft (PDX) models. Mechanistically, we found that HIF2 and EZH2, the catalytic subunit of polycomb repressive complex 2, cooperate at favoring EZH2-mediated deposition of the repressive histone mark H3K27me3 on the regulatory regions of myeloid differentiation genes. Additionally, we demonstrate that HIF2 is positively regulated by the pro-differentiation agent all-trans retinoic acid (ATRA), and its inhibition cooperates with ATRA in triggering AML cell differentiation. In conclusion, we report evidence of a new role of HIF2 in the pathogenesis of AML, by promoting an undifferentiated state via EZH2-mediated epigenetic silencing of myeloid differentiation genes. We propose that HIF2 inhibition may open new therapeutic avenues for AML treatment by licensing AML differentiation and synergizing with ATRA towards leukemia exhaustion.

overall_design: Total RNA sequencing analysis of 18 samples. The experiment was conducted in two human AML cell lines, HL60 and Kasumi1, as follows: cells were stably transfected with shRNAs against HIF1a, HIF2a or scambled shRNA as control (shCTRL). In addition, Kasumi1 cells with shRNAs were treated with 1 micromolar ATRA for 24 hours. Two biological replicates were analyzed.

first: Daniela

last: Magliulo

first: Raffaella

last: Pini


title: Transcriptome analysis reveals differences in developmental neurotoxicity mechanism of methyl-, ethyl-, and propyl- parabens in zebrafish embryos

city: Seoul

first: Cong

middle: Minh

last: Tran

address: 232 Goneung-ro, Nowon-gu

country: South Africa

institute: Seoul National University of Science and Technology

summary: Limited studies on comparison of developmental (neuro)toxicity of parabens have been conducted and unharmonized concentrations between phenotypic observations and transcriptomic analysis hamper understanding of their differential molecular mechanism. Developmental toxicity testing was conducted with commonly used methyl- (MtP), ethyl- (EtP) and propyl-paraben (PrP) in zebrafish embryos. Based on benchmark dose 5% (BMD5), embryonic mortality based-point of departure (M-PoD) values of three parabens were determined and changes in locomotor behavior were evaluated at concentrations of 0, M-PoD/50, M-PoD/10, and M-PoD in which transcriptomic analysis was conducted to explore the underlying neurotoxicity mechanism. Higher long-chained parabens were more toxic than short-chained parabens, as determined by M-PoD values of 154.1, 72.6, and 24.2 µM for MtP, EtP, and PrP, respectively. While exposure to EtP resulted in hyperactivity, no behavior effect was observed by MtP and PrP. Transcriptomics analysis revealed that abnormal behaviors in EtP-exposed group are associated with the distinctly enriched pathways in signal, transport, calcium ion binding, and metal-binding. In contrast, exposure to MtP and PrP mainly disrupted the membrane and transmembrane, which are closely linked to abnormal embryonic development rather than neurobehavior changes. According to the changes in expression of signature mRNAs, tentative transcriptomic-based PoD (T-PoD) values for each paraben were determined as MtP (2.68 µM), EtP (3.85 µM), and PrP (1.4 µM).

overall_design:
Comparative gene expression profiling analysis of RNA-seq data for zebrafish (Danio_rerio).

This study was supported by Korea Environment Industry & Technology Institute (KEITI) through "the Technology Development Project for Safety Management of Household Chemical Products", funded by Korea Ministry of Environment (MOE) (grant number 2020002960006).

