title: (R)-2-hydroxyglutarate inhibits KDM5 histone lysine demethylases to drive tumorigenesis in IDH-mutant cancers

city: Boston

first: Julie-Aurore

last: Losman

address: 450 Brookline Ave MA448

institute: Dana-Farber Cancer Institute

summary: Oncogenic mutations in isocitrate dehydrogenase (IDH)-1 and -2 occur in a wide range of cancers, including acute myeloid leukemias (AMLs) and gliomas1-3. Mutant IDH enzymes convert 2-oxoglutarate (2OG) to (R)-2-hydroxyglutarate [(R)-2HG]4,5, an oncometabolite that induces cellular transformation by dysregulating 2OG-dependent enzymes. The only direct target of (R)-2HG known to contribute to transformation is the 5-methylcytosine hydroxylase TET2, and there is ample evidence to suggest that (R)-2HG drives leukemogenesis at least in part by inhibiting TET26,7. However, IDH mutations, but not TET2 mutations, are specifically associated with aggressive hematologic diseases, suggesting that (R)-2HG has targets other than TET2 that contribute to mutant IDH-mediated transformation. Here, we report that (R)-2HG directly inhibits KDM5 histone lysine demethylases in IDH-mutant AMLs and gliomas to induce cellular transformation. These studies provide a functional link between dysregulation of histone lysine methylation and tumorigenesis in IDH-mutant cancers.

overall_design: H3K4me3 ChIP-Seq (two biological replicates per sample and one input sample per cell line), and RNAseq (two biological replicates per sample).

first: Kathryn

last: Gunn


title: The  AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner [CnT_InVitro]

status: Public on Feb 28 2023

city: STANFORD

first: Katja

last: Pekrun

state: CA

address: 269 Campus Drive

department: Pediatrics & Genetics

country: USA

institute: Stanford School of Medicine

summary: Recombinant adeno-associated viral vectors (rAAVs) are among the most commonly used vehicles for in vivo based gene therapies. However, it is hard to predict which AAV capsid will provide the most robust expression in human subjects due to the observed discordance in vector-mediated transduction between species.  We used a primate specific capsid, AAV-LK03, and demonstrated that the limitation of this capsid towards transduction of mouse cells was unrelated to cell entry and nuclear transport but rather due to depleted histone H3 chemical modifications related to active transcription, namely H3K4me3 and H3K27ac, on the vector DNA itself. A single-amino acid insertion into the AAV-LK03 capsid enabled efficient transduction and the accumulation of active-related epigenetic marks on the vector chromatin in mouse without compromising transduction efficiency in human cells. Our study suggests that the capsid protein itself is involved in driving the epigenetic status of the vector genome, most likely during the process of uncoating. Programming viral chromatin states by capsid design may enable facile DNA  transduction between vector and host species and ultimately led to rationale selection of AAV capsids for use in humans.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Human and murine cell lines were transduced with rAAV packaged using different capsids, nuclei were isolated, and Cut&Tag analysis was performed.

first: Adriana

last: Gonzalez-Sandoval

first: Shinnosuke

last: Tsuji

first: Feijie

last: Zhang

first: King

middle: L

last: Hung

first: Howard

middle: Y

last: Chang

first: Mark

middle: A

last: Kay


title: Serine 970 of RNA helicase MOV10 is phosphorylated and controls unfolding activity and fate of mRNAs targeted for AGO2-mediated silencing [eCLIP-Seq]

city: Urbana

first: Stephanie

last: Ceman

state: IL

address: 601 S. Goodwin Ave

department: Cell and Developmental Biology

institute: University of Illinois-Urbana Champaign

summary: MOV10 is an RNA helicase that is required for organismal development and is highly expressed in postnatal brain.  MOV10 was identified as an AGO2 associated protein that is also necessary for AGO2-mediated silencing. MOV10 has been shown to be ubiquitinated, leading to its degradation and release from bound mRNAs but no other post-translational modifications with functional implications have been described. Using mass spectrometry, we show that MOV10 is phosphorylated in cells at the C-terminus, specifically at serine 970 (S970). Substitution of S970 to phospho-mimic aspartic acid (S970D) blocked unfolding of an RNA G-quadruplex, similar to when the helicase domain was mutated (K531A). In contrast, the alanine substitution (S970A) of MOV10 unfolded the model RNA G-quadruplex.  To examine its role in cells, RNA-seq analysis showed that the expression of S970D causes decreased expression of MOV10 eCLIP targets compared to WT. Introduction of S970A had an intermediate effect, suggesting that S970 was protective of mRNAs. In whole cell extracts, MOV10 and its substitutions bound AGO2 comparably; however, knockdown of AGO2 abrogated the S970D-induced mRNA degradation. Thus, MOV10 activity protects mRNA from AGO2; phosphorylation of S970 restricts this activity resulting in AGO2-mediated mRNA degradation.  S970 is positioned C-terminal to the defined MOV10-AGO2 interaction site and is proximal to a disordered region that likely modulates AGO2 interaction with target mRNAs upon phosphorylation. In summary, we provide evidence for a model whereby MOV10 phosphorylation facilitates AGO2 association with the 3’UTR of translating mRNAs that leads to their degradation.

overall_design: Cell pellets from N2A cells were flash-frozen and sent to Eclipse BioInnovations. IP was perfomed using the anti-MOV10 antibody (A301-571A; Bethyl Laboratories). The RNAs pulled down were sequenced to find MOV10 bound targets.

first: Aatiqa

last: Nawaz

first: eCLIPSE

last: Bioinnovations


title: Transcriptomes of CD34+ hematopoietic stem/progenitor cells (HSPCs) from myelodysplastic syndromes (MDS) patients cultured in normoxia or hypoxia.

status: Public on Mar 01 2023

city: London

first: Eleni

last: Maniati

address: John Vane Science Centre, Charterhouse Square

department: Barts Cancer Institute

country: United Kingdom

institute: Queen Mary University of London

type: Expression profiling by high throughput sequencing

summary: Three MDS patients (SF3B1mut -H662/H662/Q699) and three age-matched healthy donors were challenged in colony forming assay under normoxic (20% O2) and hypoxic (3% O2) conditions. RNA-sequencing was performed on the HSPC-derived colonies o examine transcriptional changes under normoxic and hypoxic conditions.

overall_design: RNA-sequencing was performed on CD34+ hematopoietic stem/progenitor cells (HSPCs) from myelodysplastic syndromes (MDS) patients or Healthy donors in normoxia and hypoxia, n = 3 per group.

first: Syed

last: Mian

first: Celine

last: Philippe

first: Jun

last: Wang

first: Kevin

last: Rouaoult-Pierre


title: The  AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner [Tn5_InVivo]

overall_design: Mice were injected with rAAV packaged using different capsids, nuclei were isolated, gDNA was extracted from nuclei, and normalization for Cut&Tag analysis was performed using the Illumina DNA prep kit.


title: Serine 970 of RNA helicase MOV10 is phosphorylated and controls unfolding activity and fate of mRNAs targeted for AGO2-mediated silencing [RNA-Seq]

overall_design: We expressed WT and Phospho-MOV10 mutants in MOV10 KO cells, and extracted total RNA. We then performed RNA-seq to look at the steady state levels of mRNAs.


title: The  AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner [Tn5_InVitro]

overall_design: Human and murine cell lines were transduced with rAAV packaged using different capsids, nuclei were isolated, gDNA was extracted from nuclei, and normalization for Cut&Tag analysis was performed using the Illumina DNA prep kit.


title: Serine 970 of RNA helicase MOV10 is phosphorylated and controls unfolding activity and fate of mRNAs targeted for AGO2-mediated silencing

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

overall_design: Refer to individual Series


title: The  AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner [CnT_InVivo]

overall_design: Mice were injected with rAAV packaged using different capsids, nuclei were isolated, and Cut&Tag analysis was performed


title: Comprehensive molecular characterization of lung tumors implicates AKT and MYC signaling in adenocarcinoma to squamous cell transdifferentiation [RNA-seq]

city: New York

first: Yingqian

middle: Ada

last: Zhan

state: New York

address: 430 East 67th Street

department: EPIGENETICS RESEARCH CENTER

institute: MSKCC

summary: To investigate molecular changes occurring during lung adenocarcinoma to squamous carcinoma transformation.

overall_design: We performed RNAseq in transforming lung adenocarcinoma and squamous carcinoma clinical specimens, together with control, never-transformed lung adenocarcinomas and squamous carcinomas.

first: Alvaro

last: Quintanal-Villalonga

first: Charles

middle: M

last: Rudin


title: Comprehensive molecular characterization of lung tumors implicates AKT and MYC signaling in adenocarcinoma to squamous cell transdifferentiation [EPIC]

type: Methylation profiling by high throughput sequencing

overall_design: We performed EPIC (methyl-capture) in transforming lung adenocarcinoma and squamous carcinoma clinical specimens, together with control, never-transformed lung adenocarcinomas and squamous carcinomas.


title: The  AAV capsid can influence the epigenetic marking of rAAV delivered episomal genomes in a species dependent manner

first: Katja

last: Pekrun


title: Sp3 is essential for normal lung morphogenesis and cell cycle progression during mouse embryonic development

city: Stanford

first: Lawrence

middle: S.

last: Prince

state: California

address: 453 QUARRY RD

department: Pediatrics - Neonatology

institute: Stanford University

summary:
Members of the Sp family of transcription factors regulate gene expression via binding GC boxes within promoter regions.
Unlike Sp1, which stimulates transcription, the closely related Sp3 can either repress or activate gene expression and is required for perinatal survival in mice.
Here we use RNAseq and cellular phenotyping to show how Sp3 regulates murine fetal cell differentiation and proliferation.
Homozygous Sp3-/- mice were smaller than WT and Sp+/- littermates, died soon after birth, and had abnormal lung morphogenesis.
RNAseq of Sp3-/- fetal lung mesenchymal cells identified alterations in extracellular matrix production, developmental signaling pathways, and myofibroblast/lipofibroblast differentiation.
The lungs of Sp3-/- mice contained multiple structural defects, with abnormal endothelial cell morphology, lack of elastic fiber formation, and accumulation of lipid droplets within mesenchymal lipofibroblasts.
Sp3-/- cells and mice also displayed cell cycle arrest, with accumulation in G0/G1 and reduced expression of numerous cell cycle regulators including Ccne1.
These data detail the global impact of Sp3 on in vivo mouse gene expression and development.
Development • Accepted manuscript  lung mesenchymal cells identified alterations in extracellular matrix production, developmental  signaling pathways, and myofibroblast/lipofibroblast differentiation. The lungs of Sp3-/- mice  contained multiple structural defects, with abnormal endothelial cell morphology, lack of elastic  fiber formation, and accumulation of lipid droplets within mesenchymal lipofibroblasts. Sp3-/-  cells and mice also displayed cell cycle arrest, with accumulation in G0/G1 and reduced  expression of numerous cell cycle regulators including Ccne1. These data detail the global  impact of Sp3 on in vivo mouse gene expression and development.

overall_design: Freshly isolated E15 fetal lung mesenchymal cells were isolated and passaged twice before being lysed in TRIzol for RNA isolation. RNA samples were then processed for RNAseq.

