title: Single-cell Transcriptome Atlas of the Human Corpus Cavernosum

status: Public on Jun 23 2022

first: Zheng

last: Li

address: 85th Wujin Road, Shanghai, China

institute: Shanghai Jiao Tong University School of Medicine

summary: Single-cell transcriptomes of corpus cavernosum from three males with normal erections and five organic erectile dysfunction (ED) patients.

overall_design: Three normal tissue samples were obtained from the tumour margin of penile carcinoma resection, all patients reported good stimulated erections and early morning erections. The five ED CC tissues were obtained from biopsy samples in artificial cavernous body implantation, all the ED patients were diagnosed with organic ED rather than psychological ED by the nocturnal penile tumescence (NPT) and intracavernosal injection (ICI) tests. The DMED patients had a at least 10-year history of type 1 diabetes and had good blood glucose control before surgery.


title: FOXR2 is an epigenetically regulated pan-cancer oncogene that activates ETS transcriptional circuits [FOXR2_IUE_RNAseq]

city: Los Angeles

first: Jessica

middle: W

last: Tsai

state: California

address: 4650 Sunset Blvd MS #57

department: Pediatrics

country: USA

institute: Children's Hospital Los Angeles

type: Expression profiling by high throughput sequencing

summary: Forkhead Box R2 (FOXR2) is a forkhead transcription factor located on the X chromosome whose expression is normally restricted to the testis. In this study, we performed a pan-cancer analysis of FOXR2 activation across more than 10,000 adult and pediatric cancer samples and found FOXR2 to be aberrantly upregulated in 70% of all cancer types and 8% of all individual tumors. The majority of tumors (78%) aberrantly express FOXR2 through a previously undescribed epigenetic mechanism that involves hypomethylation of a novel promoter, which was functionally validated as necessary for both FOXR2 expression as well as proliferation in FOXR2-expressing cancer cells. FOXR2 expression is sufficient to enhance tumor formation, and coopts ETS family transcription circuits across cancers. Taken together, this study identifies FOXR2 as potent and ubiquitous oncogene that is epigenetically activated across the majority of human cancers. The identification of ETS transcription circuit hijacking by FOXR2 extends the mechanisms known to activate ETS transcription factors and highlights a mechanism through which transcription factor families cooperate to enhance tumorigenesis.

overall_design: RNA-sequencing of in vivo mouse models generated by in utero electroporation comparing FOXR2 tumors to non-FOXR2 tumors.

first: J

first: D

middle: T

last: Jones

first: P

last: Bandopadhayay

first: T

middle: N

last: Phoenix


title: FOXR2 is an epigenetically regulated pan-cancer oncogene that activates ETS transcriptional circuits

type: Genome binding/occupancy profiling by high throughput sequencing

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Role of FGF-2 induced non-coding RNA in cancer [I]

status: Public on Jun 24 2022

city: Gothenburg

first: Subazini

last: TK

address: Medicineragatan 9A

country: Sweden

institute: Gothenburg university

summary: We show that IER3 and IER3-AS1 protein coding and non-coding genes mediates oncogenic properties in cancer cell lines when they are induced by FGF-2 which is fibroblast growth factor. Comprehensive analysis of mechanisms behind the regulation of these genes by FGF-2 induction is shown in this study.

overall_design: Functional characterization for IER3 and IER3-AS1 was done using lenti viral shRNA knockdown strategies followed by RNA-seq in Hela and HEK293 cell lines

first: Meena

last: Kanduri

first: Chandrasekhar

last: Thankaswamy Kosalai


title: Genome-wide maps of chromatin state in adrenergic and mesenchymal neuroblastoma cells

status: Public on Jun 26 2022

city: Boston

first: Rani

last: George

state: Massachusetts

address: 450 Brookline Avenue

department: Pediatric Oncology

institute: Dana Farber Cancer Institute

summary: We analyzed the chromatin occupancies of active (H3K27ac and H3K4me3) and repressive (H3K27me3) histone marks in adrenergic (SH-SY5Y parental) and mesenchymal (SH-SY5Y LDK-resistant and SH-EP) neuroblastoma cells.

overall_design: 3 neuroblastoma cell lines were probed for genome-wide occupancies of active and repressive histone marks

first: Satyaki

last: Sengupta

first: Sanjukta

last: Das

middle: E


title: Role of FGF-2 induced non-coding RNA in cancer


title: PrrA modulates Mycobacterium tuberculosis response to acidic pH and high chloride levels

first: Shumin

last: Tan

state: MA

address: 150 Harrison Ave

department: Molecular Biology and Microbiology

institute: Tufts University School of Medicine

summary: The purpose of this study was to identify genes that are differentially expressed upon prrA overexpression, to reveal the impact of PrrA on global transcription in acidic pH and/or high chloride conditions.

overall_design: Mtb strain CDC1551 harboring a tetracycline inducible prrA-FLAG construct was grown to an OD600 of ~0.6 in standing, filter-capped  T-75 flasks and treated with either ethanol (EtOH) as a carrier control or 200 ng/ml anhydrotetracycline (ATC) for 2 hours. Bacteria were then subcultured to an OD600 = 0.3 in standing, filter-capped  T-25 flasks in 7H9, pH 7 media ± 250 mM NaCl, or 7H9, pH 5.7 media ± 250 mM NaCl, with continued presence of EtOH or ATC as appropriate. Exposure to the different media conditions was for 4 hours in a humidified, 37°C, 5% CO2 incubator before RNA was extracted. RNA was prepared by removing the rRNA and library prepping with a TruSeq Stranded kit (Illumina) before high-throughput sequencing with an Illumina HiSeq 2500 (High Output v4) (100 bp single end reads, one lane). Two biological samples per condition were sequenced.

first: David

last: Giacalone


title: FOXR2 is an epigenetically regulated pan-cancer oncogene that activates ETS transcriptional circuits [FOXR2_cells_RNAseq]

overall_design: RNA-sequencing performed using isogenic models including human neural stem cells and AALE cells expressing FOXR2 relative to control.  RNA-sequencing of FOXR2-expressing cancer cell lines with FOXR2 knocked down using CRISPR interference relative to controls.


title: T cell transriptome in chromosome 22q11.2 deletion syndrome

city: Kansas City

first: Daniel

middle: Paul

last: Heruth

state: MO

address: 4th Floor PRC, 2401 Gillham Road

institute: Children's Mercy

summary: We performed transcriptome analysis via RNA sequencing of T cells isolated from participants with and without 22qDS and T cells of 22qDS participants with low or normal number of T cells.

overall_design: A single-centered study involving a convenience sample of participants along with retrospective chart review. This IRB approved study included patients referred to a multidisciplinary clinic for management of chromosome 22q11.2 deletion syndrome. Healthy controls were recruited by recruitment announcements in the hospital. Peripheral blood was collected from participants aged 5-8 years. Immune function testing was performed. RNA sequencing was completed on isolated T cells; and differential gene expression profiles of T cells between 22qDS and healthy controls were established.

first: Nikita

last: Raje

first: Janelle

middle: R

last: Noel-MacDonnell

first: Katherine

last: Shortt

middle: P


title: Natural variation in the maternal and zygotic mRNA complements of the early embryo in Drosophila melanogaster

city: Davis

first: Susan

middle: E.

last: Lott

state: CA

address: 2320 Storer Hall

department: Evolution and Ecoloty

institute: University of California, Davis

summary: Maternal gene products supplied to the egg during oogenesis drive the earliest events of development in all metazoans. After the initial stages of embryogenesis, maternal transcripts are degraded as zygotic transcription is activated; this is known as the maternal to zygotic transition (MZT). Altering the abundances of maternally deposited factors in the laboratory can have a dramatic effect on development, adult phenotypes and ultimately fitness. Zygotic transcription activation is a tightly regulated process, where the zygotic genome takes over control of development from the maternal genome, and is required for the viability of the organism. Recently, it has been shown that the expression of maternal and zygotic transcripts have evolved in the Drosophila genus over the course of 50 million years of evolution. However, the extent of natural variation of maternal and zygotic transcripts within a species has yet to be determined. We asked how the maternal and zygotic pools of mRNA vary within and between populations of D. melanogaster. In order to maximize sampling of genetic diversity, African lines of D. melanogaster originating from Zambia as well as DGRP lines originating from North America were chosen for transcriptomic analysis. Single embryo RNA-seq was performed before and after zygotic genome activation to determine which transcripts are maternally deposited and which are zygotically expressed within and between these populations. Differential gene expression analysis has been used to quantify quantitative changes in RNA levels within populations as well as fixed expression differences between populations at both stages. Generally, we find that maternal transcripts are more highly conserved, and zygotic transcripts evolve at a higher rate. We find that there is more within-population variation in transcript abundance than between populations and that expression variation is highest post- MZT between African lines. Determining the natural variation of gene expression surrounding the MZT in natural populations of D. melanogaster gives insight into the extent of how a tightly regulated process may vary within a species, the extent of developmental constraint at both stages and on both the maternal and zygotic genomes, and reveals expression changes allowing this species to adapt as it spread across the world.

overall_design: We performed RNA-Seq on single embryos from stage 2 and stage 5 (Bowne's stages), from four DGRP lines (Raleigh, NC) and four lines from Zambia, with three replicates for each. The stage 5 samples were genotyped prior to sequencing to determine sex, all stage 5 embryo samples are XY. Stage 2 embryos only contain maternal mRNAs, so the embryo sex is likely irrelevant at this stage.

first: Anna

last: Feitzinger


title: SUM159 cells grown in 2D cultures in the presence or absence of SHP2

city: Basel

first: Mohamed

last: Bentires-Alj

address: Hebelstrasse 20

department: DBM

country: Switzerland

institute: University Hospital Basel

summary: The first bona fide PTP proto-oncogene was the Src-homology 2 domain-containing phosphatase SHP2 (encoded by PTPN11), an ubiquitously expressed PTP that transduces mitogenic, pro-survival, cell fate and/or pro-migratory signals from numerous growth factor-, cytokine- and extracellular matrix receptors. In malignancies, SHP2 is hyperactivated either downstream of oncoproteins or by mutations.We provide analysis of the human mesenchymal triple-negative breast cancer cell line SUM159 and grown in 2D cultures for 15 days in the presence or absence of SHP2.

overall_design: The human mesenchymal triple-negative breast cancer cells SUM159 were transduced with a doxycycline-inducible lentiviral vector expressing a CTRL miR or SHP2 miR1 or SHP2 miR2. Cells from each group were grown in 2D cultures, and the knockdown of SHP2 was induced for 15 days. RNA was extracted for gene expression analysis.

first: Atul

last: Sethi


title: A scalable organoid model of autosomal dominant polycystic kidney disease for disease mechanism and drug discovery

first: Tracy

last: Tran

address: 1425 San Pablo

institute: University of Southern California

summary: Human pluripotent stem cell-derived organoids are models for human development and disease. We report a modified human kidney organoid system that generates thousands of similar kidney organoids, each consisting of 1 to 2 nephron-like structures. Single-cell transcriptomic profiling and immunofluorescence validation highlighted patterned nephron-like structures, utilizing similar pathways, with distinct morphogenesis, to human nephrogenesis. To examine this platform for therapeutic screening, the polycystic kidney disease genes PKD1 and PKD2 were inactivated by gene-editing. PKD1 and PKD2 mutant models exhibited efficient and reproducible cyst formation. Cystic outgrowths could be propagated for months to centimeter-sized cysts. To shed new light on cystogenesis, 247 protein kinase inhibitors (PKI) were screened in a live imaging assay identifying novel compounds blocking cyst formation but not overall organoid growth. Scaling and further development of the organoid platform will enable a broader capability for kidney disease modeling and high-throughput drug screens.

