title: Melanoblast transcriptome analysis reveals novel  pathways promoting melanoma metastasis

status: Public on Dec 06 2019

city: Bethesda

first: Maxwell

last: Lee

state: MD

address: Building 37, Room 5120

department: CCR

institute: National Cancer Institute, NIH

summary:
Purpose: We hypothesized that embryonic melanocytic pathways would be reinitiated in melanoma metatastasis. To identify nove melanoblast signaling pathways with which to explore our hypothesis we used the inducible Dct-GFP mouse model, in which GFP is expressed in both immature melanoblasts and mature melanocytes. Mouse melanoblasts/melanoytes were extracted using Fluorescence Activated Cell Sorting and transcriptomes were analyzed using RNA-sequencing.
Abstract: Cutaneous malignant melanoma is an aggressive cancer of melanocytes with a strong propensity to metastasize. We posit that melanoma cells acquire metastatic capability by adopting an embryonic-like phenotype, and that a lineage approach would uncover novel metastatic melanoma biology. Using a genetically engineered mouse model to generate a rich melanoblast transcriptome dataset, we identify melanoblast-specific genes whose expression contribute to metastatic competence and derive a 43-gene signature that predicts patient survival. We identify a melanoblast gene, KDELR3, whose loss impairs experimental metastasis. In contrast, KDELR1 deficiency enhances metastasis, providing the first example of different disease etiologies within the KDELR-family of retrograde transporters. We show that KDELR3 regulates the metastasis suppressor, KAI1, and report an interaction with the E3 ubiquitin-protein ligase gp78, a regulator of KAI1 degradation. Our work demonstrates that the melanoblast transcriptome can be mined to uncover targetable pathways for melanoma therapy.
Method: FVB/N iDct-GFP mice were used and doxycycline was administered to activate GFP expression. Multiple litters were used for each developmental stage, and embryos/pups from each stage were pooled to ensure adequate numbers of GFP+ cells. At E17.5, P1 and P7 stages, most melanocytes have reached the dermis, thus only the skin was collected from these developmental stages. Back skin was immersed in a shallow layer of 1X PBS and subcutaneous fat was scraped off until skin appeared translucent. E15.5 was the youngest age assessed due to the necessity to capture sufficient cells for RNA-sequencing. Embryos of the same developmental age that were heterozygous for the TRE-H2B-GFP gene but lacked the Dct-rtTA gene were used as negative controls. Cell doublets were excluded from the analysis. Cells were sorted based on GFP expression and SSC-A. Based on these reference sorts, gates were set so that background cells represented less than 10% of sorted cells. Cells were lysed in 10-fold TRIzol reagent (w/v), phases were separated by addition of 0.2X volume of chloroform, the aqueous phase was combined with an equal volume of 70% ethanol and applied to a RNeasy Micro column (Qiagen) and processed as per the manufacturer’s instructions. Paired-end sequencing libraries were prepared using 1 μg of purified RNA following the mRNA-Seq Sample Prep Kit according to the manufacturer’s instructions (Illumina).  RNA-Seq libraries were sequenced on two lanes each of an Illumina GAIIx Genome Analyser to a minimum depth of 49 million reads. Sequence reads were aligned to the mm9 genome using the TopHat software (https://ccb.jhu.edu/software/tophat/index.shtml). Quantified Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values were generated using the Cufflinks software (http://cole-trapnell-lab.github.io/cufflinks/). The UCSC KnownGenes gene models were used for guided alignment and quantification.
Results: DESeq2 analysis identified melanoblast-specific gene expression, which was shown by further analyses using patient data and wet lab techniques to likewise be upregulated in metastatic melanoma and facilitate metastasis.

overall_design: RNA-sequencing of mouse Embryonic day 15.5, 17.5 melanoblasts and Postnatal day 1 and day 7 melanocytes.  Multiple litters (6-10 pups each) were used for each developmental stage, and embryos/pups from each stage were pooled to ensure adequate numbers of GFP+ cells. Sequence reads were aligned to the mm9 genome using the TopHat software (https://ccb.jhu.edu/software/tophat/index.shtml). Quantified Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values were generated using the Cufflinks software (http://cole-trapnell-lab.github.io/cufflinks/). The UCSC KnownGenes gene models were used for guided alignment and quantification.