middle: M

first: Jin

last: Ra

first: Dong

middle: Y

last: Rhyu

first: Ki

last: Kim


title: Gli1 marks a sentinel muscle stem cell population for muscle regeneration

status: Public on Aug 06 2023

city: Shanghai

first: jiayin

last: Peng

address: Yueyang Road

institute: Center for Excellence in Molecular Cell Science

summary: Adult skeletal muscle regeneration is mainly driven by muscle stem cells (MuSCs), which are highly heterogeneous. Although recent studies have started to characterize the heterogeneity of MuSCs, whether a subset of cells with distinct exists within MuSCs remains unanswered. Here, we found that a population of MuSCs, marked by Gli1 expression, is required for muscle regeneration. The Gli1+ MuSC population displayed advantages in proliferation and differentiation both in vitro and in vivo.  Depletion of this population led to delayed muscle regeneration, while transplanted Gli1+ MuSCs supported muscle regeneration more effectively than Gli1- MuSCs. Further analysis revealed that even in the uninjured muscle, Gli1+ MuSCs had elevated mTOR signaling activity, increased cell size and mitochondrial numbers compared to Gli1- MuSCs, indicating Gli1+ MuSCs are displaying the features of primed MuSCs. Moreover, Gli1+ MuSCs greatly contributed to the formation of GAlert cells after muscle injury. Collectively, our findings demonstrate that Gli1+ MuSCs represent a distinct MuSC population which is more active in the homeostatic muscle and enters the cell cycle shortly after injury. This cell population functions as the tissue-resident sentinel that rapidly responds to injury and initiates muscle regeneration.

overall_design: we separately collected tdT+ (Gli1+) and tdT- (Gli1-) MuSCs for RNA-Seq analysis.

first: Jiayin

first: Lili

last: Han

first: Biao

last: Liu

first: Yun

last: Zhao


title: The landscape of circular RNAs in neuroblastoma [V]

city: Berlin

first: Steffen

last: Fuchs

address: Augustenburger Platz 1

department: Pediatric Oncology

institute: Charité - Universitätsmedizin Berlin

summary: Circular RNAs (circRNAs), a noncoding RNA class originating from alternative splicing, are highly abundant in neural tissues and were shown to regulate gene expression e.g. by interacting with microRNAs and RNA-binding proteins. Neuroblastoma is an embryonal neoplasia, which arises from undifferentiated neural crest cells. Here, we aimed to explore, whether circRNAs influence the pathogenesis of high-risk neuroblastoma. We performed whole-transcriptome sequencing of 104 primary neuroblastoma samples of all risk-groups and identified 5,203 unique circRNAs involving 2,302 genes. Candidate circRNA expression did not correlate with host gene expression, indicating independent regulatory mechanisms. circRNAs were significantly downregulated in the MYCN-amplified high-risk tumors. These findings were independently reproduced in a tetracycline-inducible MYCN-overexpression system based on a non MYCN-amplified neuroblastoma cell line, suggesting that MYCN drives this global circRNA repression. We identified the RNA helicase DHX9 as a mediator of this global suppressive effect of MYCN on circRNAs. Comparing our RNA sequencing data with other cancers and controls revealed a circRNA subset specifically upregulated in neuroblastoma that included a circRNA derived from the ARID1A tumor suppressor gene. Specific circARID1A knockdown resulted in reduced proliferation, cell numbers and viability, prompted apoptosis and induced a differentiated phenotype. Neither knockdown, nor overexpression of circARID1A influenced ARID1A mRNA and protein levels significantly. To dissect the potential mode of function, we performed a pulldown assay with subsequent mass spectrometry. We identified the RNA-binding protein KHSRP as an interaction partner that participates in the mechanism of action of circARID1A. In summary, this study highlights an important role of circRNAs in neuroblastoma biology and may establish this RNA class as a future therapeutic target and biomarker.