overall_design: We performed single-cell RNA sequencing to examine the cellular composition of kidney organoids at various time points along the differentiation: day 8, day 10, day 14, day 16 and day 28

first: Andrew

last: McMahon

first: Cheng

middle: J

last: Song


title: FOXR2 is an epigenetically regulated pan-cancer oncogene that activates ETS transcriptional circuits [FOXR2_ChIP]

first: Jessica

last: Tsai

overall_design: ChIP-sequencing data of FOXR2 and non-FOXR2 expressing cancer cell lines, profiling different chromatin marks, and ChIP-sequencing of human neural stem cells expressing HA-tagged FOXR2.

first: J

first: D

last: Jones

first: P

last: Bandopadhayay

first: T

last: Phoenix


title: Transcriptome analysis of nascent hematopoietic stem cells (HSCs) induced by the ectopic expression of Evi1

status: Public on Jun 22 2022

city: Kumamoto

first: Tomomasa

last: Yokomizo

address: 2-2-1 Honjo, Chuo-ku

country: Japan

institute: Kumamoto University

summary:
To examine the role of Evi1 in HSC specification, we generated Tie2-Cre::ROSA-Evi1 mice, which can induce Evi1 in endothelial cells. We observed five-fold increase of HSCs In Tie2-Cre::ROSA-Evi1 embryo.
To characterize the induced HSCs in Tie2-Cre::ROSA-Evi1 embryos, we analyzed the gene expression profile of HSCs.

overall_design: HSCs were isolated from Tie2-Cre::ROSA-Evi1 and ROSA-Evi1 fetal livers, and were subjected to RNA-seq analysis.

last: Suda


title: RNA-seq analysis of NHR-23-depleted, SPE-44-depleted, and NHR-23+SPE-44-depleted C. elegans

city: Santa Cruz

first: Jordan

middle: David

last: Ward

address: 1156 HIGH ST, UCSC THIMANN RECEIVING, SINSHEIMER 327

department: Molecular, Cell, and Developmental Biology

institute: University of California, Santa Cruz

summary: We performed RNA-seq analysis of control, NHR-23-depleted, SPE-44-depleted and NHR-23+SPE-44 depleted adult male animals, to identify genes regulated by NHR-23 and SPE-44.

overall_design: Three biological replicates of the following depletion regimes were analyzed: i) Control; ii) NHR-23-depletion; iii) SPE-44 depletion; iv) NHR-23+SPE-44 depletion

middle: M

last: Ragle

first: A

last: Rechtsteiner

middle: D


title: SF3B1 promotes glycolysis and tumor malignancy through splicing-independent activation of HIF1α [RNA-Seq]

city: Zurich

first: Patrik

last: Simmler

address: Otto-Stern-Weg 7

institute: ETH Zurich

summary: Splicing factor 3b subunit 1 (SF3B1) is one of the most frequently mutated spliceosomal components in cancer. Recent studies additionally imply an involvement of the wildtype protein in tumor progression, but the underlaying mechanism remains elusive.  Here we report that SF3B1 acts as a coactivator for hypoxia-inducible factor (HIF) 1α. SF3B1 directly interacts with HIF1a, augments HIF1a-HIF1b heterodimer binding to hypoxia response elements in vivo and enhances transactivation of HIF target genes. In human patients, primary patient cells and in a mouse model of pancreatic ductal adenocarcinoma (PDAC), efficient HIF1a-signalling relies on SF3B1. Monoallelic deletion of Sf3b1 in this mouse tumor model severely delays tumor initiation and development and diminishes HIF target gene transcription, glucose uptake and proliferation in corresponding tumor organoids in hypoxia, while splicing remains functional. Here, we define a subset of aggressive chromophobe renal cell carcinomas (chRCCs) able to transcriptionally compensate monoallelic SF3B1 loss, a feature that correlates with poor prognosis and lymph node spread and we show that the oncogenic hotspot mutation SF3B1K700E has reduced co-activation efficiency. Also, we report increased sensitivity of cells with low levels of SF3B1 to the HIF-inhibitor PX-478. These findings reveal a splicing-independent, tumor promoting function of SF3B1, important in the pathogenesis of PDAC and other cancers.

overall_design: Panc1 cells were treated either with control siRNA (siCtrl) or siRNA targeting SF3B1 (siSF3B1). siCtrl cells were exposed to 1% O2 or 21% O2, siSF3B1 cells to 1% O2 for 8 hours.


title: Transcriptome analysis of nascent hematopoietic cells II

summary:
During ontogeny, HSCs or progenitors are generated from endothelial cells through the process known as endothelial-to-hematopoietic transition (EHT). After EHT, hematopoietic cells form cell aggregates, called hematopoietic clusters.
To obtain mechanistic insight into HSC specification, we compared the gene expression profiles of hematopoietic clusters between caudal half region and yolk sac.

overall_design: Hlf+ nascent hematopoietic cells (c-Kit+CD45-) were isolated from embryonic day (E)10.5 caudal half and yolk sac, and were subjected to RNA-seq analysis.


title: The TFEB – TGIF1 axis regulates EMT in mouse epicardial cells

city: Candiolo

first: Davide

last: Cora'

state: TO

department: Dept. of Oncology

country: Italy

institute: Piemonte Orientale University and IRCCS Candiolo Cancer Institute

summary: Epithelial-mesenchymal transition (EMT) is a complex and pivotal process involved in organogenesis and is related to several pathological processes, including cancer and fibrosis. During heart development, EMT mediates the conversion of epicardial cells into vascular smooth muscle cells and cardiac interstitial fibroblasts. Here, we show that the oncogenic transcription factor EB (TFEB) is a key regulator of EMT in epicardial cells and that its genetic overexpression in mouse epicardium is lethal due to heart defects linked to impaired EMT. TFEB specifically orchestrates the EMT-promoting function of transforming growth factor (TGF) β, and this effect results from activated transcription of thymine-guanine-interacting factor (TGIF)1, a TGFβ/Smad pathway repressor. The Tgif1 promoter is activated by TFEB, and in vitro and in vivo findings demonstrate its increased expression when Tfeb is overexpressed. Furthermore, Tfeb overexpression in vitro prevented TGFβ-induced EMT, and this effect was abolished by Tgif1 silencing. Tfeb loss of function, similar to that of Tgif1, sensitized cells to TGFβ, inducing an EMT response to low doses of TGFβ. Together, our findings reveal an unexpected function of TFEB in regulating EMT, which might provide new insights into injured heart repair and control of cancer progression.

overall_design: ChIP seq of overexpressed TFEB S142A and IgG in mouse epicardial cells

first: Elena

last: Astanina

first: Gabriella

last: Doronzo

first: Francesco

last: Neri

first: Salvatore

last: Oliviero

first: Federico

last: Bussolino


title: YAP9/A20 Complex Suppresses Proinflammatory Responses and Provides Novel Anti-inflammatory Therapeutic Potentials

city: Guangzhou

first: Jongdae

last: Lee

state: Guangdong

address: No.195, Dongfengxi Road,

department: School of Basic Medicines

country: China

institute: Guangzhou Medical University

summary: Innate anti-inflammatory mechanisms are essential for immune homeostasis and can present opportunities to intervene inflammatory diseases. In this report, we found that YAP isoform 9 (YAP9) is an essential negative regulator of the potent inflammatory stimuli such as TNFα, IL-1β, and LPS. YAP9 constitutively interacts with another anti-inflammatory regulator A20 (TNFAIP3) to suppress inflammatory responses, but A20 and YAP can function only in the presence of the other. YAP9 uses a short stretch of amino acids in the proline-rich domain (PRD) and transactivation domain (TAD) suppress the inflammatory signaling while A20 mainly uses the zinc finger domain 7 (ZF7). Cell-penetrating synthetic PRD, TAD, and ZF7 peptides act as YAP9 and A20 mimetics respectively to suppress the proinflammatory responses at the cellular level and in mice. Our data uncover a novel anti-inflammatory axis and anti-inflammatory agents that can be developed to treat acute or chronic conditions where TNFα, IL-1β, or LPS plays a key role in initiating and/or perpetuating inflammation.

overall_design: To investigate the effect of skeleton stress' effect on cell gene expression, FLS were cultured in plastic tissue culture plate (2D) for 2 days or Vitrogel for 7 days and stimulated with TNFα (1ng/ml) for 2h in duplicates. Total RNAs were collected and subjected to RNAseq.


title: Comparison of mesenchymal stem/stromal cells (MSCs) between human iPS-derived MSC and human primary cultured MSCs

city: Fujisawa

first: Daisuke

last: Kamiya

state: Kanagawa

address: 26-1 Muraoka-Higashi 2-chome

department: CiRA

institute: Kyoto University

summary: To characterize and compare  XF-iMSC (XenoFree-induced Mesenchymal stem/stromal cells) and various types of MSCs (Adipose-, Bone marrow-, Unbilical cord-derived), we performed a transcriptome analysis of these MSCs

overall_design: XF-iMSC and human primary cultured MSCs were cultured for 2 weeks for acclimation and their expression patterns were  analyzed using NGS

first: Teppei

last: Akaboshi

first: Makoto

last: Ikeya


title: SF3B1 promotes glycolysis and tumor malignancy through splicing-independent activation of HIF1α

overall_design: Refer to individual Series


title: The ParB clamp docks onto Smc for DNA loading via a joint-ParB interface [ChIP-seq]

city: Ecublens VD

first: Hon-Wing

last: Liu

address: Le Biophore, CH-1015 Lausanne

institute: Université de Lausanne

summary: Chromosomes readily unlink from one another and segregate to daughter cells during cell division highlighting a remarkable ability of cells to organize long DNA molecules. SMC complexes mediate chromosome folding by DNA loop extrusion. In most bacteria, SMC complexes start loop extrusion at the ParB/parS partition complex formed near the replication origin. Whether they are recruited by recognizing a specific DNA structure in the partition complex or a protein component is unknown. By replacing genes in Bacillus subtilis with orthologous sequences from Streptococcus pneumoniae, we show that the three subunits of the bacterial Smc complex together with the ParB protein form a functional module that can organize and segregate chromosomes when transplanted into another organism. Using chimeric proteins and chemical cross-linking, we find that ParB binds to the Smc subunit directly. We map a binding interface to the Smc joint and the ParB CTP-binding domain. Structure prediction indicates how the ParB clamp presents DNA to the Smc complex to initiate DNA loop extrusion.

overall_design: Chromatin-immunoprecipitation (ChIP) sequencing of ScpB in Bacillus subtilis ParB variants

first: Hon

first: Florian

last: Bock

first: Stephan

last: Gruber


title: Beiging of perivascular adipose tissue regulates its inflammation and vascular remodeling

status: Public on Jun 21 2022

city: Tokyo

first: Yusuke

last: Adachi

state: Outside US/Canada

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

department: Department of Cardiovascular Medicine

institute: The University of Tokyo

summary: Although inflammation plays critical roles in the development of atherosclerosis, its regulatory mechanisms remain incompletely understood. Perivascular adipose tissue (PVAT) has been reported to undergo inflammatory changes in response to vascular injury. Here, we showed that vascular injury induced the beiging (brown adipose tissue-like phenotype change) of PVAT, which fine-tunes inflammatory response and thus vascular remodeling as a protective mechanism. In a mouse model of endovascular injury, macrophages accumulated in PVAT, causing beiging phenotype change. Inhibition of PVAT beiging by genetically silencing PRDM16, a key regulator to beiging, exacerbated inflammation and vascular remodeling following injury. Conversely, activation of PVAT beiging attenuated inflammation and pathological vascular remodeling. Single-cell RNA sequencing revealed that beige adipocytes abundantly expressed neuregulin 4 (Nrg4) which critically regulated alternative macrophage activation. Importantly, significant beiging was observed in the diseased aortic PVAT in patients with acute aortic dissection. Taken together, vascular injury induced the beiging of adjacent PVAT with macrophage accumulation, where NRG4 secreted from the beige PVAT facilitated alternative activation of macrophages, leading to the resolution of vascular inflammation. Our study demonstrated the pivotal roles of PVAT in vascular inflammation and remodeling and will open a new avenue for treating atherosclerosis.