first: Kerrie

last: Marie

first: Antonella

last: Sassano

first: Howard

last: Yang

first: Aleksandra

last: Michalowski

first: Helen

last: Michael

first: Theresa

last: Guo

first: Yien

middle: C

last: Tsai

first: Allan

last: Weissman

first: Lisa

last: Jenkins

first: M

last: Zaidi

first: Eva

last: Pérez-Guijarro

first: Chi-Ping

last: Day

first: Kris

last: Ylaya

first: Stephen

last: Hewitt

first: Heinz

last: Arnheiter

first: Sean

last: Davis

first: Paul

middle: S

last: Meltzer

first: Glenn

last: Merlino

first: Pravin

middle: J

last: Mishra


title: Genome wide-binding profile of NRF2 in A549 NSCLC cells [ChIP-seq]

status: Public on Dec 05 2019

city: HANGZHOU

first: XIUWEN

last: TANG

address: YUHANGTONG ROAD

department: BIOCHEMISTRY AND GENETICS

country: China

institute: ZHEJIANG UNIVERSITY SCHOOL OF MEDICINE

type: Genome binding/occupancy profiling by high throughput sequencing

summary: This study aimed to elucidate the relationships between NRF2 and disease progression and provide insight into NRF2-mediated cancer progression/tumorigenesis by identifying novel genes and pathways regulated by NRF2 in A549 NSCLC cells

overall_design: NRF2 ChIP-seq of A549 NSCLC cells, control library contains the input DNA

first: Liu

last: Kehua

first: Tang

last: Xiuwen


title: Sex chromosomes affect gene expression of healthy CD4+ T cells in SJL.

status: Public on Dec 10 2019

city: Los Angeles

first: Yuichiro

last: Itoh

state: California

address: 635 Charles E. Young Drive South

department: Neurology

country: USA

institute: UCLA

type: Expression profiling by high throughput sequencing

summary: To determine the role of sex chromsomes in sex differences of autoimmune disease, we used high throughput RNA sequencing to analyze the transciptomes of CD4+ T cells stimuated with anti-CD3/CD28 from XX and XY- mice using the “four core genotypes” mouse model. We found higher expression of several X genes in the XY- genotype compared to XX, suggesting a difference in DNA methylation of these genes between Xp and Xm. In a hypothesis driven targeted approach, we investigated two immunomodulatory X genes, Tlr7 and FoxP3 and found that the promoter regions of both genes had more DNA methylation on the Xp than Xm, and that both genes had higher expression in autoantigen stimulated CD4+ T cells from XY- compared to XX mice. This study provides new insights regarding how sex chromsomes affect gene expression and cause sex differences in autoimmune disease.

overall_design: Female mice from the "four core genotypes" mouse model (XX and XY-) were gonadectomized at 4-6 weeks old. At 8-12 weeks old, auxillary, brachial, and inguinal draining lymph nodes were collected and processed into single cell suspensions and sorted for CD4+ T cells using the EasySep Mouse CD4+ T cell isolation kit (STEMCELL). Cells were stiumulated with  anti-CD3/CD28 Dyna Beads for 36 hours and RNA was isolated for analysis by high throughput RNA sequencing.

last: Golden

first: Noriko

first: Rhonda

last: Voskuhl


title: RNA-seq analysis of PRMT5-regulated genes in irradiated/non-irradiated LNCaP cells

city: West Lafayette

first: Chang-Deng

last: Hu

state: IN

address: 201 S. University St, Hansen Life Sciences Building, Room 401A

department: Department of Medicinal Chemistry and Molecular Pharmacology

institute: Purdue University

summary: DNA Double-strand break (DSB) repair is critical for cell survival and genome integrity. Upon recognition of DSBs, repair proteins are transiently upregulated to facilitate repair through homologous recombination (HR) or non-homologous end joining (NHEJ). We present evidence that PRMT5 cooperates with pICln to function as a master epigenetic activator of DNA damage response (DDR) genes involved in HR, NHEJ, and G2 arrest (including RAD51, BRCA1, and BRCA2) to upregulate gene expression upon DNA damage. Contrary to the predominant role of PRMT5 as an epigenetic repressor, our results demonstrate that PRMT5 and pICln can activate gene expression, potentially independent of PRMT5’s obligate cofactor MEP50. Targeting PRMT5 or pICln hinders repair of DSBs in multiple cancer cell lines, and both PRMT5 and pICln expression positively correlates with DDR genes across 32 clinical cancer data sets. Thus, targeting PRMT5 or pICln may be explored in combination with radiation or chemotherapy for cancer treatment.