overall_design: Induction of MYCN expression in the neuroblastoma cell line SK-N-AS for 10d

first: Joerg

last: Fallmann

first: Johannes

middle: H

last: Schulte


title: Acetylation reprograms MITF target selectivity and residence time

city: Oxford

first: Pakavarin

last: Louphrasitthiphol

address: Old Road Campus Research Building (off Roosevelt Drive)

department: Nuffield Department of Clinical Medicine

institute: Ludwig Institute for Cancer Research

summary: Key to effective gene regulation in development and homeostasis is the ability of transcription factors to discriminate between different classes of binding sites. Yet how this is achieved is poorly understood. The microphthalmia-associated transcription factor MITF is a lineage-survival oncogene that plays a crucial role in melanoma and melanocyte development where it suppresses invasion but promotes both proliferation and differentiation. How MITF distinguishes between differentiation and proliferation-associated targets is unknown. Unexpectedly we show here that differentiation-associated target genes are characterized by low affinity MITF binding sites. MAPK signaling, a key driver of melanomagenesis downstream from BRAF and NRAS, promotes p300/CBP-mediated MITF acetylation at K206 to reduce preferentially DNA binding affinity at differentiation-associated targets. The results reveal a MAPK-driven acetylation switch that can trigger de-differentiation of melanocytes and provide a mechanistic explanation of why a human K206Q MITF mutation is associated with Waardenburg’s syndrome characterized by pigmentation abnormalities.

overall_design: 501mel melanoma cell lines engineered to express ectopic-doxycycline-inducible-HA-epitope-tag-murine Mitf-WT or K206Q or K206R were treated with 20ng Dox prior to fixation and ChIP-sequencing

first: Colin

middle: R

last: Goding


title: The long non-coding RNA LINDA restrains cellular collapse following DNA damage in Arabidopsis thaliana

status: Public on Aug 05 2023

city: Gent

first: Josephine

last: Herbst

address: Technologiepark 71

country: Belgium

institute: VIB-UGent Center for Plant Systems Biology

summary: The genomic integrity of every organism is endangered by various intrinsic and extrinsic stresses. To maintain the genomic integrity, a sophisticated DNA damage response (DDR) network is activated rapidly after DNA damage. Notably, the fundamental DDR mechanisms are conserved in eukaryotes. However, knowledge about many regulatory aspects of the plant DDR is still limited. Important, yet little understood, regulatory factors of the DDR are the long non-coding RNAs (lncRNAs). In humans, 13 lncRNAs functioning in DDR have been characterized to date, whereas no such lncRNAs have been characterized in plants yet. By meta-analysis, we identified the long intergenic non-coding RNA induced by DNA damage (LINDA) that responds strongly to various DNA double-strand break-inducing treatments, but not to replication stress induced by mitomycin C. After DNA damage, LINDA is rapidly induced in an ATM- and SOG1-dependent manner. Intriguingly, the transcriptional response of LINDA to DNA damage is similar to that of its flanking hypothetical protein-encoding gene. Phylogenetic analysis of putative Brassicales and Malvales LINDA homologs indicates that LINDA lncRNAs originate from duplication of a flanking small protein-encoding gene followed by pseudogenization. We demonstrate that LINDA is not only needed for the regulation of this flanking gene, but also for fine-tuning of the DDR after the occurrence of DNA double-strand breaks. Moreover, Δlinda mutant root stem cells are unable to recover from DNA damage, most likely due to hyper-induced cell death.

overall_design: We performed RNA-Seq experiments on wildtype (Col-0) and  Δlinda mutants after induction of DNA damage by treatment of 14-day-old seedlings with either 80 µg/ml Zeocin or 2 kJ UV-C. Mock treated seedlings or seedlings grown under normal light conditions were used as controls for Zeocin or UV-C treatment, respectively. Samples were harvested 3 hours after induction of DNA damage. We harvested four samples per genotype per treatment.

first: Solveig

last: Nagy

first: Ilse

last: Vercauteren

first: Lieven

last: De Veylder

first: Reinhard

last: Kunze


title: Selective IL-27 production by intestinal regulatory T cells permits gut-specific regulation of Th17 immunity