first: Alyssa

last: McCoy

first: Omar

last: Lakhdari

first: Sayane

last: Shome

first: Kaitlin

last: Caoili

first: Gilberto

middle: E

last: Hernandez

first: Nima

last: Aghaeepour

first: Lindsay

middle: D

last: Butcher

first: Kathleen

last: Fisch

middle: S


title: Comprehensive molecular characterization of lung tumors implicates AKT and MYC signaling in adenocarcinoma to squamous cell transdifferentiation


title: Blood endothelial Alk1-BMP4 signaling axis ensures adult hair follicle stem cell timely activation

city: Ithaca

first: Kefei

middle: Nina

last: Li

state: NY

address: 153 VETERANS PL E408

department: Molecular Biology and Genetics

institute: Cornell University - Ithaca, NY

summary: To investigate the gene expression changes in the hair follicle stem cells (bulge cells, HFSCs) after perturbing the vascular niche by knocking out Alk1 from the endothelial cells using Cdh5CreERT2 driver

overall_design: Comparative gene expression profiling analysis of PD20 bulge cells from PD17-induced Cdh5CreERT2-driven Alk1 knockout mouse skin and littermate Cre-negative control mouse skin

middle: N

first: Tudorita

last: Tumbar


title: Small RNA sequencing of term whole placenta from normal weight and obese pregnancies

first: Perrie

last: O’Tierney-Ginn

state: MA

address: 800 Washington Street #394

department: Mother Infant Research Institute

institute: Tufts Medical Center

type: Non-coding RNA profiling by high throughput sequencing

summary: Context: An increase in maternal insulin resistance (IR) during pregnancy is essential for normal fetal growth. The mechanisms underlying this metabolic adaptation to pregnancy are poorly understood. Placenta factors are believed to instigate and maintain these metabolic changes, as IR decreases shortly after delivery. Methylation of placental gene loci that are common targets for miRNAs, are associated with maternal IR.   Objective: We hypothesized that placental miRNAs targeting methylated loci are associated with maternal IR during late pregnancy.   Methods: Placental miRNA expression was measured via small RNA-sequencing in a subset of 40 placentas selected by maternal pre-gravid BMI and neonatal adiposity.   Results: Median maternal age was 27.5 years, with a median pre-pregnancy BMI of 24.7 kg/m2, and median HOMA-IR of 2.9. Among the five selected miRNA, near delivery maternal HOMA-IR correlated with the placental expression of miRNA-371b-3p (r=0.25; p=0.008) and miRNA-3940-3p (r=0.32; p=0.0004) across the 132 individuals. After adjustment for confounding variables, placental miRNA-3940-3p expression remained significantly associated with HOMA-IR (β=0.16; p=0.03).   Conclusion: Placental miRNA may have an autocrine or paracrine effect - regulating placental genes which play a role in modulating maternal IR.

overall_design: Whole placenta miRNA profiles at full-term elective ceasarean delivery in women with and without obesity with high and low adiposity neonates. Neonatal % body fat was measured within 48 hrs of delivery. Maternal self-reported race (African American (AA), White (cauc)) and neonatal sex was recorded.

middle: F

last: O'Tierney-Ginn

first: Tianjiao

last: Chu

first: Yoel

last: Sadovsky

first: Patrick

last: Catalano


title: Gene expression in murine T-ALL following MYC inactivation

first: Benjamin

middle: AH

last: Smith

address: 290 Jane Stanford Way


title: Time course of gene expression in murine T-ALL following MYC inactivation

summary: To understand changes to gene expression over time in murine T-ALL following MYC inactivation, T-ALL cells were isolated from EμSRα-tTA/tet-O-MYC/FVB/N mice and treated with doxycyline to turn MYC off for varying amounts of time before assessing gene expression.

overall_design: Gene expression profiling by RNA-sequencing.

first: Anja

last: Deutzmann

first: Delaney

middle: K

last: Sullivan

first: Dean

middle: W

last: Felsher

first: Carolyn

middle: R

last: Bertozzi


title: Low-intensity pulsed ultrasound-mediated blood-brain barrier opening increases anti programmed death-ligand 1 delivery and efficacy in Gl261 mouse model

city: PARIS

first: Isaias

last: Hernandez-Verdin

state: Ile-de-France

address: GH PITIE SALPETRIERE ICM IHU A, 47 Boulevard DE L'HOPITAL

department: Genetics and Development of Nervous System Tumors

country: France

institute: Institut du cerveau

summary: Therapeutic antibodies targeting immune checkpoints have shown limited efficacy in clinical trials in glioblastoma (GBM) patients. Ultrasound-mediated blood-brain barrier opening (UMBO) using low-intensity pulsed ultrasound improved drug delivery to the brain. We explored the safety and the efficacy of UMBO plus immune checkpoint inhibitors in preclinical models of GBM. A Blood-brain barrier (BBB) opening was performed using a 1 MHz preclinical ultrasound system in combination with 10µl/g microbubbles. Brain penetration of immune checkpoint inhibitors was determined, and immune cell populations were evaluated using flow cytometry.The impact of repeated treatments on survival was determined. In syngeneic GL261-bearing immunocompetent mice, we showed that UMBO safely and repeatedly open the BBB. BBB opening was confirmed visually and microscopically using Evans’s blue dye and magnetic resonance imaging. UMBO plus anti-PDL-1 was associated with a significant improvement of the overall survival compared to anti-PD-L1 alone. Using mass spectroscopy, we showed that the penetration of therapeutic antibodies can be increased when delivered intravenously compared to non-sonicated brains. Furthermore, we observed an enhancement of activated microglia percentage when combined with anti-PD-L1. Here, we report that the combination of UMBO and anti-PD-L1 dramatically increases GL261-bearing mice's survival compared to their counterparts treated with anti-PD-L1 alone. Our study highlights the BBB as a limitation to overcome to increase the efficacy of anti-PD-L1 in GBM and supports clinical trials combining UMBO and in GBM patients.

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of 5 different mice with two different treatments (conditions)

first: Mohammed

last: Ahmed

first: Emie

last: Quissac

first: Nolwenn

last: Lemaire

first: Coralie

last: Guerin

first: Lea

last: Guyonnet

first: Noël

last: Zahr

first: Laura

last: Mouton

first: Mathieu

last: Santin

first: Alexandra

last: Petiet

first: Charlotte

last: Schmitt

first: Guillaume

last: Bouchoux

first: Michael

last: Canney

first: Marc

last: Sanson

first: Maite

last: Verreault

first: Alexandre

last: Carpentier

first: Ahmed

last: Idbaih


title: The single cell transcriptional landscape of esophageal adenocarcinoma and its modulation by neoadjuvant chemotherapy

city: Birmingham

first: Paul

last: Moss

state: West Midlands

address: Vincent Drive

department: Immunology and Immunotherapy

institute: University of Birmingham

summary: Immune checkpoint blockade has recently proven effective in a subset of patients with esophageal adenocarcinoma (EAC) but little is known regarding the EAC immune microenvironment. We determined the single cell transcriptional profile of EAC in patients who were treatment naive or had received neoadjuvant chemotherapy. Analysis of 52,387 cells revealed 10 major cell subsets of tumor, immune and stromal cells. Prior to chemotherapy tumors were characterised by infiltration with effector T cells showing markers of exhaustion together with large T regulatory cell infiltrates. Plasmacytoid dendritic cells were markedly expanded whilst two dominant cancer-associated fibroblast populations were observed. Pathological remission following chemotherapy was associated with broad reversal of immune abnormalities together with an increase in endothelial and myofibroblast populations although a chemoresistant epithelial stem cell population was apparent. These findings reveal features that underlie and limit the response to current immunotherapy and reveal a range of novel opportunities for targeted therapy.

overall_design: Single Cell RNA profile of cells in the EAC tumor microenvironment pre and post neoadjuvant chemotherapy treatment

first: Wayne

last: Croft

first: Richard

middle: P

last: Evans

first: Hayden

last: Pearce

first: Mona

last: Elshafie

first: Ewen

last: Griffiths


title: Gene expression in murine T-ALL following titrated MYC inactivation

first: Benjamin

last: Smith

summary: To understand changes to gene expression in murine T-ALL following MYC inactivation, T-ALL cells were isolated from EμSRα-tTA/tet-O-MYC/FVB/N mice and treated with varying concentrations of doxycyline for 48 hours to place cells in the MYC on, MYC intermediate, or MYC off state.

first: Anja

last: Deutzmann

first: Delaney

last: Sullivan

first: Dean

last: Felsher

first: Carolyn

last: Bertozzi


title: Impact of pCER270 on Bacillus cereus sensu lato gene expression

city: JOUY-EN-JOSAS

first: Alicia

last: Nevers

address: Domaine de Vilvert

department: Micalis

institute: Institut national de la recherche agronomique (Inrae)

summary: Transcriptome analyses after the transfer of pCER270-spec megaplasmid in B. cereus F4810-72 (previously cured for its plasmid), B. thuringiensis HD73, and B. weihenstephanensis KBAB6 strains. We determined differentially expressed genes in pCER270+ strains in comparison with pCER270- strains.

sample_organism:
Bacillus cereus
Bacillus mycoides
Bacillus thuringiensis

overall_design: Total RNAs from B. cereus F4810-72 pCER270- (triplicate), B. cereus F4810-72 pCER270+ (triplicate), B. thuringiensis HD73 pCER270- (triplicate), B. thuringiensis HD73 pCER270+(triplicate), B. weihenstephanensis KBAB6 pCER270- (triplicate) and B. weihenstephanensis KBAB6 pCER270+ (triplicate) were analyzed from LB and HCT samples, by high-throughput sequencing

first: Markus

last: Kranzler

first: Stephane

last: Perchat

first: Michel

last: Gohar

first: Alexei

last: Sorokin

first: Didier

last: Lereclus

first: Monika

last: Ehling-Schulz

first: Vincent

last: Sanchis-Borja


title: Short tandem repeats are important contributors to silencer elements in T cells

city: Marseille

first: Nori

last: SADOUNI

address: 169 Avenue Luminy

institute: TAGC INSERM U1090

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: Analysis of Dnase Hypersensitive Site (DHS) of P5424 mouse cell line using High-throughput reporter assay

first: Saadat

last: Hussain

last: Sadouni

first: Dominic

last: Van Essen

first: Lan

middle: T

last: Dao

first: Quentin

last: Ferre

first: Guillaume

last: Charbonnier

first: Magali

last: Torres

first: Charles-Henri

last: Lecellier

first: Tom

last: Sexton

first: Simona

last: Saccani

first: Salvatore

last: Spicuglia


title: WWOX P47T loss-of-function mutation induces epilepsy, progressive neuroinflammation, and cerebellar degeneration

city: La Plata

first: Martin

middle: Carlos

last: Abba

state: Buenos Aires

country: Argentina

institute: School of Medical Sciences - UNLP

summary: WWOX gene loss-of-function (LoF) has been associated with neuropathologies resulting in developmental, epileptic, and ataxic phenotypes of varying severity based on the level of WWOX dysfunction. WWOX gene biallelic germline variant p.Pro47Thr (P47T) has been causally associated with a new form of autosomal recessive cerebellar ataxia with epilepsy and intellectual disability (SCAR12). This mutation affects the WW1 protein binding domain of WWOX, impairing its ability to interact with canonical proline-proline-X-tyrosine motifs in partner proteins. We generated a mutant knock-in mouse model of Wwox P47T that phenocopies SCAR12. WwoxP47T/P47T mice displayed epilepsy, profound social behavior and cognition deficits, and poor motor coordination. These deficits progressed with age, and mice became practically immobile, suggesting severe cerebellar dysfunction. WwoxP47T/P47T  mice exhibited signs of progressive neuroinflammation with elevated astro-microgliosis that increased with age. The cerebellar cortex displayed significantly reduced molecular and granular layer thickness and a strikingly reduced number of Purkinje cells with degenerated dendrites. Transcriptome profiling from various brain regions from these Wwox LoF mice highlighted widespread changes in neuronal and glial pathways, enrichment of bioprocesses related to neuroinflammation and severe cerebellar dysfunction, activation of pathways compatible with compensatory neurogenesis along with major suppression of gene networks associated with neuronal cell differentiation and brain development. Our results show significant pathobiological effects and potential mechanisms through which Wwox LoF leads to cerebellar neurodegeneration, neuroinflammation, and ataxia.

overall_design: To gain insight into transcriptional changes associated with Wwox P47T mutation phenotypes, we first performed bulk RNA sequencing (RNA-seq) on brain tissue regions:  prefrontal cortex (PFC), parietal cortex (CTX), hippocampus (HPC) and cerebellum (CB) from WwoxWT/WT (n=5) and WwoxP47T/P47T (n=5) mice.