overall_design: RNA-seq analysis of both non-irradiated and irradiated LNCaP cells with and without PRMT5 knockdown

first: Jake

middle: L

last: Owens

first: Jun

last: Wan


title: U1 snRNP regulates cancer cell migration and invasion in vitro

city: Philadelphia

first: Gideon

last: Dreyfuss

state: PA

address: 415 curie blvd

institute: University of Pennsylvania

summary: This project looks into how U1 levels affect cancer cell phenotype in vitro.

overall_design: Knockdown of U1 by antisense morpholino or overexpression of U1 snRNP by plasmid transfection followed by examining transcriptome changes by RNA-seq or examining cancer phenotype by established assays.

first: Jung-Min

last: Oh


title: Maternal Pregnancy Diet Effect on Fetal Brain Transcriptome

city: Bilthoven

first: Jeroen

last: Pennings

address: A. van Leeuwenhoeklaan 9

country: Netherlands

institute: Natl. Inst. Public Health & Environment

summary:
Maternal over- and undernutrition in pregnancy plays a critical role in fetal brain development and function. The effects of different maternal diet compositions on intrauterine programming of the fetal brain in the absence of maternal obesity or maternal undernutrition is a lesser-explored area.  The goal of this study was to investigate the impact of two different maternal diets on fetal brain gene expression signatures, fetal/neonatal growth, and neonatal behavior in a mouse model.
Female C57Bl/6J mice were fed one of two commercially-available chow diets (pelleted vs. powdered) with differing micronutrient and carbohydrate compositions throughout pregnancy and lactation.  The powdered chow diet was richer in carbohydrates and lower in micronutrients than the pelleted chow diet, among other differences. On embryonic day 15.5, embryos were weighed and measured. Fetal brains were snap frozen. RNA was extracted from fetal forebrains for five fetuses per diet group and hybridized to whole genome expression microarrays.
Functional analyses identified significant upregulation of canonical pathways and upstream regulators involved in cell cycle regulation, synaptic plasticity, and sensory nervous system development in the fetal brain, and significant downregulation of pathways related to cell and embryo death. Pathways related to DNA damage response, humoral and cell-mediated immune response, carbohydrate and lipid metabolism, small molecule biosynthesis, and amino acid metabolism were also dysregulated.
Maternal dietary content is an important variable for researchers evaluating fetal brain development and offspring behavior to consider. Selection of a chow diet matched for micronutrients is crucial to avoid unexpected or undesired effects on offspring brain development and behavior.

overall_design: 10 samples, 2 groups of 5 biological replicates

first: Andrea

last: Edlow


title: Integrin-specific hydrogels modulate transplanted human mesenchymal stem cell survival, engraftment, and reparative activities