overall_design: Tissue: Outer tissues surrounding the murine femoral arteries; 2 conditions: sham vs injury; 2 replicate for each condition.

first: Kazutaka

last: Ueda

first: Kaoru

last: Ito


title: PrrA modulation of Mycobacterium tuberculosis response to nitric oxide is critically regulated by serine/threonine protein kinases

first: Shumin

last: Tan

summary: The purpose of this study was to understand how prevention of serine/threonine protein kinase (STPK) phosphorylation of PrrA impacts PrrA modulation of M. tuberculosis transcriptional response to nitric oxide.

overall_design: Mtb strain CDC1551 with a deletion of the native copy of prrA, and harboring a prrA-FLAG-DAS4 (wild type prrA allele - prrA-DUC/∆prrA) or prrA-T6A-FLAG-DAS4 (T6A STPK-phosphoablative prrA allele - prrA-T6A-DUC/∆prrA) construct were grown in aerated conditions to mid-log phase (OD600 ~0.6), before the bacteria were subcultured to an OD600 = 0.3 in filter-capped  T-75 flasks laid flat, in 12 ml 7H9, pH 7 media ± 100 µM DETA NONOate (an NO donor). Exposure to DETA NONOate was for 4 hours in a humidified, 37°C, 5% CO2 incubator before RNA was extracted. RNA was prepared by removing the rRNA and library prepping with a TruSeq Stranded kit (Illumina) before high-throughput sequencing with an Illumina HiSeq 2500 (High Output v4) (100 bp single end reads, one lane). Two biological samples per condition were sequenced.

first: David

last: Giacalone


title: The histone variant macroH2A modulates inflammatory response in cancer [UMI-4C]

city: Badalona

first: Roberto

last: Malinverni

state: Barcelona

address: Josep Carreras Building, Ctra de Can Ruti,

country: Spain

institute: Josep Carreras Leukaemia Research Institute

summary: MacroH2A histone variants have a major role in nuclear organization and large-scale 3D chromatin architecture. How these alterations impinge on the behaviour of cancer cells is not known. Here, we describe the analysis of the total macroH2A loss of function phenotype in a model of hepatoblastoma, the main childhood liver cancer. Performing transcriptomic analyses in xenografts and cell cultures, we find that macroH2A modulated the response of cancer cells to paracrine inflammatory signalling. Specifically, ablation of macroH2A ablation neutralized the induction of a large subset of genes by TNFα and led to the hyperactivation of another subset of genes. Among the top macroH2A-sensitive genes we find the cancer-related gene DKK1 . Depletion of macroH2A rendered the DKK1 gene hypersensitive to TNFα signalling boosting the secretion of DKK1 protein. On the gene regulation level, this was mediated by an alteration of the local chromatin structure and facilitated activation of distal enhancer elements. The study of human samples of hepatoblastoma showed that DKK1 is strongly upregulated in tumors and associated with poor patient outcome. Taken together our results suggest that the regulation of 3D chromatin architecture by macroH2A histone variants has a central role in the response and regulation of paracrine signalling that might be relevant for the interaction of cancers with immune cells and other cells in their microenvironment.

overall_design: UMI-4C assays performed in HepG2 cell lines stably expressing either control shRNAs (CTL) or shRNAs targeting macroH2A1 and macroH2A2 (DKD)

last: Corujo

first: Marcus

last: Buschbeck


title: Transcriptome analysis of the gene expression profiles in Vibrio alginolyticus SCSIO 43097 and its variants V1-V3.

status: Public on Jun 20 2022

first: Pengxia

last: Wang

address: 164 Xingang West Rd

department: South China Sea Institute of Oceanology

institute: Chinese Academy of Sciences

summary: To explore the mechanism underlying the phenotypic and genetic changes in the variants of SCSIO 43097, we conducted an RNA-seq analysis of the wild-type strain and the variants.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for SCSIO 43097 and its variants.

first: Weiquan

first: Kaihao

last: Tang

first: Xiaoxue


title: ID2 controls differentiation of entero-endocrine cells in mouse small intestine

city: Mainz

first: Frank

last: Rühle

address: Ackermannweg 4

department: Bioinformatics Core Facility

country: Germany

institute: Institute of Molecular Biology (IMB)

summary:
Aim: The mammalian gut is the largest endocrine organ. Dozens of hormones secreted by enteroendocrine cells regulate a variety of physiological functions of the gut but also of the pancreas and brain. Here, we examined the role of the helix-loop-helix transcription factor ID2 during the differentiation of intestinal stem cells along the enteroendocrine lineage.
Methods: To assess the functions of ID2 in the adult mouse small intestine, we used single-cell RNA sequencing, genetically modified mice, and organoid assays.
Results: We found that in the adult intestinal epithelium Id2 is predominantly expressed in enterochromaffin and peptidergic enteroendocrine cells. Consistently, the loss of Id2 leads to the reduction of Chromogranin A-positive enteroendocrine cells. In contrast, the numbers of tuft cells are increased in Id2 mutant small intestine. Moreover, ablation of Id2 elevates the numbers of Serotonin+ enterochromaffin cells and Ghrelin+ X-cells in the posterior part of the small intestine. Finally, Id2 acts downstream of BMP signalling during the differentiation of Glucagon Like Peptide-1+ L-cells and Cholecystokinin+ I-cells towards Neurotensin+PYY+ N-cells.
Conclusion: Id2 plays an important role in cell fate decisions in the adult small intestine. Firstly, ID2 suppresses the differentiation of secretory intestinal epithelial progenitors towards tuft cell lineage and thus controls host immune response on commensal and parasitic microbiota. Next, ID2 is essential for establishing a differentiation gradient for enterochromaffin and X-cells along the anterior-posterior axis of the gut. Finally, ID2 is necessary for the differentiation of N-cells thus ensuring a differentiation gradient along the crypt-villi axis.

overall_design: Single cell RNA-sequencing of 120-day old mouse small intestinal epithelium

first: Natalia

last: Soshnikova


title: RNA-Seq analysis of the stage 51 hind limb bud of Xenopus laevis

city: Dunedin

first: Caroline

last: Beck

state: Otago

address: 340 Great King Street, North Dunedin

department: Zoology

country: New Zealand

institute: University of Otago

summary: Reporting data obtained from dividing the stage 51 Xenopus laevis hind limb bud into thirds along the proximal distal axis (proximal, medial, distal) and sequencing the extracted RNA to investigate differences in gene expression

overall_design: 30 hind limbs per replicate were used for each RNA extraction, there are two biological replicate pools for each segment of the hind limb, labelled as Black or Red

first: Daniel

last: Hudson

first: Robert

last: Day


title: RNA-sequencing of injured cultured dorsal root ganglion neurons over time

city: Athens

first: Oshri

last: Avraham

state: GA

address: 120 Cedar St

department: Cellular Biology

institute: University of Georgia

summary: Injured sensory neurons activate a transcriptional program necessary for robust axon regeneration and eventual target reinnervation. Understanding the transcriptional regulators that govern this axon regenerative response may guide therapeutic strategies to promote axon regeneration in the injured nervous system. Here, we used cultured dorsal root ganglia neurons to identify pro-regenerative transcription factors. Using RNA sequencing, we first characterized this neuronal culture and determined that embryonic day 13.5 DRG (eDRG) neurons cultured for 7 days are similar to e15.5 DRG neurons in vivo and that all neuronal subtypes are represented. This eDRG neuronal culture does not contain other non-neuronal cell types. Next, we performed RNA sequencing at different time points after in vitro axotomy. Analysis of differentially expressed genes revealed upregulation of know regeneration associated transcription factors, including Jun Atf3 and Rest, paralleling the axon injury response in vivo. Analysis of transcription factor binding sites in differentially expressed genes revealed other known transcription factors promoting axon regeneration, such as Myc, Hif1a, Pparg, Ascl1a, Srf, as well as other transcription factors not yet characterized in axon regeneration. We next tested if overexpression of known and novel candidate transcription factors alone or in combination promote axon regeneration in vitro. Our results demonstrate that expression of Ctcf with Yy1 or E2f2 enhances in vitro axon regeneration.  Our analysis reveals that pairs of transcription factors can functionally synergize to promote axon regeneration and highlight that transcription factor interaction play an important role as a regulator of axon regeneration.

overall_design: Examine transcriptional changes in DRG neurons after axon injury

first: Valeria

last: Cavalli


title: Single-cell sequencing of T cells harvested 4 hours after preparation by different methods

city: Shanghai

first: Jiqin

last: Zhang

address: No.500 Dongchuan Road

institute: East China Normal University

summary: We successfully develop a two-in-one approach to generate non-viral genome specific targeted CAR T cells through CRISPR/Cas9. In the adoptive therapy for relapsed/refractory aggressive B-cell non-Hodgkin lymphoma, we observed durable responses without serious adverse events and complete remission in patients treated with these PD1 knockout CAR T cells. Here we applied single-cell sequencing to analyze the characteristcis of T cells harvested 4 hours after preparation by different methods.

overall_design: Examination of T cells prepared by different methods


title: Single cell RNASeq of murine bone marrow derived macrophages (BMDM) treated with non-polymerizing Tamm-Horsfall Protein

city: Indianapolis

first: Kaice

last: LaFavers

state: IN

address: 950 W Walnut

institute: Indiana University School of Medicine

summary: To elucidate changes in macrophage subpopulations in the presence of non-polymerizing Tamm-Horsfall Protein, we performed single cell RNAseq on mouse bone marrow derived macrophages following 18 hour treatment with either a non-polymerizing form of Tamm-Horsfall Protein or vehicle. After treatment, cells were harvested and dead cells removed prior to sorting on a 10X Chromium platform version 3 and generation of libraries. Sequencing was perfomred on an Illlumina NovaSeq 6000.

overall_design: Mouse bone marrow derived macrophages (isolated from WT 129Sv mice) were treated with 1 ug/ml non-polymerizing Tamm-Horsfall protein or vehicle (5% dextrose) for 18 hours

first: K

middle: A

first: T

last: El-Achkar


title: SF3B1 promotes glycolysis and tumor malignancy through splicing-independent activation of HIF1α [ChIP-Seq]

first: Patrik

last: Simmler

overall_design: Panc1 cells were treated either with control siRNA (siCtrl) or siRNA targeting SF3B1 (siSF3B1). siCtrl cells were exposed to 1% O2 for 8 hours. ChIP was performed on these samples with a HIF1a antibody, the retrieved ChIP DNA was sequenced with NGS after spike-in of drosophila DNA

first: Lisa

last: Koch


title: Single-nuclei RNA-seq of the nucleus tractus solitarii and area postrema at for baseline and LPS-activated cell types

status: Public on Jun 18 2022

city: New York

first: Anoj

last: Ilanges

state: NY

address: 1230 York Avenue, Co Box 89

institute: Rockefeller University

summary: To identify LPS activated cell types in the NTS and AP we region we used single-nuclei RNA seq comparing baseline cell types and cell types labeled using the TRAP2 mouse line crossed to the INTACT mouse line after labeling during LPS treatment.