first: C

last: Aldaz

first: Hussain

last: Tabish


title: Xeno-free cultured Mesenchymal Stromal Cells release Extracellular Vesicles with a “therapeutic” miRNA cargo ameliorating cartilage inflammation in vitro

city: Genova

first: simona

last: coco

address: L.go R. Benzi, 10

country: Italy

institute: IRCCS Ospedale Policlinico San Martino

summary: Mesenchymal Stromal Cells (MSCs)-derived Extracellular Vesicles (EVs) emerged as an innovative strategy for the treatment of osteoarthritis (OA). Biological activity of EVs is generally driven by their cargo, which might be influenced by microenvironment. Therefore, pre-conditioning strategies, including modifications in culture conditions or oxygen tension could directly impact on MSCs paracrine activity.   Methods: A xeno-free supplement (XFS) was used for isolation and expansion of MSCs and compared to conventional fetal bovine serum (FBS) culture. Bone Marrow-derived MSCs (BMSCs) were pre-conditioned under normoxia (20% O2) or under hypoxia (1% O2) and EVs production was evaluated. Anti-OA activity was evaluated by using an in vitro inflammatory model. miRNA content was also explored, to select putative miRNA that could be involved in a biological function.  Results: Modulation of IL-6, IL-8 and COX-2 was evaluated on hACs simultaneously treated with IL-1α and BMSC-derived EVs. FBS-sEVs exerted a blunt inhibitory effect, while a strong anti-inflammatory outcome was achieved by XFS-sEVs. Interestingly, in both cases hypoxia pre-conditioning allowed to increase EVs effectiveness. Analysis of miRNA content showed the upregulation in XFS-hBMSC-derived EVs of miRNA known to have a chondroprotective role, such as let-7b-5p, miR-17, miR-145, miR-21-5p, miR-214-3p, miR-30b-5p, miR-30c-5p. Activated pathways and target genes were investigated in silico and most of the upregulated miRNAs were found to be involved in TGF-beta and Wnt signalling pathways, by targeting genes related to cartilage homeostasis.  Conclusions: XFS medium was found to be suitable for isolation and expansion of MSCs, secreting EVs with a therapeutic cargo. The application of cells cultured exclusively in XFS overcomes issues of safety associated with serum-containing media and makes ready-to-use clinical therapies more accessible.

overall_design: Samples from two different primary cultures were analyzed, in 4 different conditions. Samples from 1 to 4 are treatments, samples from 5 to 8 are controls

first: Maria

last: Palamà

last: Coco

first: Giovanna

last: Chiorino

first: Chiara

last: Gentili


title: Latexin regulates hematopoiesis in a sex-dependent manner by differential expression of sex chromosome-specific microRNA 98-3p and thrombospondin 1

city: Lexington

first: Ying

last: Liang

state: KY

address: 760 press avenue

institute: University of Kentucky

summary: To investigate the molecular mechanism underlying gender-specific regulation of hematopoiesis by Lxn. RNA sequencing was performed on LSK cells that were isolated from male and female WT and Lxn-/- mice.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for male WT, male Lxn-/-, female WT, and female Lxn-/-.

first: Xiaojing

last: Cui

first: Cuiping

last: Zhang


title: Identify mRNAs binding with IGF2BP1 in PLC-8024 cells [RIP-seq]

city: Guangzhou

first: Binkui

last: Li

address: 651 Dongfeng East Road

country: China

institute: Sun Yat-sen University Cancer Center

summary: To investigate mRNAs binding with IGF2BP1 in HCC, we employed the RIP-seq

overall_design: IGF2BP1 immunoprecipitation was performed and the strand-specific libraries were sequenced on Illumina Novaseq 6000 system with paired-end 2×150 bp read length.

first: Yunxing

last: Shi

first: Xin

last: Feng


title: Effect of depletion of PRMT3 and IGF2BP1 on gene expression in PLC-8024 cells [PRMT3 IGF2BP1 KD RNA-seq]

summary: To investigate the  function PRMT3 and IGF2BP1 in HCC, we established PLC-8024 cell lines in which each target gene has been knocked down by shRNA.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for PLC-8024 cells and its KD derivatives.


title: Effect of Cdc50 knockout on gene expression in Candida glabrata

city: Shanghai

first: Ke-Zhi

last: Chen

address: 197 Ruijin 2nd Road

department: Department of laboratory medicine

institute: Ruijin Hospital, Shanghai Jiao Tong University School of Medicine

summary: To estiamte the role lipid flippase subunit Cdc50 plays in Candida glabrata, a Δcdc50 null mutant was constructed based on standard strain ATCC2001 (wild type, WT) through homologous recombination. Then, gene expression profiling analysis was performed using data obtained from RNA-seq of WT and Δcdc50 mutant.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for ATCC2001 yeasts and its Δcdc50 null mutant.

first: Lu-Ling

last: Wang

first: Jin-Yan

last: Liu

first: Jun-Tao

last: Zhao


title: Single-nucleus sequencing deciphers developmental trajectories in rice pistils

city: Singapore

first: Hao

last: Yu

address: 16 Science Drive 4

country: Singapore

institute: National University of Singapore

summary:
Angiosperms possess a characteristic life cycle with an alternation of sporophyte and gametophyte generations, which happens in plant organs like pistils. Rice pistils contain ovules and receive pollen for successful fertilization to produce grains. The cellular expression profile in rice pistils is largely unknown. Here we show a cell atlas of rice pistils before fertilization by droplet-based single-nucleus RNA sequencing. The Ab initio marker identification validated by in situ hybridization assists the cell-type annotation, revealing cell heterogeneity between ovule and carpel lineages.

This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Effect of CRISPR/Cas9 activating library on oxaliplatin resistance during oxaliplatin treatment of HepG2 cells [CRISPR cas9]

summary: To identify key drivers of oxaliplatin, Cas9 protein was overexpressed in HepG2 cells. Next, we treated the cell pool with vehicle or OXA for 3 days to enable positive and negative screening

overall_design: We performed gene expression profiling analysis using data obtained from groups


title: The NLRomes of Zea mays NAM founder lines and Zea Luxurians Display Presence Absence Variation, Integrated Domain Diversity and Mobility

city: Johnston

first: Shawn

last: Thatcher

state: Iowa

address: South Drive

institute: Corteva Agriscience

summary: We analyzed the nucleotide-binding leucine-rich repeat receptors (NLRs) of 26 recently sequenced diverse founder lines from the maize nested association mapping (NAM) population and compared them to the R gene complement present in a wild relative of maize, Zea luxurians.

overall_design: RNA-seq quantification of NLRs of the maize NAM founder lines


title: RNA-seq of two Kaposi’s sarcoma tissue samples and the corresponding two normal tissue samples

first: Zhengzhou

last: Lu

state: Guangdong province

address: 74 Zhongshan Second Road

institute: Sun Yat-Sen University

summary: We have reported that KSHV infection reprograms oral mesenchymal stem cells (MSCs) and transforms them to Kaposi sarcoma-like cells through mesenchymal-to-endothelial transition. To explore the mechanism of KSHV infection of MSCs, we compared the gene expression profiles of Kaposi’s sarcoma tissue and KSHV non-susceptible cells to explore potential host factors that contribute to KSHV infection.

overall_design: RNA-seq of two Kaposi’s sarcoma tissue samples (P2 Tumor and P4 Tumor) and the corresponding two normal tissue samples (P2 Normal and P4 Normal)

first: Mu-Sheng

last: Zeng

first: Yan

last: Yuan


title: Identify mRNA with m6A modification in PLC-8024 cells [MeRIP-seq]

summary: To investigate the m6A mapping in HCC, we employed MeRIP-seq to identify mRNAs with m6A modification

overall_design: m6A immunoprecipitation was performed and the strand-specific libraries were sequenced on Illumina Novaseq 6000 system with paired-end 2×150 bp read length.


title: Transcriptional Regulation by NFATc1 during murine thymocyte development [gammadelta_T_cells RNA-seq]

city: Mainz

first: Matthias

last: Klein

address: Langenbeckstrasse 1

department: Inst. for Immunology

country: Germany

institute: University Medical Center Mainz

summary: In thymus hematopoietic precursor cells differentiate into αβ T cells, γδ T cells, mucosa-associated invariant T cells (MAIT), and natural killer T (NKT) cells. We show that both ablation of NFATc1 or its induction during the DN stages of thymocyte development leads to an almost normal thymocyte development but a marked increase in γδ T cells. The γδ cells deficient for NFATc1 acquire an NKT γδ cell phenotype that exhibits the expression of CD4 co-receptor, the NK1.1 marker, the augmented usage of the Vγ1.1 and Vδ6.3 segments, and an increased in IL4 and IFN-γ production.

overall_design: Transcriptom analysis of isolated γδ thymocytes. Nfatc1-fl/fl (WT) express normal levels of NFATc1 while in Rag1cre-Nfatc1-fl/fl NFATc1 expression is ablatedby Rag1 driven Cre activity (KO). For both settings triplicates were analysed

first: Stefan

last: Klein-Heßling

first: Sabrina

last: Giampaolo


title: Transcriptional Regulation by NFATc1 during murine thymocyte development [BirA RNA-seq]

overall_design: Transcriptom analysis of isolated CD4-CD8- double negative thymocytes. BirA cells express normal levels of NFATc1 while Nc1Bio overexpress NFATc1 from one copy of a BAC transgene containing the whole murine Nfatc1 gene locus. For both settings triplicates were analysed


title: Differentially expressed genes between oxaliplatin-resistant and oxaliplatin-non-resistant patient samples [Clinical samples-RNA-seq]

summary: To identify key drivers of oxaliplatin resistance, we collected and analyzed the DEGs between oxaliplatin-resistant and oxaliplatin-non-resistant patient samples

overall_design: Comparative gene expression profiling analysis of RNA-seq data for oxaliplatin resistant and non-resistant patients samples.


title: Transcriptional Regulation by NFATc1 during murine thymocyte development [ChIP-seq]

overall_design: DNA binding of NFATc1A in murine thymocytes (either directly ex vivo (-) or in vitro stimulated by TPA and ionomycin for 4 hours(+))  analyzed by ChIP and deep sequencing. Cells from genotype Rosa26BirA expressing BirA ligase only are used as control. Cells from genotype Nfatc1/A-Bio.Rosa26BirA express an modified NFATc1A protein from a BAC transgene that is biotinylated by BirA in vivo.


title: The efficacy of novel anti-cancer agents DpC and Dp44mT in the Treatment of Estrogen Receptor Positive Breast Cancer (BC)

city: Darlinghurst

first: Heloisa

middle: Helena

last: Milioli

state: NSW

address: 384, Victoria St

department: Cancer

country: Australia

institute: Garvan Institute of Medical Research

summary: Estrogen receptor α (ER-α) is a major driver of breast cancer (BC), being expressed in 75% of all BC cases. Agents such as tamoxifen are used to block ER-α activity and interfere with its down-stream oncogenic singling. However, a major problem associated with tamoxifen is the emergence of resistance. Resistant BC is often associated with aggressive relapse, metastasis, and high mortality rates of 80%. A key mechanism for development of resistance is activation of EGFR/HER2/HER3 signaling pathways and other hormone receptors (androgen receptor (AR), progesterone receptor (PR), prolactin receptors (PRL-R)) that can intrinsically activate ER-α. This study investigated a novel class of thiosemicarbazone anti-cancer agents, namely Dp44mT and DpC, on the expression and activation between major BC hormonal receptors, their co-factors and key pathways involved in resistance in ER-α-positive BC using RNA sequencing, immunoblotting and confocal microscopy and an in vivo orthotopic BC model. For the first time, we demonstrate that DpC and Dp44mT markedly reduce the expression of ER-α, AR, PR and PRL-R by inducing their proteasomal degradation. Further, these agents also inhibited EGFR, HER2 and HER3 activation in ER-α-positive BC cells, suggesting their potential ability to overcome development of resistance. Importantly, expression of key co-factors that promote ER-α-transcriptional activity, including SRC3, c-Myc, SP1, and c-Src, were also decreased by these agents. This study demonstrates for the first time the multi-axial inhibitory effects of the novel DpT agents in ER-positive BC invitro and in vivo. We demonstrate that Dp44mT and DpC decrease ER-α expression, transcriptional activity, and promote its proteasomal degradation. This study also demonstrated the ability of the DpT agents in inhibiting RTKs and key oncogenic pathways that are well established to promote endocrine resistance. Hence, for the first time we demonstrate that Dp44mT and DpC may be a promising therapeutic approach to treat ER-positive BC and to overcome resistance to conventional BC therapies.