status: Public on Dec 09 2019

city: Atlanta

first: Andres

middle: Jose

last: Garcia

state: GA

address: 1106 IBB Building

institute: Georgia Institute of Technology

summary: Abstract: Stem cell therapies are severely limited by poor cell survival and engraftment rates. A major hurdle to the use of materials for cell delivery is the lack of understanding of material properties that govern transplanted stem cell functionality. Here, we show that synthetic hydrogels presenting integrin-specific peptides enhance the survival, persistence, and osteo-reparative functions of human mesenchymal stem cells (hMSC) transplanted in murine bone defects. Integrin-specific hydrogels regulated hMSC adhesion, paracrine signaling, and osteoblastic differentiation in vitro. Hydrogels presenting GFOGER, a triple helical peptide targeting α2β1 integrin, prolonged hMSC survival and engraftment in a segmental bone defect and resulted in improved bone repair compared to other adhesive and control peptides. Moreover, integrin-specific hydrogels had diverse pleiotropic effects on hMSC reparative activities, modulating in vitro cytokine secretion and in vivo gene expression for effectors associated with inflammation, vascularization, and bone formation. These results demonstrate that integrin specificity can be engineered into synthetic hydrogel systems resulting in improved tissue healing by directing mesenchymal stem cell survival, engraftment, paracrine immunomodulatory potential, and reparative activities.    Radial segmental defects were treated with  hydrogels functionalized with adhesive peptide and encapsulated hMSC. The tissue within the defect space was explanted at 1 week post-transplantation and stored in RNAlater solution (Qiagen) until further processing. Samples were placed in Qiazol solution (Qiagen), lysed by probe sonication, and homogenized in QIAshredder columns (Qiagen). Total RNA was isolated using an RNAeasy Plus Micro kit (Qiagen), and RNA content and purity were measured by spectrophotometry (NanoDrop 1000). cDNA synthesis was performed on total RNA (100 ng) using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher). Quantitative PCR was performed using Fluidigm 96×96 nanofluidic arrays targeting a set of 96 transcripts (human or murine) to observe changes in bone, survival, inflammation, vascularization, and matrix markers. Primers used are listed in Supplementary Table 4. The genes were pre-amplified in a single 13-cycle PCR reaction for each sample with EvaGreen Mastermix (Fluidigm BioMark) following the manufacturer’s protocol. Sixty-three gene targets resulted in detectable qPCR results. All subsequent statistical analyses were carried out using JMP-Genomics (SAS Institute) using the basic gene expression workflow 62. Raw Ct values were imported into JMP-Genomics and normalized to mean Ct values across all genes for each sample for principal components analysis (PCA), assessment of the biological principal variance component contributions (PVCA), and hierarchical clustering using Ward’s method to identify sub-types of expression profile. Finally, one-way ANOVA was used to detect statistical differences followed by False Discovery Rate analysis using two-state linear step-up procedure of Benjamini, Krieger and Yekutieli. Results are presented as raw Ct values normalized to mean Ct values across all genes for a sample.

platform_organism:
Homo sapiens
Mus musculus

overall_design: qPCR gene expression profiling.  Following implantation of hMSC-laden hydrogels (sample size of 7-8 per adhesive peptide), tissue was explanted from the defect at 1 week and total RNA was extracted and standardized across all samples.

first: Amy

middle: Y

last: Clark


title: ribosome profiling of cell line models of human breast cancer

city: Birmingham

first: Christos

last: Vaklavas

state: Alabama

address: NP2540M 1720 2nd Ave S

department: Medicine

institute: University of Alabama at Birmingham

summary: We performed ribosome profiling in cell line models of human breast cancer. We profiled 2 representative estrogen receptor-positive (T47D and ZR75-1) and 2 representative triple negative (SUM159PT and MDA-MB-231) cell lines. Translational profiles of a nonmalignant (Human Mammary Epithelial Cells, HMECs) and a nontumorigenic (MCF10A cells) counterparts were also analyzed. We analyzed and compared translational efficiencies, variances of the translational efficiencies, differential gene and transcript expression between malignant and non-malignant cell lines. We performed discriminative motif analysis of the 5'untranslated regions of the transcripts that are commonly transcribed in nonmalignant and malignant cells but preferentially translated in malignant cells and identified common motifs. Some of these motifs are highly similar to the RNA binding motifs of a small list of RNA interacting proteins. This approach led us to identify SRSF1 as an RNA binding protein commonly expressed among all analyzed cell lines that has a pervasive impact on the translational landscape of a representative estrogen receptor-positive and -negative cell line

overall_design: Ribosome profiling of 6 different cell lines in at least 2 biological replicates

first: Scott

middle: W

last: Blume

first: William

middle: E

last: Grizzle


title: Single-cell connectomic analysis of adult mammalian lungs

status: Public on Dec 04 2019

city: New Haven

first: Micha Sam

middle: Brickman

last: Raredon

state: Connecticut

address: 10 Amistad Street

department: Biomedical Engineering

institute: Yale University

summary: Single-cell RNA sequencing (scRNAseq) has made it possible to analyze complex organs such as the lung with unprecedented resolution. We have leveraged these techniques to identify conserved patterns of cell-cell cross-talk in adult mammalian lungs, analyzing mouse, rat, pig and human pulmonary tissues. Following clustering and histological registration, we mapped putative ligand-receptor interactions within and between cell types and assessed the significance of these interactions. Our findings demonstrate specific, stereotyped functional roles for each cell type in the distal lung which emerge as top candidates for the regulation of tissue phenotype.

sample_organism:
Homo sapiens
Sus scrofa
Mus musculus
Rattus norvegicus

overall_design:
Single-cell RNA sequenceing data was generated from the lungs of 2 mice, 2 rats, 2 pigs, and 14 humans