overall_design: Nuclei were isolated using douce homogenization and stained for NeuN. FACS was used to isolate neuronal nuclei (NeuN+).

first: Jeffrey

last: Friedman


title: Targeted degradation of GSPT1 causes TP53-independent cell death in leukemia whilst sparing normal stem cells

city: Boston

first: Adam

middle: S

last: Sperling

address: 4 Blackfan Circle

department: Hematology

institute: Brigham and Women's Hospital

summary: To investigate the effect of drugs that degrade GSTP1 on leukemia cell biology.

overall_design: We performed RNAseq on MOLM13 cells that were treated with vehicle (DMSO), CC-885, or CC-90009 (CC-663) for 24 hours.

first: Rob

last: Sellar

first: Benjamin

last: Ebert


title: Single-cell sequencing of T cells prepared by different methods

summary: We successfully develop a two-in-one approach to generate non-viral genome specific targeted CAR T cells through CRISPR/Cas9. In the adoptive therapy for relapsed/refractory aggressive B-cell non-Hodgkin lymphoma, we observed durable responses without serious adverse events and complete remission in patients treated with these PD1 knockout CAR T cells. Here we applied single-cell sequencing to analyze the characteristcis of T cells prepared by different methods.


title: Effect of depletion of alt-LAMA3 on gene expression of HEK293T cells

status: Public on Jun 17 2022

city: Hangzhou

first: ZHENKUN

state: Zhejiang

address: Fucheng Road No.1

institute: Hangzhou Institute of Medicine, CAS

summary:
To investigate the depletion of altLAMA3, we established HEK293T cell lines in which alt-LAMA3 gene has been knocked out by CRISPR-Cas9.
We then performed gene expression profiling analysis using data obtained from RNA-seq of 2 different cells

overall_design: Comparative gene expression profiling analysis of RNA-seq data for HEK293T cells and its KO derivatives.

first: Zhenkun

first: Haomiao

last: Su


title: Expression profiles of MCF7-CV and MCF7-6SA

city: Taichung City

first: Chun-Te

last: Ho

address: 7F, No. 6, Xueshi Rd., North Dist.

department: Integrative Stem Cell Center

country: Taiwan

institute: China Medical University Hospital

summary:
Gene set enrichment analysis links several pathways are upregulated or downregulated in MCF7-6SA, compared to MCF7-CV.
To elucidate the downstream signaling by which 6SA-Snail suppresses colonization, we first compared the expression profiles of MCF7-CV and MCF7-6SA with the use of microarray data.  Through gene set enrichment analysis (GSEA), we found that several pathways were upregulated or downregulated in MCF7-6SA, compared to MCF7-CV.   Of these, estrogen-dependent gene expression and estrogen receptor (ESR)-mediated signaling were among the most downregulated pathways in MCF7-6SA, which may contribute to the previously observed phenotypic changes in these ER+ cancer cell lines.

overall_design: Total RNA was isolated using the RNeasy kit (Qiagen, Hilden, Germany). The generation of labelled cRNA and its hybridization to Human U133 Plus 2 GeneChip arrays (Affymetrix, Santa Clara, CA) were carried out at the genomic core facilities in the National Yang-Ming University (Taipei, Taiwan) according to the standard procedures. Functional annotation and identiﬁcation of over-represented functional themes were performed using Reactome database of GSEA.

first: Shih-Chieh

last: Hung

first: Shih-Pei

last: Lin


title: Bulk RNA-seq in cholangiocytes from mouse model of primary biliary cholangitis

first: Lindsey

last: Kennedy

address: 702 Rotary Circle

summary: Background & Aims: Late-stage primary biliary cholangitis (PBC) is defined by bile duct loss, ductular reaction, peribiliary inflammation and fibrosis. Loss of anion exchanger 2 (AE2), the ‘bicarbonate umbrella’ and ductulo-canalicular junctions (DCJ) are hypothesized to promote PBC progression. The secretin (Sct)/secretin receptor (SR) axis controls cystic fibrosis transmembrane receptor (CFTR) activation and AE2 opening, and controls choleresis. We aimed to define the impact of Sct/SR signaling on biliary secretory processes and subsequent injury in late-stage PBC mouse model and human samples. Methods: Female and male wild-type (WT) and dominant-negative transforming growth factor beta receptor II (dnTGFβRII, late-stage PBC model) at 32 wks of age were treated with Sct for 1 wk or 8 wks. Pathways mediated by Sct were identified by RNA-seq in isolated cholangiocytes. Sct/SR/CFTR/AE2 expression and MUC1 levels were evaluated in human control and late-stage PBC. Results: Biliary Sct, SR, CFTR and AE2 expression is reduced in late-stage PBC mouse models and human samples. Sct treatment decreased bile duct loss, ductular reaction, inflammation and fibrosis in late-stage PBC models, as well as hepatic bile acid release and DCJ formation. RNA-seq identified that Sct promoted mature epithelial cell marker expression, specifically anterior grade 2 (Agr2, expressed only in cholangiocytes). Late-stage PBC models and human samples had reduced biliary MUC1, which was enhanced with Sct treatment. Conclusion: Loss of the Sct/SR pathway in late-stage PBC results in a faulty ‘bicarbonate umbrella’ and reduced Agr2 and mucin production. Sct restores cholangiocyte secretory processes and DCJ formation through enhanced mature cholangiocyte phenotypes and healthy bile duct growth. The Sct/SR axis may be a therapeutic target for late-stage PBC patients.

overall_design: intrahepatic cholangiocytes analyzed from WT, WT+secretin 8 wk, PBC model (dnTGFBRII), and PBC model + secretin 8 wk. WT cholangiocytes are the control sample. We analyzed male and female samples separately. 6-8 mice per treatment group were pooled to extract cholangiocytes, and total RNA from this pooled population of cholangiocytes was sequenced in triplicate at GeneWiz using an Illumina platform.

middle: L

first: Gianfranco

last: Alpini


title: CHERP controls alternative mRNA splicing in human cell line

status: Public on Jun 19 2022

city: Kyoto

first: Seiji

last: Masuda

state: Kyoto

address: Kitashirakawa Oiwakecho, Sakyo-ku

institute: Division of Integrated Life Sciences, Graduate School of Biostudies

summary: Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP) is co-localized with inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum or peri-nuclear part, and has been considered to have a role in intracellular calcium signaling. Recently, it has recognized that CHERP structurally carries nuclear localization signal and arginine/serine-dipeptide repeats like domain, and interacts with spliceosome. In this study, we showed that poly (A)+ RNA was accumulated in the nucleus on CHERP depletion. Using the global analysis, CHERP regulated alternative mRNA splicing events through the interaction with U2 snRNP and U2 snRNP related proteins. Our analysis indicated that intron retention was most frequently observed among five alternative splicing patterns in accordance with the accumulation of poly (A)+ RNA in the nucleus. Further, intron retention and cassette exon choice were influenced by the strength of 5’ or 3’ splice site, GC content, or intron length. In addition, CHERP depletion induced the abnormality of cell cycle progression at M phase and cell division. These results suggested that CHERP was involved in the regulation of alternative splicing.

overall_design: U2OS cells were treated EGFP or CHERP#2 siRNA transfection for 48h and then harvested. Whole or cytoplasmic RNA were extracted from biological duplicate samples with Sepasol RNA I super G. RNA-seq library was prepared using Truseq Stranded mRNA Library Prep Kit and sequenced on an NextSeq High Output.

first: Y

last: Yamanaka

first: S


title: The histone variant macroH2A modulates inflammatory response in cancer

first: Roberto

last: Malinverni

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

overall_design: Refer to individual Series


title: Chromatin footprints after double-hit stress

first: Jordan

last: Marrocco

address: 1230 York Ave

institute: The Rockefeller University

summary: It was our goal to discern the epigenomic signatures of the ventral hippocampus, in male and female mice in a double hit model of acute stress: one hit of acute restraint stress followed by forced swim stress one week later.

overall_design: At 2.5 months of age, mice were randomly assigned to either acute restraint stress (ARS), forced swim stress (FSS), double-hit stress, namely mice undergoing FSS that were previously exposed to ARS, or control groups. Animals exposed to ARS were only exposed to acute stress one week prior to sacrifice. FSS mice were only exposed to acute stress on the day of sacrifice. Double-hit mice were exposed to acute stress at both time points.

first: Salvatore

middle: G

last: Caradonna


title: Foxp3-dependent and -independent functions of peripherally-induced regulatory T cells: scRNA-seq

city: Princeton

first: Yuri

last: Pritykin

state: NJ

address: 245 Carl Icahn Lab, Lewis-Sigler Institute for Integrative Genomics

institute: Princeton University

summary: Regulatory T (Treg) cells expressing the X-linked lineage-defining transcription factor (TF) Foxp3 are essential for preventing fatal autoimmunity.  During thymic differentiation, Foxp3 expression is induced in a subset of developing self-reactive thymocytes yielding thymic Treg (tTreg) cells, critical for enforcing tolerance to “self”.  In the periphery, Foxp3 induction in mature naïve CD4 T cells activated under particular conditions results in extrathymic generation of Treg (pTreg) cells, thought to contribute to tolerance against commensal microbiota and dietary antigens. While Foxp3 is indispensable for tTreg cell differentiation and function, its role in pTreg cells has remained unknown. Here, we used complementary genetic approaches to characterize pTreg cells induced by microbial colonization and elucidate a role for Foxp3 in these cells. We found that a pTreg cell-specific gene expression program was installed in the gut-draining mesenteric lymph nodes (mLN) in a Foxp3-independent manner. In contrast to tTreg cells residing in the secondary lymphoid tissues, colonic microbiota-dependent pTreg cells did not depend on Foxp3 for their fitness or lineage commitment and were capable of preventing colonic effector T cell expansion in a Foxp3-independent manner. Rather, Foxp3 acted in a cell intrinsic manner to limit IL-17 production and was critical for the suppression of rampant intestinal mastocytosis. Thus, in contrast to tTreg cells, whose functionality is entirely Foxp3-dependent, Foxp3 expression plays a notably nuanced role in pTreg cells and acts to fine-tune the activity of IL-17 producing cells with Foxp3-independent regulatory functions.

overall_design: Germ-free male Foxp3GFP and Foxp3CNS1- mice were colonized with SPF fecal microbiota or kept germ-free. 9 days post-colonization, a 50%-50% mix of GFP+ and GFP- CD4 T cells was sorted from pooled mLN of 3 mice per group and used as input for scRNA-seq analysis.

first: Joris

last: van der Veeken


title: Cullin 4b complex targets IRGM1 to regulate Wnt signaling and intestinal

city: Jinan

first: Yujia

last: Fan

address: Institute of Molecular Medicine and Genetics, School of Basic Medical Sciences, Shandong University, Jinan, Shandong, China.

institute: Shandong University

summary: Hierarchical organization of intestine relies on their stem cells by self-renew and producing committed progenitors. Although signals like Wnt are known to animate the continued renewal by maintaining intestinal stem cells (ISCs) activity, molecular mechanisms especially E3 ubiquitin ligases that modulate ISCs ‘stemness’ and supportive niche have not been well understood. Here, we investigated the role of Cullin 4B (Cul4b) in regulating ISC functions.We conclude that Cul4b is essential for ISC self-renewal and Paneth cell function by targeting Irgm1 and modulating Wnt signaling. Our results demonstrate that Cul4b is a novel ISC stemness and niche regulator.