overall_design: MCF-7 cells were treated with either control media or media containing DpC (5 μM) for 24 h. Isolation of mRNA was then performed using TRIzol® (Invitrogen) according to the manufacturers’ procedures. RNA sequencing was performed by next generation sequencing (NGS) using the Illumina NextSeq technology at the Ramaciotti Centre for Genomics. Sequences were trimmed using Trim Galore (version 0.4.4) and genome-guided alignment to a human reference (HG38) was performed using the STAR software (version 2.5). Duplicated reads were located and tagged using Picard tools (version 1.138), MarkDuplicates. Differential gene expression was computed in R (version 3.4.3) using the package Rsubread 1.28.0. The differential gene expression analysis was conducted using edgeR 3.20.1 and limma 3.34.1 packages, and the gene set enrichment analysis (GSEA) with cluterProfiler.

middle: H

first: Faten

last: Shehadeh Tout

first: Zaklina

last: Kovacevic


title: Tcf21 functions as a rate-limiting factor during visceral adipose tissue development [ATAC- and RNA-Seq]

city: Baton Rouge

first: Xing

last: Fu

state: LA

address: 229G Animal and Food Sciences laboratory building

department: School of Animal Sciences

institute: Louisiana State University

summary: The location of visceral adipose tissue (VAT) suggests specific developmental regulation. Our lineage-tracing study identified the specific expression of Tcf21 in VAT progenitor cells (PCs). Tcf21 lineage PCs (LPs) actively differentiate into adipocytes during development but not in adulthood. Bulk RNAseq identified dynamic gene expression of Tcf21 LPs, including enrichment of pro-mitotic and pro-adipogenic genes during development and increased expression of a set of fibrotic genes in obese mice. ATACseq revealed chromatin remodeling that was highly correlated with gene expression dynamics. Using a novel computational approach via integrative analysis of gene expression and chromatin accessibility profiles, an underlying gene regulatory network was reconstructed. Single-cell RNAseq identified subpopulations of Tcf21 LPs including mesothelial and interstitial subpopulations. Using an inducible Tcf21 knockout line, we identified that loss of Tcf21 promoted the adipogenesis and developmental progress of Tcf21 LPs, leading to improved metabolic health of mice after HFD challenge. Mechanistic studies showed that Tcf21 mainly targets enhancers, and the inhibitory effect of Tcf21 on adipogenesis is at least partially through the increasing Dlk1 expression.

overall_design: Bulk RNA-seq and ATAC-seq

first: Jiangwen

last: Sun

first: Mohammad

last: Shiri


title: Reconceptualising resilience within a translational framework is supported by unique and brain-region specific transcriptional signatures in mice

city: Göttingen

first: Thomas

last: Lingner

address: Rudolf-Wissell-Str. 28a

institute: Genevention GmbH

summary: Chronic social defeat (CSD) in mice has been increasingly employed in experimental resilience research. Particularly, the degree of CSD-induced social avoidance is used to classify animals into resilient (socially non-avoidant) versus susceptible (avoidant). Inspired by human data pointing to threat-safety discrimination and responsiveness to extinction training of aversive memories as characteristics of resilient individuals, we here describe a translationally informed stratification which identified three phenotypic subgroups of mice following CSD: the Discriminating-avoiders, characterised by successful social threat-safety discrimination and successful extinction of social avoidance; the Indiscriminate-avoiders, showing fear generalisation, and the Non-avoiders (absence of social avoidance) displaying impaired conditioned learning. Furthermore, and supporting the biological validity of our approach, we uncovered subgroup-specific transcriptional signatures in classical fear conditioning and anxiety-related brain regions. Our reconceptualisation of resilience in mice refines the currently used dichotomous classification and contributes to advancing future translational approaches.

overall_design: Chronic social defeat (CSD) took place for 10 days on 165 mice (Defeated) while 55 animals served as a control group. Following the last session by 24h, the Modified Social Interaction test (MSIT) was performed. Following the test, Defeated mice were divided into three subgroups in addition to the Control group. Cue discrimination test followed the MSIT 48h later on the first subset of animals; n=10 Non-avoiders, n=17 Indiscriminate-avoiders, and n=11 Discriminating-avoiders. Active avoidance task followed the MSIT 48h later on another subset of animals; n=18 Non-avoiders, n=10 Indiscriminate-avoiders, and n=9 Discriminating-avoiders. Social avoidance extinction treatment followed the MSIT 24h later and lasted for 16 consecutive days on yet another subset of animals; n=21 Indiscriminate-avoiders and n=25 Discriminating-avoiders. Following the last session by 24h, the second MSIT took place. Finally, remaining animals were sacrificed following the (first) MSIT and used for transcriptome analysis.

first: Sarah

last: Ayash

first: Soojin

last: Ryu

first: Raffael

last: Kalisch

first: Ulrich

last: Schmitt

first: Marianne

last: Mueller


title: Lipocalin-2 deficiency attenuates acute lung inflammation

city: JINJU

first: GU SEOB

last: ROH

address: 15, 816 Beon-gil, Jinju-daero

country: South Korea

institute: Gyeongsang National University

summary: Lipocalin-2 promotes acute lung inflammation

overall_design: WT(control), WT LPS(sample)

first: Gu Seob

last: Roh


title: Tcf21 functions as a rate-limiting factor during visceral adipose tissue development [single-cell RNA-seq]

overall_design: Single-cell RNA-seq


title: Tcf21 functions as a rate-limiting factor during visceral adipose tissue development

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


title: A pan-grass transcriptome reveals patterns of cellular divergence in crops

status: Public on Feb 27 2023

city: New York

first: Bruno

last: Guillotin

address: 12 Waverly Place, Floor 6

department: Biology

institute: NYU-Biology-CGSB

summary: Different plant species within the grasses were parallel targets of domestication, giving rise to crops with distinct evolutionary histories and traits. Key traits that distinguish these species are mediated by specialized cell types. Here, we compare the transcriptomes of root cells in three grass species—Zea mays (maize), Sorghum bicolor (sorghum), and Setaria viridis (Setaria). We first show that single-cell and single-nucleus RNA-seq provide complementary readouts of cell identity in both dicots and monocots, warranting a combined analysis. Cell types were mapped across species to identify robust, orthologous marker genes. The comparative cellular analysis shows that the transcriptomes of some cell types diverged more rapidly than others—driven, in part, by recruitment of gene modules from other cell types. The data also show that a recent whole genome duplication provides a rich source of new, highly localized gene expression domains that favor fast- evolving cell types. Together, the cell-by-cell comparative analysis shows how fine-scale cellular profiling can extract conserved modules from a pan transcriptome and shed light on the evolution of cells that mediate key functions in crops.

sample_organism:
Arabidopsis thaliana
Setaria viridis
Sorghum bicolor
Zea mays

overall_design: Single cell and single nuclei integration of Arabidospsis thaliana, Zea mays, Sorghum bicolor and Setaria viridis root meristems. Arabidopsis 6 cell replicates and 3 nuclei replicates. Maize 4 nuclei replicates and 3 cell replicates from (Ortiz Ramirez et al., 2021, 10.1126/science.abj2327). Sorghum 1 cell replicate and 3 nuclei replicates. Setaria 3 cell replicates and 2 nuclei replicates

first: Ramin

last: Rahni

first: Michael

last: Passalacqua

first: Mohammed

middle: A

last: Mohammed

first: Xiaosa

last: Xu

first: Sunil

last: Raju

first: Carlos

middle: O

last: Ramírez

first: David

last: Jackson

first: Simon

middle: C

last: Groen

first: Jesse

last: Gillis

first: Kenneth

last: Birnbaum


title: A proteomic and transcriptomic screening demonstrate increased mast cell-derived CCL23 in systemic mastocytosis

city: Solna

first: Joakim

last: Dahlin

address: CMM, Visionsgatan 18

department: Department of Medicine, Solna

country: Sweden

institute: Karolinska Institutet

summary: Systemic mastocytosis is a heterogeneous group of mast cell-driven diseases, diagnosed by bone marrow sampling. However, there are a limited number of available blood disease biomarkers. We performed a single-cell transcriptomics screen alongside a plasma proteomics screen to identify such potential biomarkers.

overall_design:
We performed single-cell RNA sequencing of isolated human bone marrow cells from patients with Systemic Mastocytosis. 3 datasets from 3 patients respectively in total were generated consisting of Ficoll separated cells mixed with FACS-isolated CD117+ FCɛR1+ cells (mast cells)
*** Please note that Swedish legislation and GDPR prevent unrestricted distribution of raw sequencing data and thus raw data is not provided ***

first: Stina

last: Soderlund

first: Daryl

last: Boey

first: Wouter

last: van Midden

first: Matilda

last: Kjellander

first: Kajsa

last: Ax

first: Hong

last: Qian

first: Johanna

last: Ungerstedt


title: Single-cell RNA sequencing of hematopoietic stem and progenitor cells from young and aged mice

city: Paris

first: Jason

last: Cosgrove

address: 11 Rue Pierre et Marie Curie, Institut Curie, Batiment Curie, RDC12

institute: INSTITUT CURIE

summary: Ageing is associated with changes in the cellular composition of the immune system. During ageing, hematopoietic stem and progenitor cells (HSPCs) that produce immune cells are thought to decline in their regenerative capacity.  However, HSPC function has been mostly assessed using transplantation assays, and it remains unclear how HSPCs age in the native bone marrow niche. To address this issue, we present a novel in situ single cell lineage tracing technology to quantify the clonal composition and cell production of single cells in their native niche. Our results demonstrate that a pool of HSPCs with unequal output maintains myelopoiesis through overlapping waves of cell production throughout adult life. During ageing, the increased frequency of myeloid cells is explained by greater numbers of HSPCs contributing to myelopoiesis, rather than increased myeloid output of individual HSPCs. Strikingly, the myeloid output of HSPCs remained remains constant over time despite accumulating significant transcriptomic changes throughout adulthood. Together, these results show that, unlike emergency myelopoiesis post-transplantation, aged HSPCs in their native microenvironment do not functionally decline in their regenerative capacity. This repository contains single-cell RNA sequencing datasets of hematopoietic stem and progenitor cells from young and aged mice associated with this study

overall_design: Sca1+ cKit+ HSPCs were purified from the bone marrow of DRAG mice by fluorescence-acticated cell sorting (FACS) and cells were analysed using scRNAseq using the 10X genomics platform

first: Leila

last: Perie


title: RNA Sequencing Analysis of Nicol-Het, Nicol-KO and Ros1-KO Caput Epididymal Transcriptomes.

city: Nagoya

first: Daiji

last: Kiyozumi

state: Aichi

address: Furocho, Chikusa-ku

department: Research Institute of Environmental Medicine

country: Japan

institute: Nagoya University

summary: Purpose: The goal of this study is to detect differentially expressed genes, between Nicol +/- (Nicol-Het), Nicol-/- (Nicol-KO), and Ros1-/- (Ros1-KO) caput epididymis by RNA sequencing  Methods: Caput epidiymal mRNA profiles of 14-week-old Nicol-Het, Nicol-KO, and Ros1-KO mice were generated by deep sequencing, in triplicate, using Illumina NovaSeq6000.  Results: RNA-seq data identified differentially expressed transcripts.  Conclusions: Our results show that the expression of many genes were downregulated in Nicol-KO caput epididymis compared with Nicol-Het one. The gene downregulation of Nicol-KO caput epididymis  was similar to that of Ros1-KO one.