Please note that ther are privacy concerns with the raw reads (for the human samples, GSM4050097-GSM4050115), as they are clinical in origin.  Thus, the raw data files are intended to be uploaded to dbGaP in the coming months.

first: Micha

first: Taylor

last: Adams

first: Jonas

last: Schupp


title: Investigating the mechanism by which the natural product compound puupehenone potentiates the antifungal drug caspofungin

city: University

first: Ameeta

last: Agarwal

state: MS

address: NCNPR, Room 2049

department: National Center for Natural Products Research

institute: University of Mississippi

summary: The cell wall-targeting echinocandin antifungals, although potent and well-tolerated, are inadequate in treating fungal infections due to their narrow spectrum of activity and their propensity to induce pathogen resistance. A promising strategy to overcome these drawbacks is to combine echinocandins with a molecule that improves their activity and also disrupts drug adaptation pathways. In this study, we show that puupehenone (PUUP), a marine sponge-derived sesquiterpene quinone potentiates the echinocandin drug, caspofungin (CAS) in CAS-resistant fungal pathogens. We have conducted RNA-Seq analysis, followed by genetic and molecular studies, to elucidate PUUP’s CAS-potentiating mechanism. We found that the combination of CAS and PUUP blocked the induction of CAS-responding genes required for the adaptation to cell wall stress through the cell wall integrity (CWI) pathway. Further analysis showed that PUUP inhibited the activation of Slt2 (Mpk1), the terminal MAP kinase in this pathway. We also found that PUUP induced heat shock response genes and inhibited the activity of heat shock protein 90 (Hsp90). Molecular docking studies predicted that PUUP occupies a binding site on Hsp90 required for the interaction between Hsp90 and its co-chaperone Cdc37. Thus, we show that PUUP potentiates CAS activity by a previously undescribed mechanism which involves disruption of Hsp90 activity and the CWI pathway. Given the requirement of the Hsp90-Cdc37 complex in Slt2 activation, we suggest that inhibitors of this complex would disrupt the CWI pathway and synergize with echinocandins. Therefore, the identification of PUUP’s CAS-potentiating mechanism has important implications in the development of new antifungal combination therapies.

overall_design: Gene expression profiles of Saccharomyces cerevisiae cells treated with caspofungin (CAS), puupehenone (PUUP), and caspofungin + puupehenone (CAS + PUUUP).

middle: K


title: Gene expression of  fibroblasts carrying SAMHD1 mutations or not

status: Public on Dec 03 2019

city: Padova

first: Gerolamo

last: Lanfranchi

address: Via U. Bassi, 58/B

department: CRIBI - Biotechnology Center and Biology Department

country: Italy

institute: University of Padova

summary: In mammalian cells, the catabolic activity of the dNTP triphosphohydrolase SAMHD1 sets the balance and the concentrations of the four dNTPs. Deficiency of SAMHD1 leads to unequally increased pools and marked dNTP imbalance. Although it is documented that imbalanced dNTP pool expansion increases mutation frequency in cancer cells, it is not known if the SAMHD1-induced dNTP imbalance favors accumulation of somatic mutations in non-transformed cells. Here we have investigated how fibroblasts isolated from Aicardi Goutières Syndrome (AGS) patients with mutated SAMHD1 react to the constitutive pool imbalance characterized by a huge dGTP pool. We focused on the effects on dNTP pools, cell-cycle progression, dynamics and fidelity of DNA replication, efficiency of UV-induced DNA repair. AGS fibroblasts entered senescence prematurely or upregulated genes involved in G1/S transition and DNA replication. The normally growing AGS cells exhibited unchanged DNA replication dynamics and, when quiescent, faster rate of excision repair of UV-induced DNA damages than wildtype fibroblasts. To investigate if the lack of SAMHD1 affects DNA replication fidelity we compared de novo mutations in AGS and WT cells by exome next generation sequencing. Somatic variant analysis indicated a mutator phenotype suggesting that SAMHD1 is a caretaker gene whose deficiency is per se mutagenic promoting genome instability in non-transformed cells.