overall_design:
1. Tissues-Intestinal epithelial-specific deletion of Cul4b (pVillin-Cre; Cul4bfn/Y, KO) and its wildtype control (WT)
2. Organoids- Intestinal epithelial-specific deletion of Cul4b (pVillin-Cre; Cul4bfn/Y, IKO) and its wildtype control (IWT)
3. Organoids-inducible deletion of Cul4b (CAG-CreERT2; Cul4bfn/Y, OKO) and its wildtype control (OWT).
Intestinal tissues and were organoids generated by deep sequencing, using illumina hiseq Xten.

first: Xiaohan

last: Huo

first: Beibei

last: Guo

first: Xiaohui

last: Zhang

first: Yang

last: Yang

first: Jiabei

last: Lian

first: Xinyuan

last: Meng

first: Yongxin

last: Zou

first: Haiyang

first: Haitao

last: Wang

first: Gongping

last: Sun

first: Hao

last: Dou

first: Changshun

last: Shao

first: Yaoqin

last: Gong

first: Huili

last: hu


title: RNA sequencing of zebrafish liver during development, growth and EtOH treatment

status: Public on Jun 16 2022

city: Greater Noida

first: Sumeet

middle: Pal

last: Singh

state: Uttar Pradesh

address: NH91, Tehsil Dadri

department: Life Sciences

country: India

institute: Shiv Nadar Institution of Eminence

summary: Hepatocytes were the first cell-type for which oscillations of cytoplasmic calcium levels in response to hormones were described. Since then, investigation of calcium dynamics in liver explants and culture has greatly increased our understanding of calcium signaling. A bottleneck, however, exists in observing calcium dynamics in a non-invasive manner due to the optical inaccessibility of the mammalian liver. Here we take advantage of the transparency of the zebrafish larvae to develop a setup that allows in vivo imaging of calcium flux in zebrafish hepatocytes at cellular resolution.Using this, we provide quantitative assessment of intracellular calcium dynamics during multiple contexts, including growth, feeding, ethanol-induced stress and cell ablation. Specifically, we show that synchronized calcium oscillations are present in vivo, which are lost upon starvation. Feeding recommences calcium waves in the liver, but in a spatially restricted manner. Further, ethanol treatment as well as cell ablation induces calcium flux, but with different dynamics. The former causes asynchronous calcium oscillations, while the latter leads to a single calcium spike. Overall, we demonstrate the presence of oscillations, waves and spikes in vivo. Thus, our study introduces a platform for observing diverse calcium dynamics while maintaining the native environment of the liver, which will help investigations into the dissection of molecular mechanisms supporting the intra- and intercellular calcium signaling in the liver.

overall_design: For RNA-Seq, mRNA was isolated from livers at the corresponding stages. For this, livers from 30 larvae / replicate were dissected and collected in the lysis buffer from ReliaPrep™ RNA Miniprep Systems (Promega, Z6110). mRNA was isolated from the lysed tissue by following the manufacture’s instruction. cDNA synthesis, library preparation and Illumina sequencing was performed by Eurofins Genomics Europe Sequencing GmbH.  Integrity and quantity of the starting mRNA was determined by appropriate methods, e.g. measurement of volume and quantity, gel electrophoresis and/or fluorimeter measurements. Library preparation incorporated adaptor sequence adapters and indexing compatible for Illumina sequencing technology, using proprietary methods of Eurofins Genomics Europe Sequencing GmbH. The protocol for RNA library preparation was based on NEBNext Ultra II Directional RNA Library Prep Kit for Illumina. For cDNA library preparation, after first strand synthesis, the second strand synthesis was performed using dUPT. The ends of the double stranded cDNA fragments were repaired and dATP ligated to the blunt ended fragments. Next, the sequencing adapters were ligated to the DNA fragments, and the dUTP containing second strand was removed. Sequencing was performed on the Illumina NovaSeq 6000 platform using 2x150 Sequence mode.   Raw reads were mapped using HiSAT2 against the GRCz11 zebrafish genome, and counted using FeatureCounts. For normalization, differential gene expression analysis, and GO analysis, iDEP version 0.951 was utilized with default parameters. For differential gene expression, a fold-change of 2 and false-discovery rate of 0.1 was used for cut-off.

first: Macarena

last: Pozo-Morales


title: Immune checkpoint blockade unleashes aberrant immune responses against the microbiota

city: Bethesda

first: Verena

last: Link

address: 4 Memorial Drive

department: NIAID

institute: NIH

summary: Immune checkpoint inhibitors (ICIs) targeting PD-1 and CTLA-4 are essential components of the cancer therapeutic armamentarium. The remarkable responses seen with ICIs however, are also accompanied by immune-related adverse events (irAEs). These inflammatory side-effects impact virtually all organ systems, with the most common being the skin and gastrointestinal tract. Here, we establish a mouse model of commensal bacteria-driven skin irAEs and demonstrate that immune checkpoint blockade aberrantly unleashes commensal-specific T cell responses. Such responses caused widespread immune cell infiltration and inflammation throughout the skin, recapitulating the skin irAEs seen in patients treated with ICIs. This skin inflammation was dependent on production of the cytokine IL-17 by commensal-specific T cells, which in turn induced hyperactive responses from macrophages and myeloid cells. Importantly, these responses had long-term consequences, with commensal-specific T cells elicited in the context of ICIs being highly sensitive to re-activation by commensals months later. Together, our results establish a mouse model of skin irAEs and show that immune checkpoint blockade can potentiate aberrant immunity to skin commensals with long-lasting consequences.

overall_design: Examiniation of whole ear skin tissue in mice treated with Stapholococcus epidermidids with and without checkpoint inhibitor anti-CTLA-4 treatment

first: Ian

last: Hu

first: Nicholas

last: Collins

first: Yasmine

last: Belkaid


title: The effect of CNF1 on macrophage polarization

city: Tianjin

first: Jian

middle: ming

address: No22.Qixiangtai Rd.,Heping Dist,Tianjin P.R. China

institute: Tianjin Medical University

summary: Research on the mechanism of Cytotoxic Necrotizing Factor 1 in regulating macrophage polarization.To study the molecular mechanisms through which CNF1 induces M1 macrophage polarization, we analyzed gene expression profiles of BMDMs treated with CNF1 or dialysis buffer.

overall_design: Research on gene expression profiles BMDMs treated with CNF1 or dialysis buffer

first: Xuan

first: Jianming


title: Effects of Immunecomplexes on primary human monocytes and their differentitation towards osteoclasts

city: Glasgow

first: Carl

last: Goodyear

address: 120 University Place

department: Institute of Infection, Immunity and Inflammation

country: United Kingdom

institute: University of Glasgow

summary: primary human monocytes were isolated from peripheral blood using Stemcell CD14 magnetic cell seperation and incubated over night with 10ng/ml  M-CSF and then differentiated with 100ng/ml RANKL for 24hrs in the absence or prescence of 25 mg/ml SIC (immunecomplexes consisting of SpA and IgG) or the appropriate concentration of IgG or SpA alone.

overall_design: this study includes biological triplicates for each condition (3 independent buffy coats)

first: Katja

last: Thuemmler

first: Moeed

last: Akbar

first: Martin

last: McBride


title: A regulatory axis between Epithelial Splicing Regulatory Proteins and Estrogen Receptor alpha modulates the alternative transcriptome of luminal breast cancer

city: Orbassano

first: Giulio

last: Ferrero

address: Regione Gonzole, 10

department: Dept. Clinical and Biological Sciences

institute: University of Turin

summary:
The epithelial splicing regulatory proteins 1 and 2 (ESRP1/2) control the epithelial‐to‐mesenchymal transition (EMT) splicing program in cancer. However, their exact role in Breast Cancer (BC) remains under debate. Here, we report that ESRP1, but not ESRP2, is overexpressed in luminal BCs patients with poor prognosis and correlates with Estrogen Receptor α (ERα) mRNA levels. Analysis of ERα genome binding profiles in both cell lines and primary breast tumors showed its binding on both ESRP1 and ESRP2 promoters, and the expression of these genes strongly decreased by ERα silencing in hormone-deprived conditions (apoERα). The combined knock down of ESRP1/2 in MCF-7 cells followed by RNA-Seq, revealed the dysregulation of 754 genes expression, with a widespread alteration of alternative splicing events (ASEs) of genes involved in cell signaling, metabolism, cell growth, and EMT. Functional network analysis of ASEs that correlate with ESRP1/2 expression in ERα+ BCs showed RAC1 as the hub node in the protein-protein interactions altered by ESRP1/2 silencing.
The comparison of apoERα- and ESRP-modulated ASEs revealed 63 commonly regulated events, including 27 detected both in primary BCs and endocrine resistant cell lines.
Our data support a functional implication of the ERα-ESRP1/2 axis in the onset and progression of BC by controlling the splicing patterns of related genes.

overall_design: MCF-7 transfected with siCTRL or siRNAs targeting ESRP1 and ESRP2

first: Jamal

last: Elhasnaoui

first: Valentina

last: Miano

first: Lorenzo

last: Franchitti

first: Isabella

last: Tarulli

first: Santina

last: Cutrupi

first: Michele

last: De Bortoli


title: Distinct gene expression dynamics in developing and regenerating crustacean limbs

status: Public on Jun 15 2022

city: Lyon

first: Mathilde

last: PARIS

address: 32-34 Av. Tony Garnier

country: France

institute: CNRS

summary: Regenerating animals have the ability to reproduce body parts that were originally made in the embryo and subsequently lost due to injury. Understanding whether the process of regeneration mirrors development is an open question in most regenerative species. Here we take a transcriptomics approach to examine to what extent leg regeneration shows the same temporal patterns of gene expression as leg development in the embryo, in the crustacean Parhyale hawaiensis. We find that leg development in the embryo shows stereotypic temporal patterns of gene expression. In contrast, global patterns of gene expression during leg regeneration show a high degree of variation, related to the physiology of individual animals. A major driver of this variation is the molting cycle. After dissecting the transcriptional signals of individual physiology from regeneration, we obtain temporal signals that mark distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although both processes use largely the same genes, the temporal patterns in which these gene sets are deployed are different and cannot be systematically aligned.

overall_design:
Regeneration Dataset (Rd); Regeneration dataset (Rp); Development dataset (Ef and Ed):
212 mRNAseq leg samples during time courses of development or regeneration in the Crustacean Parhyale hawaiensis: 70 samples were collected during development, 60 limbs were amputated and the most-distal and moore proximal parts of the leg were collected separately throughout regeneration, for a total of 120 samples (60 samples corresponding to the regenerating most-distal part of the leg, as well as the 60 samples corresponding to the non-regenerating most distal part of the leg). 20 entire legs were collected at different moments of the molting cycle. Finally two entire legs were also collected, with to tracking of their molting stage, for the purpose of genome annotation.

first: Chiara

last: Sinigaglia

first: Alba

last: Almazán

first: Marie

last: Lebel

last: Sémon

first: Benjamin

last: Gillet

first: Sandrine

last: Hughes

first: Eric

last: Edsinger

first: Michalis

last: Averof

last: Paris


title: Aspirin antitumoral effect on pituitary adenoma

city: Budapest

first: István

last: Likó

address: Üllői út 26.

country: Hungary

institute: Semmelweis University

summary: Aspirin, is a commonly used non-steroidal anti-inflammatory drug with antipyretic, analgesic, anti-inflammatory, and anti-thrombotic effect. Its antitumoral effect was also demonstrated in several cancer types. In this study our aim was to investigate the genomic  effects of ASA in pituitary adenomas using high-throughput profiling approaches and validate our findings by targeted methodologies, using in vitro functional assays. Whole transcriptome profile of RC4-B/C and GH3 pituitary cell lines upon Aspirin treatment was investigated.