city: La Jolla

first: Li-Fan

last: Lu

state: CA

address: 9500 Gilman Dr., NSB 5314

institute: University of California, San Diego

summary: Regulatory T cells (Treg cells) are instrumental in establishing immunological tolerance. However, the precise effector mechanisms by which Treg cells control a specific type of immune response in a given tissue remains unresolved. By simultaneously studying Treg cells from different tissue origins under systemic autoimmunity, in the present study we show that interleukin (IL)-27 is specifically produced by intestinal Treg cells to regulate helper T17 cell (TH17 cell) immunity. Selectively increased intestinal TH17 cell responses in mice with Treg cell-specific IL-27 ablation led to exacerbated intestinal inflammation and colitis-associated cancer, but also helped protect against enteric bacterial infection. Furthermore, single-cell transcriptomic analysis has identified a CD83+CD62Llo Treg cell subset that is distinct from previously characterized intestinal Treg cell populations as the main IL-27 producers. Collectively, our study uncovers a new Treg cell suppression mechanism crucial for controlling a specific type of immune response in a particular tissue and provides further mechanistic insights into tissue-specific Treg cell-mediated immune regulation.


title: Neural plate progenitors give rise to both anterior and posterior pituitary cells

city: Rehovot

first: Gil

last: Levkowitz

state: Rehovot

address: Wolfson Building of Biological Sciences, Department of Molecular Cell Biology, Weizmann Institute of Science, 234 Herzl Street

department: Molecular Cell Biology

country: Israel

institute: Weizmann Institute of Science

summary: The pituitary is the master neuroendocrine tissue, which regulates body homeostasis. It consists of the adenohypophysis (AH), which harbors hormones producing cells and the neurohypophysis (NH), which relays the direct passage of hormones from the brain to the periphery. We previously characterized the mouse NH cell identities (Chen Q et al., 2020). However, the zebrafish NH cell types remained unknown owning to surgical inaccessible and limited amount of the tissue. Using Mars-Seq single-cell sequencing (Jaitin et al., 2014), we characterized the cell types of the neurohypophyseal enriched population from the juvenile and adult zebrafish pituitaries, which laid the foundation for further lineage and functional research in this study.

overall_design: 3 pools of 37 days post fertilization, 5-month and 7-month old Tg(pomca:EGFP) zebrafish were prepared for Mars-seq single-cell RNA-Seq. Two 348-well plates were collected for each age group.

first: Qiyu

last: Chen

first: Dena

last: Leshkowitz

first: Hanjie

last: Li

first: Ido

last: Amit


title: Effect of adseverin deletion on gene expression in the articular cartilage of 10 week old mice

city: Toronto

first: Byron

middle: Sze Kwong

last: Chan

state: ON

address: 25 Orde Street, RM#5-1013

institute: University of Toronto

summary: To investigate the alterations in the transcriptome profile due to adseverin deletion in articular chondrocytes, differential gene expression profiling analysis was performed using data obtained from RNA sequencing of articular chondrocytes in the native cartilage of 10 week old mice

overall_design: Differential gene expression profiling analysis of RNA sequencing data for wildtype and adseverin knockout articular cartilage

first: Rita

last: Kandel

first: Jeffrey

last: Wrana

first: Kin


title: Single-nucleus transcriptional profiling of chronic kidney disease after cisplatin nephrotoxicity

city: Augusta

first: Zhengwei

last: Ma

state: GA

address: 1460 Laney walker Blvd.

institute: Augusta University

summary: Cisplatin induces both acute and chronic nephrotoxicity during chemotherapy in cancer patients. Here, we report the study of single-nucleus sequencing (snRNA-seq) of cisplatin-induced nephrotoxicity.

overall_design: Male C57BL/6 mice were given 4 consecutive weekly injections of 8 mg/kg cisplatin. Kidney tissues were collected at 9 weeks after the first cisplatin injection for single-nucleus RNA sequencing (snRNA-seq)

first: Xiaoru

last: Hu

first: Han-Fei

last: Ding

first: Zheng

last: Dong


title: PAX4 loss of function alters human endocrine cell development and influences diabetes risk.