overall_design: This experiment was designed to understand if mutations on SAMHD1 gene interfere with gene expression. We used one primary cell line as control (WT) and four primary cell line carrying SAMHD1 mutantions (AGS patients). The four AGS cell lines are named P1 (AGS282 carring the mutation R145X and R164X), P2 (AGS295 carring a deletion of exons 12-16), P3 (AGS165 carring the deletion of AA 120_123), P4 (AGS128 carring the mutation Q149X). Each cell line was tested in at least three biological replicates using one color design.

first: Stefano

last: Cagnin

first: Chiara

last: Rampazzo

first: Beniamina

last: Pacchioni

first: Caterina

last: Millino


title: Imprinted Cdkn1c Genomic Locus Cell-Autonomously Promotes Cell Survival in Cerebral Cortex Development

city: Klosterneuburg

first: Florian

middle: M

last: Pauler

address: Am Campus 1

country: Austria

institute: Institute of Science and Technology Austria (IST Austria)

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

overall_design: Refer to individual Series


title: JMJD3 and UTX Determine Fidelity and Lineage Specification of Human Neural Progenitor Cells [ChIP-Seq]

status: Public on Dec 02 2019

city: Guangzhou

first: Tian

last: Zhang

state: Guangdong

address: 190 Kaiyuan Avenue,Science Park

institute: GIBH

summary: Neurogenesis entails a highly orchestrated process from pluripotent to neural cell fates including progenitors (NPCs) and various neural subtypes. However, the precise epigenetic mechanisms underlying the fate decision remain poorly understood. Here, we deleted KDM6s (JMJD3 or/and UTX), the demethylases for H3K27me3, in human embryonic stem cells (hESCs) and show that their deletion does not impede NPC generation from hESCs. However, KDM6-deficient NPCs exhibit poor proliferation and fail to become neurons and glia. Mechanistically, KDM6 deficiency leads to H3K27me3 accumulation and blockade of DNA accessibility at loci essential for neurogenesis, thus hinder the fate transition from NPCs to neural subtypes. Our findings uncover an essential role of KDM6s in neurogenesis and highlight the contribution of individual epigenetic regulators in specifying lineage fidelity and fate decision during human development.

overall_design: We compared the JMJD3 binding sites of  NPCs by ChIP-seq.

first: Yongli

last: Shan

first: Guangjin

last: Pan


title: Heat-shock influence on gene expression in HEK293T human cells.

city: Moscow

first: Nickolai

last: Tchurikov

address: 32, Vavilova str.

department: Department of Epigenetic Mechanisms of Gene Expression Regulation

country: Russia

institute: Engelhardt Institute of Molecular Biology

summary: RNA-Seq procedure was used for analysis of expression in HEK293T cells. Heat shock treatments was performed for 20 min at 43°C. Then heat shock treated cells were incubated for 6 h at 37°C and replicates were separately lysed with Trisol reagent. Total RNA was extracted from cell lysed with Trisol reagent and PureLink RNA Micro Kit (Invitrogen) according to manufacture instruction. RNA Quality was checked with  BioAnalyser and  RNA 6000 Nano Kit(Agilent). Poly(A)+ RNA was isolated with Dynabeads® mRNA Purification Kit (Ambion). Illumina library was prepared from poly(A)+RNA with NEBNextNEBNext® Ultra™II RNA Library Prep Kit for Illumina® (NEB) according to manual. Sequencing was performed on HiSeq1500 with 50 bp read length.

overall_design: Analysis of HEK293T gene expression levels by RNA-Seq.

middle: A

first: Yuri

middle: V

last: Kravatsky

first: Olga

last: Kretova


title: JMJD3 and UTX Determine Fidelity and Lineage Specification of Human Neural Progenitor Cells


title: RNAseq of DUX KO mouse 2-cell embryos

city: Lausanne

first: Delphine

last: Grun

address: route Cantonale

country: Switzerland

institute: EPFL

summary: Some of the earliest transcripts produced in fertilized human and mouse oocytes code for DUX, a double homeodomain protein that promotes embryonic genome activation (EGA). Deleting Dux by genome editing at the 1- to 2-cell stage in the mouse impairs EGA and blastocyst maturation. Here, we demonstrate that mice carrying homozygous Dux deletions display markedly reduced expression of DUX target genes and defects in both pre- and post-implantation development, with notably a disruption of the pace of the first few cell divisions and significant rates of late embryonic mortality. However, some Dux-/- embryos give raise to viable pups, indicating that DUX is important but not strictly essential for embryogenesis.