first: Henriett

last: Butz


title: Two-step regulation of centromere distribution by condensin II with LINC complex and nuclear lamina

city: Yokohama

first: motoaki

last: seki

address: Suehiro-cho 1-7-22, Tsurumi-ku

department: CSRS

institute: RIKEN

summary: To identify genes affected by the mutations in CAP-H2 and CRWN1/4.

overall_design: Total 9 samples were analyzed.

first: Takuya

last: Sakamoto

first: Yuki

first: Stefan

last: Grob

first: Tomoe

last: Yamashita

first: Nanami

last: Ito

first: Yuka

last: Oko

first: Tomoya

last: Sugiyama

first: Takumi

last: Higaki

first: Seiichiro

last: Hasezawa

first: Maho

last: Tanaka

first: Akihiro

last: Matsui

first: Motoaki

last: Seki

first: Takamasa

last: Suzuki

first: Ueli

last: Grossniklous

first: Sachihiro

last: Matsunaga


title: Identification of methylated sites in BT-549 cells affected by thymoquinone treatment

city: Luzhou

first: Md. Asaduzzaman

last: Khan

state: Sichuan

address: 3-319, Zhongshan road

department: The research center for preclinical medicine

institute: Southwest Medical University

type: Methylation profiling by high throughput sequencing

summary: BT-549 cells were treated with thymoquinone for 6 h and chromatin immune-precipitation (ChIP) was performed using anti- MBD1 antibody (Abcam, USA). Anti-MBD1 could precipitate specific genomic region, which are methylated. The ChIP products were sequenced by library construction and bioinformatics analysis. The gene ontology (GO) analysis for multiple pathways was performed. The genomic methylated regions affected by TQ treatment were identified.

overall_design: Investigation of thymoquinone's role on genomewide methylation

first: Md Asaduzzaman

first: Junjiang

last: Fu


title: Genome wide analysis of the chromatin state and gene expression in Wild-type, tor-es mutant, and fie mutants

first: Jen

last: Sheen

state: MA

address: 185 Cambridge st. CPZN7620

department: Molecular Biology

institute: Massachusetts General Hospital

summary:
Using Western blot, we found the level of H3K27me3, but not H3K4me3, H3K9me2 and H3K36me3, was specifically reduced in the tor-es mutant. To gain a genome-wide view of the effects of TOR activity on H3K27me3 distribution, we performed quantitative chromatin immunoprecipitation with an exogenous reference genome (ChIP-Rx) followed by deep-sequencing. We find a global reduction of H3K27me3 occupancy in tor-es, whereas the H3K9me2 level was largely unaffected. These results suggest that TOR may be a specific and direct regulator of global deposition of H3K27me3.
To investigate the function of TOR phosphorylation of FIE, we complemented the heterozygous fie/+ plants with GFP-FIE or the phosphorylation site mutant (SSTS/AAAA) under the control of the FIE promoter. To provide a parallel comparison with SSTS/AAAA/fie, we generated estradiol-inducible fie-amiR-es transgenic lines, that eliminated FIE protein. Quantitative ChIP-seq analyses revealed greatly reduced H3K27me3 levels across the genome in SSTS/AAAA and fie-amiR-es mutants. And transcriptome profiling by RNA-Seq was conducted to globally identify thousands of genes coordinately dysregulated in the shoots of SSTS/AAAA and fie-amiR-es plants. Furthermore, gene Ontology analysis of 986 TOR-FIE-PRC2 target genes revealed significant enrichment for transcription factors/regulators controlling a broad spectrum of developmental programs.

overall_design: ChIP-seq analysis of H3K2me2 and H3K27me3 in Wild-type, tor-es mutant, and fie mutants. RNA-seq analysis of FIE amiRNA mutant and FIE phosphorylation site mutant

first: Ruiqiang

last: Ye

first: Meiyue

last: Wang

first: Yijing

last: Zhang


title: A multi-omic dissection of super-enhancer driven oncogenic gene expression programs in ovarian cancer [HiC]

city: Chapel Hill

first: Michael

middle: Ross

last: Kelly

state: NC

address: 120 Mason Farm Road

department: Bioinformatics and Genetics

institute: University of North Carolina at Chapel Hill

summary: Ovarian cancer is one of the deadliest cancers among women worldwide and is the leading cause of gynecologic-related cancer deaths in the U.S. Enhancers are often repurposed by the tumor cells for the regulation of genes that allow tumor cells to become more aggressive and resistant to therapy. Recent evidence suggests that exploiting transcriptional dependence by targeting oncogenic super-enhancers may be a viable therapeutic avenue. To this end, we leveraged genomic data such as H3K27ac, BRD4, and copy number information to identify putatively oncogenic super-enhancers. We found that copy number amplification of these super-enhancers is predictive of clinical outcome and that the targets genes associated to the copy number events via chromatin eQTL analysis are involved in numerous oncogenic processes. To systematically probe the functions these super-enhancers we designed a CRISPR interference screen (dCas9-KRAB) so specifically inhibit each super-enhancer and measure the consequences on gene expression via RNA-seq. These results show pervasive gene expression changes that underlie the biology of ovarian cancer. Finally, we select two salient super-enhancers for further analysis. CRISPR-based deletion of these two super-enhancers results in in dramatic changes in gene expression and decreased cell proliferation of ovarian cancer cells. Taken together these analyses highlight the importance of BRD4-bound and copy number amplified super-enhancers in ovarian cancer oncogenic regulation.

overall_design: In this study, two super-enhancers were identified as being oncogenic in ovarian cancer. We wanted to determine if the DEGs detected from DESeq2 and the results of our biological assays were due to direct or indirect effects. Therefore, we performed Hi-C Seq to investigate interaction frequencies across the OVCAR3 genome.

first: Hector

last: Franco

first: Kamila

last: Wisniewska

first: Andrea

last: Perreault

first: Eric

last: Davis


title: Delineating A Serotonin Receptor Pathway for Appetite Suppression

city: Dallas

first: Li

last: Li

state: TX

address: 5323 Harry Hines Boulevard

institute: UT Southwestern Medical Center

summary: single-nucleus RNA sequencing (snRNA-seq) in the ARH of AgRP-Cre; Sun1-GFP mice

overall_design: We used fluorescence-activated cell sorting (FACS) to enrich GFP-positive nuclei from fifteen adult mice. Nuclei from individual mice were tagged with unique DNA barcodes (Gaublomme et al., 2019) before being pooled for library preparation and subsequent sequencing.

first: Steven

middle: C

last: Wyler

first: Chen

last: Liu


title: Enterococcus faecalis antagonizes Pseudomonas aeruginosa growth in mixed-species interactions

city: Singapore

first: Casandra

last: Tan

address: 60 Nanyang Drive

country: Singapore

institute: SCELSE

summary: Enterococcus faecalis is often co-isolated with Pseudomonas aeruginosa in mixed-species biofilm-associated infections of wounds and the urinary tract. As a defence strategy, the host innately restricts iron availability at infection sites. Despite their co-prevalence, the polymicrobial interactions of these two pathogens in low iron conditions, such as those found in the host, remains unexplored. Here we show that E. faecalis inhibits P. aeruginosa growth within macrocolony biofilms when iron is restricted. E. faecalis lactate dehydrogenase (ldh1) gives rise to L-lactate production during fermentative growth. We find that E. faecalis ldh1 mutant fails to inhibit P. aeruginosa growth. Additionally, we demonstrate that ldh1 expression is induced when iron is restricted, resulting in increased lactic acid exported and consequently, a reduction in pH. Together, our results suggest that E. faecalis synergistically impact P. aeruginosa growth negatively by decreasing environmental pH and L-lactate-mediated iron chelation. Overall, this study highlights that the microenvironment in which the infection occurs is important for understanding its pathophysiology.

sample_organism:
Pseudomonas aeruginosa
Enterococcus faecalis

overall_design: We aim to identify genes that are differentially expressed by E. faecalis and/or P. aeruginosa when grown in unchelated and iron-chelated conditions.

first: Kimberly

last: Kline


title: Detection of Cell-Cell Interactions via Photocatalytic Cell Tagging

city: Cambridge

first: Cory

middle: Haley

last: White

address: 320 Bent St

department: DAGS

institute: Merck

summary: The growing appreciation of immune cell-cell interactions within disease environments has led to significant efforts to develop highly effective protein-, and cell-based immunotherapies.  However, characterizing these complex cell-cell interactions in high resolution remains challenging.  Thus, technologies that leverage therapeutic-based modalities for profiling intercellular environments can provide unique advantages towards understanding these cellular interactions at molecular-level detail.  To address this, we introduce photocatalytic cell tagging (PhoTag), a platform for profiling cell-cell interactions that utilizes a single domain antibody (VHH) conjugated to a photoactivatable flavin-based cofactor.  Upon irradiation with visible light, the tethered flavin photocatalyst generates phenoxy radical tags for targeted labeling within cell-cell contact environments.  Using anti-PD-1 or anti-PD-L1 VHH flavin conjugates, we demonstrate that PhoTag achieves highly selective synaptic labeling in antigen presenting cell-T cell co-culture systems. By combining the high resolution transcellular biotinylation capability of PhoTag with multi-omics single cell sequencing, we interrogated transient interactions between Peripheral blood mononuclear cell (PBMC) populations and Raji PD-L1 B cells and discovered that specific T cell subtypes can transiently interact more efficiently than others.   We envision that the spatio-temporal and modular nature of PhoTag will enable its broad utilization for detailed profiling of intercellular interactions across different biological systems.

overall_design: For the single cell sequencing aspect of this study, 3 donors were used with samples from each donor split into the different treatment groups (Isotype, PD-L1, and LFA-1 inhibitor).  Isotype labeling serves as the reference samples.

middle: H

first: Tamara

last: Reyes Robles

first: Rob

last: Oslund

first: Olugbeminiyi

last: Fadeyi


title: TRPV1 SUMOylation suppresses itch by inhibiting TRPV1 interaction with H1 receptors

status: Public on Jun 14 2022

city: Shanghai

first: Yong

address: 280 South Chongqing Road

department: Department of Biochemistry and Molecular Cell Biology

institute: Shanghai Jiao Tong University School of Medicine

summary: To examine the functional significance of TRPV1 SUMOylation in vivo, we created a TRPV1K823R KI transgenic mouse line.We thus performed gene expression profiling analysis using data obtained from RNA-seq of the trigeminal ganglion of TRPV1K823R and control mouse models . Our analyses unveiled a role for TRPV1 SUMOylation in regulating gene networks related to the pain and itch pathway.

overall_design: Comparative gene expression profiling analysis of RNA-seq data of trigeminal ganglion lysates from TRPV1K823R and control mouse models at 8 weeks old.

first: Yingwei

last: Gao


title: Single-cell sequencing of infusion products and T cells after administration of non-viral PD1-targeted anti-CD19 CAR T cells

first: Jiqin

address: No.500 Dongchuan Road

institute: East China Normal University

summary: We successfully developed a two-in-one approach to generate non-viral genome specific targeted CAR T cells through CRISPR/Cas9. In the adoptive therapy for relapsed/refractory aggressive B-cell non-Hodgkin lymphoma, we observed durable responses without serious adverse events and complete remission in patients treated with PD1 knockin CAR T cells. Here we applied single-cell sequencing to analyze the characteristcis of infusion products and T cells after administration.