overall_design: Caput epididymal mRNA profiles of 14-week old Nicol-Het, Nicol-KO, and Ros1-KO mice were generated by deep sequencing, in quadplicate, using Illumina NovaSeq6000.

first: Kentaro

last: Shimada

first: Masahito

last: Ikawa


title: RRC1 is required for epidermal phyB siganling by maintaining the circadian rhythm by suppressing the alternative splicing of core clock genes.

city: Daejeon

first: hanim

last: kim

address: Daehak-ro 291

department: biological science

institute: KAIST

summary:
Purpose : The goal of this study is to identify transcriptome changes induced by rrc1 mutation under red light. We compared the transcriptome of MLB (ML1pro::phyB-GFP/phyB-9) with EMS27 grown either in darkness or continuous red light to investigate the genes that the expression and the splicing patterns are regulated by epidermal phyB and/or RRC1. The transcriptome of Col-0 and rrc1-689 was further compared to enlarge the list of putative splicing targets of RRC1.
Methods : Seedlings grown under either monochromatic red light (19 μmol/m2s) or darkness for 60 hours on 1/2 MS agar plates were sampled. Total RNAs were extracted using the Spectrum Plant Total RNA Extraction Kit (Sigma-Aldrich). RNA libraries were generated and sequenced on the Illumina NextSeq 500 platform. The analysis was performed with three biological replicates. Roughly 90% of the sequencing reads were mapped to specific loci on the Arabidopsis TAIR10 reference genome using the TopHat pipeline. Mapped sequencing files were further processed with Aspli1.9 R package for Differentially Expressed Gene (DEG) analysis and Differentially Spliced Gene (DSG) analysis.

overall_design: Epidermal phyB-induced gene expression change was identified by comparing Dark-grown MLB with Red-grown MLB, while RRC1-induced gene expression or splicing change was identified by comparing Red-grown control line (Col-0 or MLB) with Red-grown rrc1 mutant (rrc1-689 or EMS27). All samples in triplicates.

first: Hanim

last: Kim

first: Jaewook

first: Giltsu

last: Choi


title: Transgenerational impact of grand-paternal lifetime exposures to both folic acid deficiency and supplementation on genome-wide DNA methylation in male germ cells

city: Montreal

first: Jacquetta

last: Trasler

state: Quebec

address: 1001 Decarie Boul, Block E, ES1.4380

department: Pharmacology & Therapeutics; Human Genetics

country: Canada

institute: McGill University

summary:
Background: DNA methylation (DNAme) erasure and reacquisition occurs during prenatal male germ cell development; some further remodelling takes place after birth during spermatogenesis. Environmental insults during germline epigenetic reprogramming may affect DNAme, presenting a potential mechanism for transmission of environmental exposures across multiple generations.
Objectives: We investigated how germ cell DNAme is impacted by lifetime exposures to diets containing either low or high, clinically relevant, levels of the methyl donor folic acid and whether resulting DNAme alterations were inherited in germ cells of male offspring of subsequent generations.
Materials and Methods: Female mice were placed on a 7-fold folic acid deficient (7FD) and 10- or 20-fold supplemented (10FS and 20FS) diets before and during pregnancy. Resulting F1 litters were weaned on the respective diets. F2 and F3 males received control diets. Genome-wide DNAme was assessed in F1 spermatogonia, and in F1, F2 and F3 sperm.
Results: In F1 germ cells, a greater number of differentially methylated cytosines (DMCs) was observed in spermatogonia as compared with F1 sperm for all folic acid diets. DMCs were lower in number in F2 versus F1 sperm, while an unexpected increase was found in F3 sperm. DMCs were predominantly hypomethylated, with genes in neurodevelopmental pathways commonly affected in F1, F2 and F3 male germ cells. While no DMCs were found to be inherited inter- or transgenerationally, we observed over-representation of repetitive elements, particularly young LINEs.
Discussion and Conclusion: These results suggest that the prenatal window is the time most susceptible to folate-induced alterations in sperm DNAme in male germ cells. Altered methylation of specific sites in F1 sperm was not present in later generations. However, the finding of hypomethylated young LINE1 elements in sperm from F1 to F3 males provides insight into potential mechanisms of epigenetic inheritance of the effects of folate deficiency and supplementation.

overall_design: In order to examine the effects of pre-natal exposures of folic acid supplementation and deficiency,  F0 BALB/s females were placed on the different diets for 4 weeks prior to mating with Oct4-GFP +/- BALB/c males fed rodent chow.  Females were maintained on diets throughout mating, gestation and lactation. F1 Oct4-GFP +/- males were sacrificed at PND 6 for spermatogonia isolation via fluorescent activated cell sorting.  In order to study the effect of inter- and transgenerational effects of folic acid supplementation and deficiency, F0 BALB/s females were placed on the different diets for 4 weeks prior to mating with rodent chow fed males.  The females were maintained on their folic acid defined diets throughout mating, gestation and lactation.  Resultant F1 males were weaned onto their respective folic acid defined diets.  Upon sexual maturity, F1 males were mated with rodent chow fed females.  These females remained on the rodent chow diet throughout gestation and lactation. Subsequent F2 male pups were weaned and maintained on regular rodent chow until 18 weeks of age, then were mated with 10-week old female BALB/c fed with regular rodent chow to produce unexposed F3 litters.

first: Donovan

last: Chan

first: Lunid

last: Ly

first: Edgar

last: Martínez Duncker Rebolledo

first: Josée

last: Martel

first: Mylène

last: Landry

first: Marie-Pier

last: Scott-Boyer

first: Arnaud

last: Droit


title: Transcriptional Regulation by NFATc1 during murine thymocyte development

first: Matthias

last: Klein

overall_design: Refer to individual Series


title: ChIP-seq analysis for core 3' processing factors in control and U1 AMO-treated HeLa cells

city: guangzhou

first: chengguo

last: yao

address: No. 74 zhongshan 2rd road

department: school of medicine

institute: sysu

summary: To understand the effect of U1 AMO treatment on chromatin binding profiles of core 3' processing factors, we carried out ChIP-seq analysis for several core 3' processing factors after control and U1 AMO treatment

overall_design: control and U1 AMOs were transfected into HeLa cells,ChIP-seqs were carried out for core 3' processing factors

first: Yanhui

last: Deng

first: Yi

last: Ran

first: Junjie

last: Shi

first: Chengguo

last: Yao


title: wdr33 iCLIP-seq in control and U1 AMO-treated HeLa cells

summary: To understand the effect of U1 AMO treatment on wdr33 binding profile , we carried out CstF64 iCLIP-seq experiments in HeLa cells treated with either control or U1 AMO.

overall_design: Control and U1 AMO were transfected into HeLa cells,after 12 hour. wdr33 iCLIP-seq(s) were carried out using the standard iCLIP-seq protocol (König J et al. 2010)


title: poly(A+) RNAs 3'-seq analysis in the cytoplasmic RNAs of control and U1 AMO treated Hela cells

summary: To understand if U1-AMO induced truncated forms of mRNAs could be exported into cytoplasm, we performed 3'-seq analysis in the cytoplasmic RNAs of control and U1 AMO treated Hela cells

overall_design: Control and U1 AMO were transfected into Hela cells, cytoplasmic fractions were isolated and RNAs were subsequently purified by Trizol reagent. 3'-seq analysis was performed using lexogen kit (016.024)


title: ribo-seq in control and U1 AMO treated HeLa cells

summary: Control and U1 AMO were transfected into Hela cells, Ribosome-associated RNAs were purified and sequenced in Novaseq platform.To compare the translation efficiency, mRNA-seq were performed in parallel.

overall_design: To understand the ribosome-binding profile of mRNAs in control and U1 AMO treated HeLa cells, we carried out Ribo-seq analysis in control and U1 AMO treated HeLa cells. To compare the translation efficiency, we performed mRNA-seq analysis in parallel.

first: Qiumin

last: Feng


title: The U1 antisense morpholino oligonucleotide (AMO) disrupts U1 snRNP structure to promote intronic PCPA modification of pre-mRNAs


title: Sex differences in NK cells mediated by the X-linked epigenetic regulator UTX

city: Los Angeles

first: Feiyang

last: Ma

address: 610, Charles Young Dr East, TLSB 3000C

department: Molecular Biology Institute

institute: UCLA

summary: Sexually dimorphic immune responses are evident by increased susceptibility of males to viral infections. Interestingly, our results indicate that while males possess more natural killer (NK) cells, they display impaired effector function. These differences are attributed to lower male expression of X-linked UTX, an epigenetic regulator which escapes X-inactivation in females. NK cell-specific heterozygous deletion of UTX in females recapitulated male NK cell phenotypes of increased NK cells and defective effector function. Notably, mice with NK cell-intrinsic UTX deficiency show increased lethality to mouse cytomegalovirus. Integrative UTX CUT&Tag, ATAC-seq and RNA-seq analysis revealed a critical role for UTX in regulating the chromatin landscape and expression at gene loci involved in NK cell homeostasis and effector function. Collectively, these data implicate UTX as a molecular determinant of sex differences in NK cell homeostasis and effector function and suggest enhancing UTX as a strategy to boost endogenous NK effector responses.

overall_design:
Bulk RNA-seq and ATAC-seq on wild type or UTX-deficient mouse NK cells following viral infection
CUT&Tag followed by Illumina sequencing

first: Mandy

middle: I

last: Cheng

first: Joey

last: Li

first: Luke

last: Riggan

first: Bryan

last: Chen

first: Rana

last: Yakhshi Tafti

first: Scott

last: Chin

first: Matteo

last: Pellegrini

first: Haley

last: Hrncir

first: Arthur

last: Arnold

first: Timothy

middle: E

last: O'Sullivan

first: Maureen

last: Su


title: poly(A+) RNAs 3'-seq analysis in control and U1-specific proteins KD (knock-down) samples

first: chengguo

last: yao

summary: To understand the effect of U1 snRNP-specific proteins on poly(A+) RNAs profiles, we carried out control and U1A, U1C, U1-70k depletion (using synthetic siRNAs) experiments followed by poly(A+) RNA 3'-seq anlyais using lexogen kit (016.024)in HeLa cells

overall_design: siRNAs targeting U1A, U1-70K, U1C were designed and transfected into Hela cells,knock-down efficiency was confirmed by western blots. 3'-seq analysis was performed using lexogen kit (016.024)

first: Yanhui

last: Deng


title: poly(A+) RNAs 3'-seq analysis in control and FUS KD (knock-down) samples

summary: To understand the effect of FUS protein knock-down on poly(A+) RNAs profiles, we carried out control and FUS depletion (using synthetic siRNAs) experiments followed by poly(A+) RNA 3'-seq anlyais using lexogen kit (016.024)in HeLa cells

overall_design:
siRNAs targeting FUS was designed and transfected into Hela cells,knock-down efficiency was confirmed by western blots. 3'-seq analysis was performed using lexogen kit (016.024)

si-control-U1-AMO:
This experiment was designed and carried out to serve as positive control for 3'-seq under FUS knockdown condition
Negative control siRNA and U1 AMO was transfected into Hela cells, 3'-seq  was subsequently carried out to examine the polyadenylation site usage at the transcriptome level using a lexgen kit (Cat. 016.24)

first: Yi

last: Ran

first: Junjie

last: Shi


title: Spt6 directly interacts with Cdc73 and is required for Paf1C occupancy at active genes in Saccharomyces cerevisiae

status: Public on Feb 24 2023

city: Pittsburgh

first: Mitchell

last: Ellison

email: mae92@pitt.edu, mae133@pitt.edu, melliso46@gmail.com, ellisoniima@upmc.edu

state: PA

address: Langley Hall / 3477 Euler Way

department: Pathology / Biological Sciences

institute: University of Pittsburgh

summary: The Paf1 complex (Paf1C) is a conserved transcription elongation factor that regulates transcription elongation efficiency, facilitates co-transcriptional histone modifications, and impacts molecular processes linked to RNA synthesis, such as polyA site selection. Coupling of the activities of Paf1C to transcription elongation requires its association with RNA polymerase II (Pol II). Mutational studies in yeast identified Paf1C subunits Cdc73 and Rtf1 as important mediators of Paf1C association with Pol II on active genes. While the interaction between Rtf1 and the general elongation factor Spt5 is relatively well-understood, the interactions involving Cdc73 have not been fully elucidated. Using a site-specific protein cross-linking strategy in yeast cells, we identified direct interactions between Cdc73 and two components of the Pol II elongation complex, the elongation factor Spt6 and the largest subunit of Pol II. Both of these interactions require the tandem SH2 domain of Spt6. We also show that Cdc73 and Spt6 can interact in vitro and that rapid depletion of Spt6 dissociates Paf1 from chromatin, altering patterns of Paf1C-dependent histone modifications genome-wide. These results reveal interactions between Cdc73 and the Pol II elongation complex and identify Spt6 as a key factor contributing to the occupancy of Paf1C at active genes in Saccharomyces cerevisiae.