city: Singapore

first: Adrian

middle: Kee Keong

last: Teo

state: Singapore

address: 61 Biopolis Drive, Proteos

department: Stem Cells and Diabetes Laboratory

country: Singapore

institute: IMCB, A*STAR


title: PAX4 loss of function alters human endocrine cell development and influences diabetes risk [dataset 2]

summary: Diabetes is a major chronic disease with an excessive healthcare burden on society. A coding variant (p.Arg192His) in the transcription factor PAX4 is uniquely and reproducibly associated with altered risk for type 2 diabetes (T2D) in East Asian populations, whilst rare PAX4 alleles have been proposed to cause monogenic diabetes8. In mice, Pax4 is essential for beta cell formation but neither the role of diabetes-associated variants in PAX4 nor PAX4 itself on human beta cell development and/or function are known. Here, we demonstrate that non-diabetic carriers of either the PAX4 p.Arg192His or a newly identified p.Tyr186X allele exhibit decreased pancreatic beta cell function. In the human beta cell model, EndoC-βH1, PAX4 knockdown led to impaired insulin secretion, reduced total insulin content and altered hormone gene expression. Deletion of PAX4 in isogenic human induced pluripotent stem cell (hiPSC)-derived beta-like cells resulted in de-repression of alpha cell gene expression whilst in vitro differentiation of hiPSCs from carriers of PAX4 p.192His and p.186X alleles exhibited increased polyhormonal endocrine cell formation and reduced insulin content. In silico and in vitro studies showed that these PAX4 alleles cause either reduced PAX4 expression or function. Correction of the diabetes-associated PAX4 alleles reversed these phenotypic changes. Together, we demonstrate the role of PAX4 in human endocrine cell development, beta cell function and its contribution to type 2 diabetes risk.

overall_design: Using patient-derived hiPSCs to model pancreatic beta cell development to study mechanisms underlying diabetes risks.

first: Hwee

last: Lau

first: Soumita

last: Ghosh

first: Shawn

last: Hoon

first: E

last: Tai

middle: K


title: PAX4 loss of function alters human endocrine cell development and influences diabetes risk [dateset 1]

first: Nicole

last: Krentz

first: Hanice

last: Sun


title: Impaired age-associated mitochondrial translation is mitigated by exercise and PGC-1α

status: Public on Aug 04 2023

city: Basel

first: Laura

middle: Maria

last: de Smalen

state: Basel-Stadt

address: Spitalstrasse 41

department: Biozentrum

country: Switzerland

institute: University of Basel

summary: To investigate age-related changes in muscle mitochondrial proteostasis, as well as the effect of higher and lower levels of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in this paradigm, we isolated RNA from M. gastrocnemius muscle tissue of muscle-specific PGC-1α gain- and loss-of-function mouse models, at the ages of three and twenty four months.

overall_design: We performed gene expression profiling analysis using RNA-seq, which was performed with 5-6 biological replicates for each condition.

first: Anastasiya

last: Börsch

first: Aurel

last: Leuchtmann

first: Jonathan

middle: F

last: Gill

first: Danilo

last: Ritz

first: Mihaela

last: Zavolan

first: Christoph

last: Handschin


title: Epigenetic control of training adaptation in skeletal muscle

first: Regula

last: Furrer

summary: Skeletal muscle has an enormous plastic potential and adapts to various stimuli such as mechanical stress, nutrients and hormones. While morphological changes observed in endurance trained muscles are well described, the molecular underpinnings of training adaptation is poorly understood. Therefore, the aim of our study was to define the molecular signature of a trained muscle and unravel the transcriptional responses of untrained and trained muscles to an acute bout of endurance exercise. Our results reveal that even though at baseline, the transcriptome of trained and untrained muscles is very similar, training status substantially affects the transcriptional response to an acute challenge, both quantitatively and qualitatively. Therefore, we provide novel insights into the complex molecular processes that are induced in response to exercise in a temporal- and training status-dependent manner. In addition, our study reveals that the epigenetic landscape contributes to the transcriptional memory of a trained muscle.