overall_design: Examination of infusion products and T cells after administration in three patients


title: EZH2 KO RNA-seq in HSC-5 cells

city: Cold Spring Harbor

first: Seamus

last: Balinth

address: 1 Bungtown road

institute: Cold Spring Harbor Lab

summary: CRISPR was used to deplete EZH2 in HSC-5 cells. RNA-seq was then carried out to identify changes in the transcriptome following EZH2 loss in these cells. Critically, we identified TP63 to be downregulated in the EZH2-depleted cells, and also RUNX3 to be significantly upregulated.

overall_design: Cas9 was stably expressed in HSC-5 cells via lentiviral transduction. The cells were then transduced with sgRNAs targeting EZH2. G418 was used to select for HSC-5 cells that had taken up the sgRNA. Following selection, EZH2 depletion was confirmed via western blotting and RNA was immediately collected for RNA-seq. 2 different sgRNAs were used. Samples were run in triplicate. One control was omitted from final analysis as it did not cluster with the others. Significantly changed genes were confirmed in follow up studies through RT-qPCR analysis.


title: Failure of human rhombic lip differentiation constitutes medulloblastoma

city: Toronto

first: Liam

last: Hendrikse

state: Ontario

address: 610 University Ave

department: Princess Margaret Cancer Centre

country: Canada

institute: University Health Network

summary: Single-nucleus RNA sequencing was performed on Group 3 medulloblastoma tumorspheres expresssing high vs. low levels of OTX2

overall_design: OTX2 levels were knocked down in HDMB03 and MB3W1 Group 3 MB cells (2X105 cells/well) as previously described (Zagozewski et al., Nature Comm, 2020; Stromecki et al., Molecular Oncology, 2018). Briefly, OTX2 was silenced using Silencer Select siRNAs 9931 or 9932 (Life Technologies) at a concentration of 30 nM. A non-silencing (scramble) served as the negative control. Samples were collected at 72 hours for RNA extraction and sequencing.

first: Tamra

last: Werbowetski-Ogilvie

first: Michael

last: Taylor


title: p63 ChIP-seq in HSC-5 cells

summary: p63 was IP'd from HSC-5 cells to identify target genes

overall_design: p63 ChIP-seq in HSC-5 cells


title: EZH2 Regulates a SETDB1/dNp63a Axis Via RUNX3 to Drive a Cancer Stem Cell Phenotype in Squamous Cell Carcinoma

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

overall_design: Refer to individual Series


title: Precocious expression of Zelda does not initiate early zygotic genome activation

city: Madison

first: Melissa

last: Harrison

email: mharrison3@wisc.edu, ykaur@wisc.edu, torreszelada@wisc.edu, mmfreund2@wisc.edu

state: WI

address: 420 Henry Mall

department: Biomolecular Chemistry

institute: University of Wisconsin Madison

summary: During the first stages of development, the fertilized germ cells rapidly transition to totipotency. Maternally deposited mRNAs encode the proteins necessary for reprogramming the transcriptionally quiescent zygotic genome during this maternal-to-zygotic transition (MZT). The transcription factor Zelda is essential for this reprogramming in the Drosophila embryo. Zelda is necessary for transcriptional activation of the zygotic genome, and the absence of Zelda leads to embryonic lethality during the MZT. Excess Zelda activity is also lethal to the embryo, demonstrating that Zelda levels must be precisely controlled during early development. Because Zelda is encoded by a maternally deposited mRNA, Zelda levels in the embryo are controlled at the level of translation. To understand how levels of this essential reprogramming factor were regulated to allow for embryonic development and zygotic genome activation, we investigated the factors that regulated translation of zelda. Brain Tumor (BRAT) is a translational regulator that was previously shown to bind to zelda mRNA in the embryo. We showed that BRAT functions to repress zelda translation, as embryos deficient for maternal BRAT activity prematurely express Zelda. We further showed that in the larval brain, BRAT similarly regulates Zelda levels and identified specific BRAT-binding sites that mediate these effects. Thus, BRAT regulates Zelda in multiple tissues. Because both too little and too much Zelda are lethal to the embryo, we hypothesized that precocious expression of this transcriptional activator might be capable of driving precocious activation of the zygotic genome, leading to embryonic lethality. To test this hypothesis, we performed single embryo RNA-seq at distinct nuclear cycles throughout zygotic genome activation (NC10, NC12, NC13, and NC14) in control and brat-mutant embryos. Our results conclusively demonstrated that in embryos lacking functional BRAT, Zelda target genes were not prematurely activated. Rather, these genes were expressed normally, but become significantly upregulated at nuclear cycle 14, when the division cycle slows. Our data support a model in which zygotic genome activation requires precise coordination between expression of reprogramming factors, such as Zelda, and the slowing of the cell cycle.

overall_design: RNA-seq from single late stage oocytes and single embryos (NC10, NC12, NC13, early NC14 and later NC14) colleted in triplicate or quadruplicate from bratDf/fs1, brat Df or brat fs1, and His2AVRFP flies.

first: Elizabeth

last: Larson

first: Hideyuki

last: Komori

first: Zoe

last: Fitzpatrick

first: Samuel

last: Krabbenhoft

first: Cheng-Yu

last: Lee


title: Gene expression profile at single cell level of monocytes (MOs) and T cells from the lungs and from broncho-alveolar lavage fluids (BALF) from Mock and MuHV-4 infected  WT and CCR2 KO mice.

city: Liège

first: Bénédicte

last: Machiels

state: Liège

address: Avenue de Cureghem 7b, B43b

department: DMI

country: Belgium

institute: Université de Liège

summary: Gammaherpesviruses (γHVs) have co-evolved with their hosts over millions of years, leading to remarkably high infection prevalence and establishment of symbiotic relationship. Therefore, the lifelong persistence of γHVs appear to broadly shape host immunity. Here, we show that pulmonary infection with Murid herpesvirus 4 (MuHV-4), a mouse γHV, drives the recruitment in the airways of Ly6Chi monocytes (MOs) that control the host immune response. Indeed, the absence of those MOs elicits a severe virus-induced immunopathology associated with the systemic release of inflammatory mediators. Mechanistically, MuHV-4 imprinted MOs recruit CD4 T cells in the airways and trigger immunosuppressive signalling pathways through the PDL1/PD-1 axis, thereby dampening the deleterious activation of cytotoxic CD4 T cells. These results uncover a role for Ly6Chi MOs in the modulation of CD4 T cell functions, and reveal key pathways that could be targeted to interfere with the detrimental immunopathological cascade during respiratory viral infections.

overall_design: Lung MOs and CD4 T cells from C57BL/6 mice were isolated by Fluorescence-activated cell sorting (FACS) according to the presence or absence of surface markers.

first: Céline

last: Maquet

first: Jérôme

last: Baiwir

first: Pauline

last: Loos

first: Lucia

last: Rodriguez-Rodriguez

first: Justine

last: Javaux

first: Rémy

last: Sandor

first: Fabienne

last: Perin

first: Padraic

last: Fallon

first: Matthias

last: Mack

first: Didier

last: Cataldo

first: Laurent

last: Gillet


title: A multi-omic dissection of super-enhancer driven oncogenic gene expression programs in ovarian cancer [ChIP-seq]

first: Michael

last: Kelly

country: USA

overall_design: In this study, two super-enhancers were identified as being oncogenic in ovarian cancer. The genomic coordinates for super-enhancer 60 (SE60) are chr20:52352051-52368920 using the Hg19 human genome reference. The genomic coordinates for super-enhancer 14 (SE14) are chr1:16493671-16516686 using the Hg19 human genome reference.  Each super enhancer was inhibited using CRISPR-dCas9-KRAB (CRISPRi) in the ovarian cancer cell line OVCAR3. In order to verify the integrity of our guide process we then performed ChIP-Seq for H3K9me3 on both the SCRAMBLE controls and the SE60 CRISPRi targeted cells. This was performed by ActiveMotif.

first: Hector

last: Franco

first: Kamila

last: Wisniewska

middle: R


title: Foxp3 Dependent and Independent Functions of Peripherally Induced Regulatory T Cells: colonic scRNA+TCR-seq

city: Princeton

first: Yuri

last: Pritykin

address: 245 Carl Icahn Lab, Lewis-Sigler Institute for Integrative Genomics

institute: Princeton University

summary: Regulatory T (Treg) cells expressing the transcription factor Foxp3 are an essential suppressive CD4 T cell lineage of dual origin: Foxp3 induction in thymocytes and mature CD4 T cells gives rise to thymic (tTreg) and peripheral (pTreg) Treg cells, respectively. While tTreg cells primarily suppress autoimmunity, pTreg cells are thought to enforce tolerance to food and commensal microbiota. However, the role of Foxp3 in pTreg cells and the mechanisms underlying their differentiation and function remain poorly understood. Here, we used genetic lineage tracing to unambiguously identify microbiota-induced pTreg cells and found that many of their distinct features were Foxp3-independent. Lineage-committed, microbiota-dependent pTreg-like cells persisted in the colon in the absence of Foxp3 protein. While Foxp3 was critical for the suppression of a Th17 cell program, colitis and rampant mastocytosis, pTreg cells suppressed colonic effector T cell expansion in a Foxp3-independent manner. Thus, Foxp3 and the tolerogenic signals that precede and promote its expression independently confer distinct facets of pTreg functionality.

overall_design: Approximately 5,000 sorted tdTomato+GFP+ and tdTomato-GFP- colonic lamina propria CD4 T cells were used as input for each replicate. Biological triplicates from the same group were labeled with TotalSeq-C reagents and multiplexed on one lane of 10X Chromium, aiming for a total of 15,000 cells per sample.

first: Joris

last: van der Veeken


title: Remote ischemic preconditioning causes transient cell-cycle arrest and renal protection by a NF-kB-dependent Sema5B pathway

city: Münster

first: Jan

last: Rossaint

address: Albert-Schweitzer-Campus 1, Bldg. A1

department: Dpt. of Anesthesiology

institute: University of Münster

summary: Our study investigates how RIPC modulates the transcriptomic profile of neutrophils and renal tubular epithelial cells in the kidney during acute kidney injury.

overall_design: Acute kidney injury was induced by ischemia-reperfusion injury (IRI) in mice. Single neutrophils were isolated from the blood and renal tubular epithelial cells were isolated from the kidney 24 hours after induction of AKI.

first: Tobias

last: Tekath


title: RNA sequencing of Escherichia coli K12 substr. MG1655 (MTCC 1302) treated with silver nanoparticles

city: Columbia

first: Sathesh

middle: K

last: Sivasankaran

state: MO

address: Bond Life Sciences Center Suite 106, 1201 Rollins Street

institute: University of Missouri

summary: The current study deals to decipher the antibacterial mechanism of lysozyme coated silver nanoparticles (L-Ag NPs) (coated with lysozyme) against a Gram negative modal organism Escherichia coli K12 (MTCC 1302). Hence, the whole transcriptome profiling of E. coli K12 was done by exposing it to the MIC75 concentration of L-Ag NPs for 5 and 30 min., by RNA sequencing (RNAseq) analysis. The obtained results were utilized to understand all the metabolic pathways, signaling and molecular functions in bacterial cells under the stress of L-Ag NPs. RNAseq showed a high number of total reads along with significant ratio of high-quality reads, which confirmed the excellent quality and quantity of the obtained RNAseq data. Controlled release of ions from the surface of L-Ag NPs allowed the bacterial cells to function normally till the accumulation of threshold amount of silver ions which triggered the action of defence system, thus, reducing the chances of resistance development in bacteria. In long term, such treatment may force the bacterial machinery to induce changes in their genome to counteract the situation and develop resistance against silver ions, similar to the well-known antibiotic resistance problem. The obtained results advocate that L-Ag NPs can be used as effective antibacterial agent.