overall_design: ChIP-seq analysis of proteins involved in transcription elongation, histones, and histone modifications in an SPT6-AID strain with and without acute depletion of Spt6 conducted in biological duplicate

first: Sanchirmaa

last: Namjilsuren

first: Margaret

last: Shirra

first: Matthew

middle: S

last: Blacksmith

first: Rachel

last: Schusteff

first: Eleanor

middle: M

last: Kerr

first: Fei

last: Fang

first: Yufei

last: Xhiang

first: Karen

last: Arndt


title: Systematic fine-mapping and functional studies of prostate cancer risk variants [4C]

status: Public on Feb 25 2023

city: Tianjin

first: Wange

last: Lu

address: 94 Weijin Road

institute: Nankai University

summary: To date, genome-wide association studies (GWAS) have revealed over 200 genetic risk loci associated with prostate cancer; yet, true disease-causing variants in gene regulatory regions remain elusive. Identification of causal variants and their targets from association signals relevant to prostate cancer is complicated by high linkage disequilibrium and limited availability of functional genomics data for specific tissue/cell types. Here, we integrated statistical fine-mapping and functional annotation from prostate-specific epigenomic profiles, high resolution 3D genome features, and quantitative trait loci data to distinguish causal variants from associations and identify target genes they regulate. Our fine-mapping analysis yielded 1,892 likely causal variants, and multiscale functional annotation linked them to 406 target genes. We prioritized rs10486567, located in an enhancer, as a genome-wide top-ranked SNP and predicted HOTTIP as its target. Deletion of the rs10486567-associated enhancer in prostate cancer cells decreased their capacity for invasive migration. HOTTIP overexpression in an enhancer-KO cell line rescued defective invasive migration. Furthermore, we found that rs10486567 regulates HOTTIP through allele-specific long- range chromatin interaction.

overall_design: Allele-specific 4C-seq of rs10486567 in 22Rv1

first: Yuyang

last: Qian


title: RNA localization to protrusions of mouse melanoma YUMM1.7 cells.

city: Dallas

first: Megan

middle: L

last: Norris

state: TX

address: 5323 Harry Hines Boulevard

department: Molecular Biology

institute: UT Southwestern Medical Center

summary: Subcellular localization of messenger RNA (mRNA) is a widespread phenomenon that can impact the regulation and function of the encoded protein. In non-neuronal cells, a subset of mRNAs localize to cell protrusions and proper mRNA localization is required for cell migration. However, the mechanisms by which mRNA localization regulates protein function in this setting remain unclear. Here, we examined the functional consequences of localization of the mRNA encoding KIF1C. KIF1C is a kinesin motor protein required for cell migration and mRNA trafficking, including trafficking of its own mRNA. We show that Kif1c mRNA localization does not regulate KIF1C protein abundance, distribution, οr ability to traffic other mRNAs. Conversely, robust Kif1c mRNA localization is required for directed cell migration. We used mass spectrometry to identify binding partners of endogenous KIF1C, which revealed dramatic dysregulation of the number and identity of KIF1C interactors in response to Kif1c mRNA mis-localization. These results therefore uncovered a mechanistic connection between mRNA localization to cell protrusions and the specificity of protein-protein interactions. We anticipate that this mechanism is not limited to Kif1c and is likely to be a general principle used by protrusion-enriched mRNAs in non-neuronal cells.

overall_design: Comparative gene expression analysis of RNA-seq data from cell protrusions and cell bodies of YUMM1.7 cells

first: Joshua

last: Mendell


title: Systematic fine-mapping and functional studies of prostate cancer risk variants [ChIP-seq]

overall_design: histone modifications of 22Rv1


title: pDC-like cells are pre-DC2 and require Klf4 to control homeostatic CD4 T cells

city: Basel

first: DBM Bioinformatics

last: Core Facility

state: BS

address: Hebelstrasse 20

department: Departement of Biomedicine

country: Switzerland

institute: University of Basel

summary: Plasmacytoid dendritic cells (pDCs) have been shown to play an important role during immune responses, ranging from initial viral control through the production of type I interferons (IFNs) to antigen presentation. However, recent evidence uncovered unexpected heterogeneity among pDCs. Notably we identified a new subset, referred to as pDC-like cells, that resembles pDCs but shares cDC features. Here we show that this subset is a circulating progenitor distinct from common DC progenitors (CDP), with prominent cDC2 precursor potential. This precursor subset responds to homeostatic cytokines, such as macrophage colony stimulating factor (M-CSF) by expanding and differentiating into cDC2 that efficiently prime Th17 cell. Development of pDC-like cells into CX3CR1+ESAM- cDC2b but not CX3CR1- ESAM+ cDC2a requires the transcription factor KLF4. Finally, we show that under homeostatic conditions this developmental pathway regulates the immune threshold at barrier sites, by controlling the pool of Th17 cells within the skin draining lymph nodes.

overall_design: Bulk RNA Sequencing of sort-purified DC subsets derived from different tissues.

first: Patrick

middle: F

last: Rodrigues

first: Robert

last: Ivanek

first: Roxane

last: Tussiwand


title: pH regulates hematopoietic stem cell potential via polyamines

city: Ulm

first: Medhanie

middle: Assmelash

last: Mulaw

address: Albert-Einstein-Allee 11

institute: University of Ulm

summary: Identification of a culture condition for maintaining the potential of HSCs ex vivo.

overall_design: We performed gene expression profiling analysis using data obtained from RNA-seq of HSCs cultured in 2 different pH conditions pH7.4 and pH 6.9.  Data was collected from 6 individual mice.


title: snRNA-seq data on human subcutaneous and omental white adipose tissue

status: Public on Feb 23 2023

city: Stockholm

first: Jiawei

last: Zhong

address: Blickagången 16, Flemingsberg

department: Department of Medicine, Huddinge

summary: We generated single nucleus transcriptomic atlas for human subcutaneous and omental white adipose tissue (WAT).

overall_design: Fresh or frozen WAT was used. The nuclei were extracted and snRNA-seq was performed.

first: Lucas

last: Massier

first: Pamela

last: Nono Nankam

first: Tongtong

last: Wang


title: INPP5D modulates TREM2 loss-of-function phenotypes in a β-amyloidosis mouse model

city: Hongo

first: Akihiro

last: Iguchi

state: Bunkyo-ku

department: Pharmaceutical Sciences

institute: The University of  Tokyo

summary: We crossed Tyrobp-deficient and Inpp5d-deficient mice with App(NL-G-F/NL-G-F) Alzheimer model mice (hereafter, NLGF) and generate Tyrobp-/-;Inpp5d+/-;NLGF mice to assess the role of Inpp5d in Tyrobp-/- mice. We isolated microglia from NLGF, Tyrobp-/-;NLGF, and Tyrobp-/-;Inpp5d+/-;NLGF mice and analyzed microglial transcriptome using RNA-sequencing. Tyrobp-/- microglia showed substantially different transcriptome compared with NLGF microglia. Previously described "disease asscociated microglia" genes were enriched in the differentially expressed genes in Tyrobp-/- microglia. On the other hand, Tyrobp-/-;Inpp5d+/-;NLGF microglia showed similar transcriptome to Tyrobp-/- microglia except several genes (e.g., Wdfy1, Mamdc2, Klrb1f). These data suggest Inpp5d modulates microglial functions without affecting disease associated microglial gene expression in Tyrobp-/-;NLGF micrglia.

overall_design: Microglial transcriptome of 9-month-old NLGF (N=3), Inpp5d+/-;NLGF (N=3), Tyrobp-/-;NLGF (N=2), Tyrobp-/-;Inpp5d+/-;NLGF (N=3) mice. CD11b+/CD45+ live cells were collected as microglia.

first: Sho

last: Takatori

first: Shingo

last: Kimura

first: Yukiko

last: Hori

first: Junko

last: Sasaki

first: Takashi

last: Saito

first: Takaomi

last: Saido

first: Tsuneya

last: Ikezu

first: Toshiyuki

last: Takai

first: Takehiko

first: Taisuke

last: Tomita

first: Hiroki

last: Muneto

first: Kai

first: Hayato

last: Etani

first: Genta

last: Ito

first: Haruaki

last: Sato


title: Granulosa cell mevalonate pathway abnormalities contribute to oocyte meiotic defects and aneuploidy

status: Public on Feb 26 2023

city: Shanghai

first: Qian

last: Bian

state: Shanghai

address: Pudong New District, Jinzun Rd, No.115

department: Ninth People’s Hospital

institute: Shanghai Jiao Tong University School of Medicine

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


title: Transcriptomic profileing of iPSC-derived engineered cardiac tissue in which cMyBP-C have been ablated in the heterozygous and homozygous states on an isogenic background

city: Madison

first: Willem

middle: Jacobus

last: DE LANGE

state: Wisconsin

address: Room 8553, 1111 Highland Av

department: Pediatrics

institute: University of Wisconsin-Madison

summary: Truncation mutations in cardiac myosin binding protein C (cMyBP-C), are common causes of hypertrophic cardiomyopathy (HCM). Heterozygous carriers present with classical HCM, while homozygous carriers present with early onset HCM that rapidly progress to heart failure. We used CRISPR-Cas9 to introduce heterozygous (cMyBP-C+/-) and homozygous (cMyBP-C-/-) frame-shift mutations into MYBPC3 in human iPSCs. Cardiomyocytes derived from these isogenic lines were used to generate engineered cardiac tissue constructs (ECTs). RNAseq analysis on 14 day old ECTs revealed enrichment of differentially expressed hypertrophic, sarcomeric, Ca2+-handling and metabolic genes in cMyBP-C+/- and cMyBP-C-/- ECTs.

overall_design: We used CRISPR-Cas9 to introduce heterozygous (cMyBP-C+/-) and homozygous (cMyBP-C-/-) frame-shift mutations into MYBPC3 in human iPSCs. Cardiomyocytes were derived from these isogenic lines using the GiWi protocol. On day 30 post differentiation, cardiomyocytes were mixed with isogenic iPSC-derived fibroblasts and used to generate engineered cardiac tissue constructs (ECTs). These ECTs were cultured for 2 weeks prior to RNA extraction and RNA-seq analysis.

middle: J

last: De Lange

first: Emily

last: Farrell

first: J

last: Ralphe


title: Systematic fine-mapping and functional studies of prostate cancer risk variants [RNA-seq]

first: Wange

last: Lu

overall_design: 22Rv1 mRNA profiles of wild type (WT) and rs10486567 Knockout (KO)

first: Yuyang

last: Qian


title: Systematic fine-mapping and functional studies of prostate cancer risk variants [Hi-C]

overall_design: HiC experiments of 22Rv1


title: CstF64 iCLIP-seq in control and U1 AMO-treated HeLa cells

first: chengguo

last: yao

summary: To understand the effect of U1 AMO treatment on CstF64 binding profile , we carried out CstF64 iCLIP-seq experiments in HeLa cells treated with either control or U1 AMO.