overall_design: Quadriceps muscle RRBS of wildtype and PGC-1alpha muscle-specific knockout and transgenic mice; either in sedentary condition, after 4 weeks of training or after an acute bout of exhaustion exercise (untrained) , 12 groups, 5 replicates/mice per group

first: Barbara

last: Heim-Kupr

first: Karl

middle: V

last: Nordström

first: Svenia

last: Schmid

first: Jörn

last: Walter


title: Transcriptional control of training adaptation in skeletal muscle

overall_design: Quadriceps muscle RNA-seq of wildtype and PGC-1alpha muscle-specific knockout and transgenic mice; either in sedentary condition, after 4 weeks of training or after an acute bout of exhaustion exercise (untrained or trained) , 28 groups, 5 replicates/mice per group

middle: J


title: Manipulating the 3D organization of the largest synthetic yeast chromosome [RNAseq]

city: New York

first: Weimin

last: Zhang

state: NY

address: 435 E 30th street

department: Institute for Systems Genetics

institute: NYU Langone Health

summary: Whether synthetic genomescan power life has attracted broad interest in the synthetic biology field, especially when the synthetic genomes are extensively modified with thousands of designer features. Here we reportde novosynthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bpSaccharomyces cerevisiaechromosome resulting from extensive genome streamlining and modification. During the construction ofsynIV, we developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction and facilitated chromosome debugging. In addition to the drastic sequence changes made to synIV by rewriting it, we further manipulated the three-dimensional structure of synIV in the yeast nucleus to explore spatial gene regulation within the nuclear space. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of the largest synthetic yeast chromosome shed light on higher-order architectural design of the synthetic genomes.

overall_design: For synIV multipoint tethering to the inner nuclar membrane assays, WT or synIV strains contain HEH1 and HEH2 serve as control, WT or synIV strains contain HEH1-ZF and HEH2-ZF are the experimental group. Two or three biological replicates were includes for each genotype.

first: Jef

middle: D

last: Boeke


title: Short term exposure to alcohol induces transcriptional changes in non-tumorigenic breast cells

city: Boulder

first: Jennifer

last: Kugel

state: CO

address: 596 UCB

department: Biochemistry

institute: University of Colorado Boulder

summary: Breast cancer is the second leading cause of cancer-related deaths in women (*). Many genetic and behavioral risk factors can contribute to the initiation and progression of breast cancer, one being alcohol consumption. Numerous epidemiological studies have established a positive correlation between alcohol consumption and breast cancer; however, the molecular basis for this link remains ill defined. Elucidating ethanol-induced changes to global transcriptional programming in breast cells is important to ultimately understand how alcohol and breast cancer are connected mechanistically. We investigated early induced transcriptional changes in response to cellular exposure to moderate levels of alcohol. We treated the non-tumorigenic breast cell line MCF10A, and the tumorigenic breast cell lines MDA-MB-231 and MCF7, with ethanol for 6 h, then captured the changes to ongoing transcription using 4-thiouridine metabolic labeling followed by deep sequencing. Only the MCF10A cell line exhibited statistically significant changes to ongoing transcription in response to ethanol treatment. Further experiments revealed some ethanol-upregulated genes are sensitive to the dose of alcohol treatment, while others are not. Gene ontology and biochemical pathway analyses revealed that ethanol-upregulated genes in MCF10A cells are enriched in biological functions that could contribute to cancer development.

overall_design: 4sU-seq in MCF10A, MCF7, and MDA-MB-231 cells before and after 6h treatment with 0.3% ethanol. Metabolic labeling with 4-thiouridine occurred the last 2h of the ethanol treatment. 4sU-seq under all conditions was performed in biological triplicate.

first: Georgiana

last: Salant

first: Tyler

last: Brant

first: James

middle: A

last: Goodrich


title: Manipulating the 3D organization of the largest synthetic yeast chromosome

type: Genome variation profiling by high throughput sequencing

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

overall_design: Refer to individual Series