overall_design: RNA sequencing was carried out for E. coli K12 (MTCC 1302) exposing it to the MIC75 concentration of L-Ag NPs for 5 and 30 min., duplicate samples were sequenced for each stage.

first: Vikram

last: Pareek

first: Rinki

last: Gupta

first: Stephanie

last: Devineau

first: Arpit

last: Bhargava

first: Mohd

last: Khan

first: Shabarinath

last: Srikumar

first: Séamus

last: Fanning

first: Jitendra

last: Panwar


title: Two-step regulation of centromere distribution by condensin II with LINC complex and nuclear lamina [36 samples]

first: motoaki

last: seki

state: Kanagawa

summary: To identify genes affected by the mutations in CAP-H2 and CRWN1/4 under the DNA damaging condition

overall_design: Total 36 samples were analyzed.

first: Takuya

last: Sakamoto

first: Yuki

first: Stefan

last: Grob

first: Tomoe

last: Yamashita

first: Nanami

last: Ito

first: Yuka

last: Oko

first: Tomoya

last: Sugiyama

first: Takumi

last: Higaki

first: Seiichiro

last: Hasezawa

first: Maho

last: Tanaka

first: Akihiro

last: Matsui

first: Motoaki

last: Seki

first: Takamasa

last: Suzuki

first: Ueli

last: Grossniklous

first: Sachihiro

last: Matsunaga


title: Total transcriptome assays were performed on ovules and developing seeds derived from two seeded (Italia and Red globe) and two seedless (Thompson Seedless and Autumn Royal) table grape varieties

city: VR

first: Alessandra

last: Amato

address: strada le grazie 15

institute: University of Verona

summary:
VviAGL11, the Arabidopsis SEEDSTICK homolog, has been proposed to have a causative role in grapevine stenospermocarpy. An association between a mutation in the coding sequence (CDS) and the seedless phenotype was reported, however, no working mechanisms have been demonstrated yet.
We performed a deep investigation of the full VviAGL11 gene sequence in a collection of grapevine varieties belonging to several seedlessness classes that revealed three different promoter-CDS combinations. By investigating the expression of the three VviAGL11 alleles, and by evaluating their ability to activate the promoter region, we observed that VviAGL11 self-activates in a specific promoter-CDS combination manner.
Furthermore, by transcriptomic analyses on ovule and developing seeds in seeded and seedless varieties and co-expression approaches, candidate VviAGL11 targets were identified and further validated through luciferase assay and in situ hybridization. We demonstrated that VviAGL11 Wild Type CDS activates Methyl jasmonate esterase and Indole-3-acetate beta-glucosyltransferase, both involved in hormone signaling and Isoflavone reductase, involved in secondary metabolism. The dominant-negative effect of the mutated CDS was also functionally ectopically validated in target induction. VviAGL11 was shown to co-localize with its targets in the outer seed coat integument, supporting its direct involvement in seed development, possibly by orchestrating the crosstalk among MeJA, auxin, and isoflavonoids synthesis.
In conclusion, the VviAGL11 expression level depends on the promoter-CDS allelic combination, and this will likely affect its ability to activate important triggers of the seed coat development. The dominant-negative effect of the mutated VviAGL11 CDS on the target genes activation was molecularly validated. A new regulatory mechanism correlating VviAGL11 haplotype assortment and seedlessness class in grapevine is proposed.

overall_design: Ovules and developing seeds transcriptome of two seeded (Italia and Red globe) and two seedless (Thompson seedless and Autumn royal) table grape varietas. Four developing stages were considered (S1-S4; sheet 2) and each sample have been tested in biological triplicate (48 samples in total). For each probe, the fluorescence values is indicated.

first: Maria

middle: F

last: Cardone

first: Nallatt

last: Ocarez

first: Fiammetta

last: Alagna

first: Benedetto

last: Ruperti

first: Chiara

last: Fattorini

first: Riccardo

last: Velasco

first: Nilo

last: Mejia

first: Sara

last: Zenoni

first: Carlo

last: Bergamini


title: The Impact of Monosomies, Trisomies and Segmental aneuploidies on Chromosomal stability

city: Amsterdam

first: Dorine

last: Hintzen

address: Plesmanlaan 121

department: Cell Biology

country: Netherlands

institute: Netherlands Cancer Institute

summary: Aneuploidy and chromosomal instability are both commonly found in cancer. Chromosomal instability leads to karyotype heterogeneity in tumors and is associated with therapy resistance, metastasis and poor prognosis. It has been hypothesized that aneuploidy per se is sufficient to drive CIN, however due to limited models and heterogenous results, it has remained controversial which aspects of aneuploidy can drive CIN. In this study we systematically tested the impact of different types of aneuploidies on the induction of CIN. We generated a plethora of isogenic aneuploid clones harboring whole chromosome or segmental aneuploidies in human p53-deficient RPE-1 cells. We observed increased segregation errors in cells harboring trisomies that strongly correlated to the number of gained genes. Strikingly, we found that clones harboring only monosomies do not induce a CIN phenotype. Finally, we found that an initial chromosome breakage event and subsequent fusion can instigate breakage-fusion-bridge cycles. By investigating the impact of monosomies, trisomies and segmental aneuploidies on chromosomal instability we further deciphered the complex relationship between aneuploidy and CIN.

overall_design: 6 different clones with whole chromosome aneuploidies (WA) are analyzed compared to parental P53KD background to obtain differentially expressed genes in these clones. Each sample contains two replicates.

first: Jonne

last: Raaijmakers

first: Michael

last: Schubert

first: Roel

middle: J

last: Kluin


title: The intrinsic and extrinsic effects of Tet proteins during gastrulation

city: Rehovot

first: Markus

last: Mittnenzweig

state: Shfela

address: 234 Herzl St.

country: Israel

institute: Weizmann Institute of Science

summary:
To isolate cell-autonomous effects of TET loss during gastrulation, we utilized a chimeric embryo platform in which fluorescently tagged Tet-deficient and control mouse embryonic stem cells (mESCs) were either injected into tetraploid (4N) or diploid (2N) blastocysts, and allowed to develop in utero. In 4N complemented embryos, the resulting embryonic compartment is solely comprised of the injected mESCs derivatives (hereinafter denoted as whole-embryo chimera), whereas in chimeras obtained using 2N host blastocysts, the embryonic compartment contains both wild-type (WT) and injected cell derivatives (hereinafter denoted as mixed chimera).
We performed single-cell RNA-seq for each whole-embryo and mixed chimera embryo, and compared the transcriptomes to a defined single-cell/single-embryo atlas. We further performed methylation analysis using post-bisulfite adaptor tagging (PBAT) for sorted Tet-TKO mutant and control from E8.5 mixed chimeras. Our work demonstrates an unbiased approach for defining intrinsic and extrinsic embryonic gene function based on temporal differentiation atlases, and disentangles the intracellular effects of the demethylation machinery from its broader tissue-level ramifications.

overall_design:
We individually imaged each chimeric embryo and performed index-sorting for them. To map the developmental potential for Tet-TKO cells in the whole-embryo chimera context, embryos were sampled at E7.5-8.5. Mixed chimera embryos were collected at E7.5. We then perform combined analysis of timed chimeric embryos in the context of a precise single embryo/single-cell temporal gastrulation atlas.
To map the impact of Tet deficiency on embryonic DNA methylation while focusing on intrinsic effects, we injected a mixture of TKO and control cells to 2N blastocysts, generating chimeric embryos that were harvested at E8.5 when substantial differentiation is already established. We then sorted apart Tet-TKO mutant and control cells for analysis using PBAT protocol.

first: Saifeng

last: Cheng

first: Yoav

last: Mayshar

first: Aviezer

last: Lifshitz

first: Marko

last: Dunjić

first: Yoach

last: Rais

first: Raz

last: Ben-Yair

first: Stephanie

last: Gehrs

first: Elad

last: Chomsky

first: Zohar

last: Mukamel

first: Katharina

last: Schlereth

first: Netta

last: Reines

first: Ayelet

last: Orenbuch

first: Amos

last: Tanay

first: Yonatan

last: Stelzer


title: Transcriptome profiling of the ventral pallidum reveals a role for pallido-thalamic neurons in cocaine reward

status: Public on Jun 13 2022

city: Bordeaux cedex

first: Michel

last: Engeln

address: Zone nord Bat 2 2ème étage

institute: Institut de Neurosciences Cognitives et Intégratives d'Aquitaine (INCIA)

summary: Using RNAseq, we evaluated transcriptomic changes in the ventral pallidum (VP) of mice 24h following 10 days of cocaine self-administration.

overall_design: C57BL/6 mice self-administered cocaine (0.5mg/kg/infusion) or saline for 10 consecutive days. Twenty-four hours following the last session, fresh ventral pallidum has been collected. RNA has been extracted to perform RNA sequencing.

first: Ramesh

last: Chandra

first: Mary

last: Lobo


title: Donor T-cell DNMT3a regulates alloreactivity in murine models of hematopoietic stem cell transplantation.

city: Baltimore

first: Yiouli

last: Ktena

state: MD

address: 1800 Orleans Street

department: SOM Onc Pediatric Oncology

institute: JHU; School of Medicine


title: A multi-omic dissection of super-enhancer driven oncogenic gene expression programs in ovarian cancer [CRISPRi RNA-seq]

city: Chapel Hill

first: Michael

last: Kelly

address: 120 Mason Farm Road

institute: University of North Carolina at Chapel Hill

summary: Ovarian cancer is one of the deadliest cancers among women worldwide and is the leading cause of gynecologic-related cancer deaths in the U.S. Enhancers are often repurposed by the tumor cells for the regulation of genes that allow tumor cells to become more aggressive and resistant to therapy. Recent evidence suggests that exploiting transcriptional dependence by targeting oncogenic super-enhancers may be a viable therapeutic avenue. To this end, we leveraged genomic data such as H3K27ac, BRD4, and copy number information to identify putatively oncogenic super-enhancers. We found that copy number amplification of these super-enhancers is predictive of clinical outcome and that the targets genes associated to the copy number events via chromatin eQTL analysis are involved in numerous oncogenic processes. To systematically probe the functions these super-enhancers we designed a CRISPR interference screen (dCas9-KRAB) so specifically inhibit each super-enhancer and measure the consequences on gene expression via RNA-seq. These results show pervasive gene expression changes that underlie the biology of ovarian cancer. Finally, we select two salient super-enhancers for further analysis. CRISPR-based deletion of these two super-enhancers results in in dramatic changes in gene expression and decreased cell proliferation of ovarian cancer cells. Taken together these analyses highlight the importance of BRD4-bound and copy number amplified super-enhancers in ovarian cancer oncogenic regulation.

overall_design: In this study, two super-enhancers were identified as being oncogenic in ovarian cancer. The genomic coordinates for super-enhancer 60 (SE60) are chr20:52352051-52368920 using the Hg19 human genome reference. The genomic coordinates for super-enhancer 14 (SE14) are chr1:16493671-16516686 using the Hg19 human genome reference.  Each super enhancer was inhibited using CRISPR-dCas9-KRAB (CRISPRi) in the ovarian cancer cell line OVCAR3. The guides RNAs used for deletion and the genotyping results are described in the associated publication. For each super enhancer, two knockdown replicates were generated. RNA was purified from asynchronous cells for each clone. Library prep was done using the Illumina TruSeq mRNA Library Kit following the manufacturer's protocol. RNA was sequenced at 150 cycles, paired end, on an Illumina NextSeq 550. The super-enhancer locations, gRNA locations, and gRNA sequences are located in one of the supplimentary tables from the associated manuscript.

first: Hector

last: Franco

first: Kamila

last: Wisniewska