overall_design: Control and U1 AMO were transfected into HeLa cells,after 12 hour. CstF64 iCLIP-seq(s) were carried out using the standard iCLIP-seq protocol (König J et al. 2010)

first: Yanhui

last: Deng

first: Yi

last: Ran

first: Junjie

last: Shi

first: Chengguo

last: Yao


title: Gene expression response to irradiation and social environments in the reproductive tracts of male seed beetles, Callosobruchus maculatus

city: Uppsala

first: Mareike

last: Koppik

address: Norbyvägen 18D

department: Department of Ecology and Genetics

institute: Uppsala University

summary: We studied the effect of social environment (presence of conspecific males and/or females) and gamma irradiation on gene expression in the male reproductive tract (aedeagus, ejaculatory bulb, 2 bilobed testes and 3 pairs of smaller accessory glands; the two pairs of larger accessory glands were removed) in a full factorial design.

overall_design: The experiment was replicated in two lines (MA1 & MA3) and over two days, resulting in 4 replicate samples per irradiation/social environment combination. Both lines are experimental evolution lines evolved under intense sexual selection with natural selection removed (Baur and Berger, 2020, Nature Ecology and Evolution 4, 737-744, https://doi.org/10.1038/s41559-020-1140-7). Males were held in one of four social environments (virgin: alone, mated: with one conspecific female, male-male: in groups of 5 conspecific males, mixed: in groups of 5 conspecific males and 5 conspecific females) for 24 hours. Afterwards males were isolated and either irradiated (a dose of 25 Gray of gamma radiation from a caesium-137 source over 35 min) or kept as controls. After a 2 hour recovery period males were flash-frozen and stored at -80 °C until dissections. Per sample, we pooled the reproductive tracts from 9-10 males within each line/day/treatment combination resulting in a total of 32 samples.

first: Julian

last: Baur

first: David

last: Berger


title: Precise control of GATA3 expression in CD4+ T cell differentiation via selective activation of primed enhancers

city: La Jolla

first: Graham

last: McVicker

state: CA California

address: 10010 N Torrey Pines Rd

department: Integrative Biology Laboratory

institute: Salk Institute for Biological Studies

summary: GATA3 is a master regulator with expression that must be precisely controlled during differentiation of naïve CD4+ T cells into diverse subtypes to ensure correct immune responses. The regulatory landscape of GATA3 is complex with dozens of potential enhancers in different states. Here, we use thousands of tiling deletions to systematically map regulatory sequences that affect GATA3 expression. We identify and characterize 34 regulatory sequences in naïve CD4+ T cells, including 4 active enhancers and, unexpectedly, 10 primed enhancers. Focusing on the differentiation of naïve CD4+ T cells into T helper 2 cells where GATA3 expression is highest, we find that upregulation of GATA3 is achieved by retaining active enhancers and activating poised and primed enhancers, including one which harbors candidate causal genetic variants for allergic diseases.

overall_design: We focused on the 2.02Mbp GATA3 region (hg19 chr10: 7,096,667-9,117,164) to identify regulatory elements for GATA3 in human T cells. We designed 14,769 pairs of single guide RNAs (sgRNAs) to tile ~1kb deletions across the 2 Mb genome region centered on GATA3. The targeted deletions have a median size of 1043 bp and median step size of 96 bp, such that each base in the screened region is covered by a median of 8 programmed deletions. We cloned oligos encoding the sgRNAs and generated a lentiviral library to deliver and constitutively express both the sgRNA pairs and spCas9. After transducing the lentiviral library into Jurkat T cells, which we use as a model for naïve CD4+ T cells, we selected for cells with viral genome integration and flow sorted them into pools based on GATA3 protein expression. Finally, we performed deep sequencing of the sgRNA pairs in each expression pool. We performed four biological replicates of the screen, sorting into three expression level pools in the first two replicates and seven expression level pools in the other two replicates.

first: Hsiuyi

middle: V

last: Chen

first: Patrick

last: Fiaux

first: Arko

last: Sen

first: Ishika

last: Luthra

first: Aaron

last: Ho

middle: R

first: Karthik

last: Guruvayurappan

first: Michael

last: Lorenzini

first: Carolyn

last: O’Connor


title: PDGF receptors and signaling are required for 3D-structure formation and differentiation of human iPSCs-derived hepatic spheroids

city: Tokyo

first: Syusaku

last: Tsuzuki

address: 4-6-1, Shirokanedai, Minato-Ku

department: Center for Stem Cell Biology and Regenerative Medicine

institute: The University of Tokyo Institute of Medical Science Division of Regenerative Medicine

summary: Human iPSC-derived liver organoids (LO) or hepatic spheroids (HS) have attracted widespread interest and the numerous studies on them have recently provided various production protocols. However, the mechanism by which the 3D structures of LO and HS are formed from the 2D-cultured cells and the mechanism of the LO and HS maturation remain largely unknown. In this study, we demonstrate that PDGFRA is specifically induced in the cells that are suitable for HS formation and that PDGF receptors and signaling are required for HS formation and maturation. Additionally, in vivo, we show that the localization of PDGFRα is in complete agreement with mouse E9.5 hepatoblasts which begin to form the 3D-structural liver bud from the single layer. Our results present that PDGFRA play important roles for 3D structure formation and maturation of hepatocyte in vitro and in vivo, and provide a clue to elucidate the hepatocyte differentiation mechanism.

overall_design: During differentiation of hepatocyte-like cells derived from hiPSC, each sample was extracted on day8, 10, 12, and 14 in d4DE or d6DE protocol (total 8 samples).

first: Hideki

last: Taniguchi


title: MeCP2 and H3K27me3 binding in Mecp2-knockout and MECP2-transgenic hippocampus

city: Houston

first: Sameer

middle: Subhash

last: Bajikar

address: 1250 Moursund St

department: Molecular and Human Genetics

institute: Baylor College of Medicine

summary: MeCP2 globally regulates gene expression in the brain. One example is Growth-differentiation factor 11 (Gdf11), which is down-regulated upon loss of MeCP2 and up-regulated upon gain of MeCP2. To assess if MeCP2 binds near the Gdf11 locus we performed CUT&RUN for MeCP2 in Mecp2-knockout or MECP2-transgenic hippocampus. To assess if a repressive histone modification (H3K27me3) was concordantly changed we profiled H3k27me3 in these same matched tissues.

overall_design: CUT&RUN for MeCP2, H3K27me3, and Rabbit IgG from the nuclei of Mecp2-knockout, MECP2-transgenic, and associated wild-type littermates


title: Canonical Wnt signaling regulates soft palatal development through mediating ciliary homeostasis

city: Alhambra

first: Eva

last: Janeckova

address: 2250 Alcazar street

institute: USC, CCMB

summary: The goal of this study is to compare transcriptome profiling (RNA-seq) of soft palatal tissue at E14.0 between control and Osr2-Cre;b-cateninfl/fl mice

overall_design: Methods: mRNA profiles of control and Osr2-Cre;b-cateninfl/fl mice were generated by deep sequencing, in triplicate, using Illumina Nextseq 500 platform. The quality of RNA samples was determined using an Agilent 2100 Bioanalyzer and all samples for sequencing had RNA integrity numbers >9.


title: Systematic fine-mapping and functional studies of prostate cancer risk variants

first: Wange

last: Lu

overall_design: Refer to individual Series


title: A unified model for the surveillance of translation in diverse noncoding sequences

city: New York

first: Xuebing

last: Wu

state: NY

address: 630 West 168th Street, P&S 10-401

institute: Columbia University

summary: Translation is pervasive outside of canonical coding regions, occurring in lncRNAs, UTRs, and introns. While the resulting polypeptides are often non-functional, translation in noncoding regions is nonetheless necessary for the birth of new coding regions. The mechanisms underlying the surveillance of translation in diverse noncoding regions and how escaped polypeptides evolve new functions remain unclear. Intriguingly, noncoding sequence-derived functional peptides often localize to membranes. Here, we show that the intrinsic nucleotide bias in the noncoding genome and in the genetic code frequently results in polypeptides with a hydrophobic C-terminal tail, which is captured by the ribosome-associated BAG6 membrane protein triage complex for either proteasomal degradation or membrane targeting. In contrast, canonical proteins have evolved to deplete C-terminal hydrophobic residues. Our results uncovered a fail-safe mechanism for the surveillance of unwanted translation from diverse noncoding regions and suggest a possible biochemical route for the preferential membrane localization of newly evolved proteins.

overall_design: Massively parallel reporter assays for the translation of noncoding sequences in HEK293T cells and BAG6 KO cells

first: Jordan

last: Kesner

first: Ziheng

first: Alexis

last: Aparicio


title: Open ocean and coastal strains of the N2-fixing cyanobacterium UCYN-A have distinct transcriptomes

city: Santa Cruz

first: Jonathan

middle: David

last: Magasin

state: CA

address: 1156 High Street

department: Ocean Sciences

institute: UC Santa Cruz

summary: Although N2 fixation can occur in free-living cyanobacteria, the unicellular endosymbiotic cyanobacterium Candidatus Atelocyanobacterium thalassa (UCYN-A) is considered to be a dominant N2-fixing species in marine ecosystems. Four UCYN-A sublineages are known from partial nitrogenase (nifH) gene sequences. However, few studies have investigated their habitat preferences and regulation by their respective hosts in open-ocean versus coastal environments. Here, we compared UCYN-A transcriptomes from oligotrophic open-ocean versus nutrient-rich coastal waters. UCYN-A1 metabolism was more impacted by habitat changes than UCYN-A2. However, across habitats and sublineages genes for nitrogen fixation and energy production were highly transcribed. Curiously these genes, critical to the symbiosis for the exchange of fixed nitrogen for fixed carbon, maintained the same schedule of diel expression across habitats and UCYN-A sublineages, including UCYN-A3 in the open-ocean transcriptomes. Our results undersore the importance of nitrogen fixation in UCYN-A symbioses across habitats, with consequences for community interaction and global biogeochemical cycles.

overall_design: From 6-15 April 2015 at Station ALOHA in the North Pacific Subtropical Gyre, whole-community water samples were collected from 45m for metatranscriptome analysis of the UCYN-A sublineages A1, A2 and A3.  Samples covered a full light-dark cycle with 8 time points and 3 h intervals. In total there were  16 samples (two replicates per time point). Time points were as follows: 9:00-L3, 12:00-L6, 15:00-L9, 18:00-L12, 21:00-D3, 00:00-D6, 03:00-D9, 06:00-2D12, 09:00-2L3 and 12:00-2L6, where L and D stand for light and dark period, respectively, 2L and 2D the second light-dark cycle, and the number the corresponding hours into the light or dark period.

first: María

last: del Carmen Muñoz-Marín

last: Zehr


title: Sodium ferrous citrate and 5-aminolevulinic acid improve type 2 diabetes by maintaining muscle and mitochondrial health

city: Fujisawa

first: Naho

last: Kitamura

state: Kanagawa

department: Graduate School of Media and Governance

institute: Keio University

summary:
Objective
Improving mitochondrial function is a promising strategy for intervention in type 2 diabetes mellitus. This study investigated the preventive effects of sodium ferrous citrate (SFC) and 5-aminolevulinic acid phosphate (ALA) on several metabolic dysfunctions associated with obesity because they have been shown to alleviate abnormal glucose metabolism in humans.

Methods
Six-week-old male C57BL/6J mice were fed with a normal diet, a high-fat diet, or a high-fat diet supplemented with SFC and ALA for 15 weeks.

Results
The simultaneous supplementation of SFC + ALA to high-fat diet-fed mice prevented loss of muscle mass, improved muscle strength, and reduced obesity and insulin resistance. SFC + ALA prevented abnormalities in mitochondrial morphology and reverted the diet effect on the skeletal muscle transcriptome, including the expression of glucose uptake and mitochondrial oxidative phosphorylation-related genes. In addition, SFC + ALA prevented the decline in mitochondrial DNA copy number by enhancing mitochondrial DNA maintenance and antioxidant transcription activity, both of which are impaired in high-fat diet-fed mice during long-term fasting.

Conclusions
These findings suggest that SFC + ALA supplementation exerts its preventive effects in type 2 diabetes mellitus via improved skeletal muscle and mitochondrial health, further validating its application as a promising strategy for the prevention of obesity-induced metabolic disorders.

overall_design: Male C57BL/6J mice of 6 weeks of age were fed with a normal diet (Control;10% kcal fat), a high fat diet (HF; 60% kcal fat), or a high fat supplemented with 0.023% w/w sodium ferrous citrate (SFC) and 0.2% w/w 5-aminolevulinic acid phosphate (HF+SFC+ALA) for 15 weeks. Control diet (D12450B) contained 10 kcal% fat, 20 kcal% protein, and 70 kcal% carbohydrate, whereas the HF diet (D12492) contained 60 kcal% fat, 20 kcal% protein, and 20 kcal% carbohydrate (Research Diets, Inc., New Brunswick, NJ, USA). Datas were generated using pooled samples within each group.

first: Shiyang

last: Zhang

first: Jean-David

last: Morel

first: Utana

last: Nagano

first: Tanon

last: Taworntawat

first: Shotaro

last: Hosoda

first: Anna

last: Nakamura

first: Yoko

last: Ogawa

first: Johan

last: Auwerx

first: Kazuo

last: Tsubota

last: Yokoyama

first: Mitsuhiro

last: Watanabe


title: Dual-action host-guest chemistry suppresses virulence in Pseudomonas aeruginosa

city: Helsinki

first: Päivi

last: Saavalainen

address: Haartmaninkatu 8

department: TRIMM

country: Finland

institute: University of Helsinki

summary: This study describes the influence of selected synthetic macrocycle on the gene expression in Pseudomonas aeruginosa PAO1. In this work, we propose two distinct functionalities responsible for a dual mechanism of action. We identified a water-soluble pillararene whose inner cavity tightly binds to specific homoserine lactones (HSLs) thereby interfering with interbacterial signalling, leading to effective synchronized suppression of exotoxins and biofilms in P. aeruginosa. An additional concerted mechanism of action is suggested that involves the cationic functional side groups on the pillararene that disrupt both the bacterial outer membrane and biofilms, to increase the penetration and efficacy of intracellular antibiotics.

overall_design: Bacterial cells were grown with (P5A samples) or without (Ctrl samples) the addition of 2.5 mM pillararene for 24 h.

first: Katarzyna

last: Leskinen

first: Christopher

last: Jonkergouw

first: Markus

last: Linder


title: Differentially Expressed MiRNAs in Epithelial Ovarian Cancer Tissue and Serum Samples by MiRNA Microarray Analysis

city: Istanbul

first: Tuba

last: Gunel

state: Fatih

address: Balabanaga mahallesi sehzadebasi cad.

country: Turkey

institute: Istanbul University

summary: Epithelial ovarian cancer (EOC) is the type of ovarian cancer having the highest mortality rate. Because of the asymptomatic characteristic and a few diagnostic tests, it is mostly diagnosed at advanced stages. Therefore, this study aims to gain the predictive and/or early diagnostic novel circulating miRNAs for EOC. Firstly, microarray analysis of miRNA expression level is performed on plasma samples from pathologically confirmed EOC patients and matching healthy individuals; EOC tissue samples and benign ovarian neoplastic tissues. 31 significantly dysregulated miRNAs in serum and 3 miRNAs in tissue were identified by microarray. The results were validated using RT-qPCR. KEGG database was used for the target gene and pathway analysis. Three miRNA from EOC serum (hsa-miR-1909-5p, hsa-miR-885-5p, hsa-let-7d-3p) and one microRNA from tissue samples (hsa-miR-200c-3p) were validated as significant enough to distinguish EOC patients from healthy individuals. KEGG pathway enrichment analysis showed 7 significant pathways, which are prion diseases, proteoglycans in cancer, oxytocin signaling pathway, hippo signaling pathway, adrenergic signaling in cardiomyocytes, oocyte meiosis, and thyroid hormone signaling pathway, in which our validated miRNAs have a role. It supports our hypothesis that several miRNAs, in our case validated 4 miRNAs, can have the potential to be a biomarker of EOC diagnosis and treatment.

overall_design: Microarray analysis of miRNA expression level is performed on a total of 32 plasma samples from pathologically confirmed 8 EOC patients and 8 matching healthy individuals; 8 EOC tissue samples and 8 benign ovarian neoplastic tissues. 31 significantly dysregulated miRNAs in serum and 3 miRNAs in tissue were identified by microarray.

first: Ece

last: Gumusoglu-Acar

first: Mohammad

middle: K

last: Hosseini


title: TCF21high pericyte subpopulation promotes cancer metastasis by remodeling perivascular matrix

city: Guangzhou

first: Lipeng

last: Mao

state: Guangdong

address: No.601, West Huangpu Avenue

department: Department of Microbiology and Immunology

institute: Jinan University

summary: The role of perivascular cell heterogeneity in tumor metastasis remains unknown. Here, we dissected the heterogeneity of perivascular cells by single-cell RNA sequencing and defined 13 subpopulations of pericytes within human colorectal cancers (CRC). Among them, an extracellular matrix (ECM)-rich subpopulation of TCF21high tumor pericytes (TPCs) was identified, which highly correlated with metastatic potentials in human CRC patients. Pericyte-specific conditional deletion of Tcf21 in mice mitigated CRC metastasis by decreasing perivascular ECM resmodeling, maintaining the integrity of the basement membrane, and reducing circulating cancer cells. Conversely, co-injection of CRC cells with TCF21high TPCs markedly promoted metastasis. Mechanistically, high expression of TCF21 in TPCs altered ECM stiffness, collagen deposition/rearrangement, and basement membrane degradation, which collectively instigated tumor cell intravasation and metastasis. Compelling experimental evidence showed that the loss of integrin α5 activated the FAK/PI3K/AKT axis, impaired DNA hypermethylation of TCF21, leading to high expression of TCF21 in TPCs. Our data provide new mechanistic insights into functions of a specific subpopulation of perivascular cells in promoting cancer metastasis and therapeutic targeting of metastasis-promoting TPCs may provide a new paradigm for cancer therapy.

overall_design: Colon cancer vascular pericytes--CCVC-1 (lymphatic metastasis), colon cancer perivascular cells--PCCVC-1 (lymphatic metastasis), rectal cancer vascular pericytes--RCVC-1 (non-metastasized), rectal cancer perivascular cells Pericytes--PRCVC-1 (non-metastatic), sigmoid colon cancer vascular pericytes--CCVC-2 (liver metastasis), parasigmoid vascular pericytes--PCCVC-2 (liver metastasis), 3 human cancer tissues and cancer cells beside  of (six tissues combined) 10x single-cell sequencing

first: Xiaobo

last: Li


title: Single-cell RNA-seq profiling of early murine adrenogonadal development

city: Genève

first: Serge

last: Nef

address: Rue Michel-Servet 1

department: Genetic Medicine and Development

institute: University of Geneva

summary: To generate a gene expression atlas of early adrenogonadal development, we performed single-cell transcriptome profiling using a high throughput droplet-based system (10X Genomics).

overall_design: Posterior trunks (embryonic day (E) 9.0), urogenital ridges (E10.5 and E11.5), gonads and adrenal glands (E12.5) were harvested from XX and XY Nr5a1 Tg mouse embryos (for adrenals glands, only XY embryos were analyzed). Tissues were enzymatically dissociated and single-cell RNA-Seq libraries were prepared using the Chromium Single Cell 3′ v2 Reagent Kit (10x Genomics) according to manufacturer’s protocol. Each condition (organ, sex and developmental stage) was performed in two independent replicates.

first: Yasmine

last: Neirijnck

first: Pauline

last: Sararols

first: Chloé

last: Mayère


title: Re-generation of cytotoxic γδT cells with distinctive signatures from human γδT-derived iPSCs

city: Kobe City

first: Michiyo

last: Koyanagi-Aoi

address: Chuo-ku, Kusunoki-cho 7-5-1

institute: Kobe University

summary: The goal of this study was to compare gene expressions on the single cell level in (i) freshly isolated PBMCs (no stimulation and no sorting), (ii) PBγδT cells; PBMCs were stimulated with HMBPP in vitro and CD3(+) γδTCR(+) cells were sorted and (iii) iγδT cells; differentiated cells from γδT-iPSC clone were stimulated with HMBPP and CD3(+) γδTCR(+) cells were sorted or not sorted.

overall_design: Single-cell RNA sequencing (scRNA-seq) was performed on following seven samples; S1 (freshly isolated PBMCs, Sample Tag01), S2 (CD3+gdTCR+ sorted PB-γδT cells, Sample Tag02), S3 (CD3+gdTCR+ sorted γδ Tcells differentiated from iPSCs, Exp137 d42), S4 (CD3+gdTCR+ sorted γδ Tcells differentiated from iPSCs, Exp138 d41), S5 (CD3+gdTCR+ sorted γδ Tcells differentiated from iPSCs, Exp139, d36), S6 (non-sorted γδ Tcells differentiated from iPSCs, Exp53-5A, d38) and S7 (non-sorted γδ Tcells differentiated from iPSCs, Exp54-5A, d34). Freshly isolated PBMCs, PBγδT cells and iγδT cells were captured and processed using the BD Rhapsody platform, and messenger RNA profiles of 397 genes (BD Rhapsody Immune response panel Hs) were generated by deep sequencing.

first: Nobuyuki

last: Murai

first: Takashi

last: Aoi


title: Expression data from HDLEC treated with melanosome

city: Tel Aviv

first: Guy

last: Shapira

address: Chaim Levanon 30

country: Israel

institute: Tel Aviv University

summary: We are investigating the effect of the melanosomes on endothelial cells located in the periphery of the melanoma tumor.

overall_design: Human Dermal Lymphatic Endothelial Cells (HDLEC) were treated with melanoma melanosomes or liposomes. RNA profile was analazed on llumina Human Sentrix-12 v4 BeadChip

first: Shoshana

last: Greenberger

first: Carmit

last: Levy


title: Characterization of cell subsets in lung tissue from mice treated with or without MSDC-0602K following influenza virus infection

city: Rochester

first: Bibo

last: Zhu

state: MN

address: 200 1st ST SW

department: Pulmonary and Critical Care Medicine

country: USA

institute: Mayo Clinic

summary: To understand how a new insulin sensitizer MSDC-0602K affects lung phenotype in vivo, lungs from WT mice treated with DMSO or a new insulin sensitizer MSDC-0602K at day 4 post influenza virus infection were utilized for single-cell RNA sequencing analysis. Dimensionality reduction analysis of combined DMSO or MSDC-0602K-treated lungs revealed 25 clusters based on their gene expression pattern, and cells from those two groups showed similarity as visualized in UMAP plot. Interestingly, MSDC-0602K treatment diminished infiltration of proinflammatory cell types including monocytes or neutrophils while increased percentage of alveolar macrophages or Type I epithelial cells compared to DMSO treatment. Moreover, a number of proinflammatory gene expression was decreased in alveolar macrophages in lungs from MSDC-0602K treated mice.

overall_design: Influenza virus infected-WT mice were administrated with DMSO or MSDC-0602K daily from day 1 to day 3 post viral infection.  Lung cell suspensions were obtained at day 4 post influenza virus infection for sequencing.

first: Xiaoqin

last: Wei

first: Jie

last: Sun