overall_design: RNA-sequencing of mouse 2-cell embryos from WT, heterozygous and homozygous DUX KO mice crossings

first: Alberto

last: De Iaco


title: Comparative transcriptome analysis of melatonin or auxin treated-Arabidopsis

city: Melbourne

first: Sajal

middle: Fatima

last: Zia

state: VIC

address: Agribio,  5 Ring Rd, Bundoora VIC 3083

department: Animal, Plant and Soil Sciences

country: Australia

institute: La Trobe University

summary:
Objective: Melatonin and auxin are both tryptophan-derived indole molecules. Much attention has been given to proposed auxin-like activities of melatonin (regulating growth concentration-dependently). However, it still largely remains unclear whether melatonin and auxin regulate signalling pathways in a similar fashion. The purpose of this study is to directly compare the transcriptome response of Arabidopsis with melatonin or auxin.
Method: mRNA profiles of 12-day old rosettes treated for a further of 3 days with +/- melatonin (5µM, 1005µM) or NAA(4.5µM) were generated by RNA-sequencing in triplicates (three independent biological experiments), using Illumina NextSeq 550 technology.
Results: Comparative global transcriptome analysis conducted on Arabidopsis rosette treated with melatonin or NAA under exact same set of experimental conditions revealed differential number of genes and type of pathways. While auxin (4.5µM) regulated a large number of genes and elicited a diverse response, melatonin (100µM) showed a modest effect on transcriptome with only few genes significantly regulated whereas none regulated at approximately equimolar concentration with NAA (5µM) as compared to untreated solvent control (0.1% EtOH). Interestingly, the most prominent category of genes regulated by melatonin trended towards biotic stress defense pathways.
Conclusions: These findings indicate that melatonin and auxin act quite differently toward signaling pathways in Arabidopsis. Melatonin has its own set of mechanisms to exert its functions, with strong inclination toward biotic defense pathways.

overall_design: mRNA profiles of +/- melatonin (5µM or 100µM) or NAA(4.5 µM)-treated Arabidopsis rosettes were generated by RNA-sequencing in triplicates (three independent biological experiments), using Illumina NextSeq 550.

middle: F

first: Frank

last: Bedon

first: Oliver

last: Berkowitz

first: James

last: Whelan

first: Ashley

last: Franks

first: Kim

last: Plummer


title: The Stearyl CoA desaturase is a gatekeeper that protects human ß cells against lipotoxicity and maintains their identity

city: Paris

first: Masaya

last: OSHIMA

address: 123 boulevard de Port Royal

country: France

institute: INSERM U1016

summary: Here, we used the human ß cell line EndoC-ßH1 and analyzed its sensitivity to a lipo- and gluco-lipotoxic (high palmitate with or without high glucose) insult, as a way to model human ß cells in a T2D environment.

overall_design: siSCD vs. siCTRL

last: Oshima

first: Raphael

last: Scharfmann


title: Effect of ET-2 expression of innate Lymphocyte precursors (ILCPs)

city: OKLAHOMA CITY

first: jose

last: alberola-ila

state: OK

address: 825 N.E. 13th Street, Room E407

institute: OMRF

summary: Innate Lymphoid Cells (ILCs) are tissue-resident lymphoid cells that reside mostly at barrier surfaces and participate in the initial response against pathogens. They are classified into different effector programs that parallel T helper subsets based on cytokine production and transcription factor expression. They all derive from the Common Lymphoid Precursor, but the molecular mechanisms regulating ILC subset development into effector programs is not well understood. Experiments using Id2 knockout mice have previously shown that E-protein activity inhibition is an absolute requirement for development of all ILC subsets. Here, we use a genetic approach to demonstrate that small increases in E protein activity during ILC development selectively inhibit ILC2 development. ILC1 are mostly unperturbed, and ILC3 show only a minor inhibition. This effect is first evident at the ILC2P stage and is ILC intrinsic. Therefore, our results demonstrate that modulation of E protein activity can bias cell fate decisions in developing ILCs.

overall_design: ILCPs (Lin-;IL7Rα+;α4β7+;CD90+;CD25-) expressing ET-2 (GFP+) or not (GFP-) were sorted from Rag1-cre;ET-2 mice bone marrow. Three biological replicates, each sorted from 4-5 pooled mice were processed

first: Alberola-Ila

last: Jose

first: Berrett

last: Hannah


title: Neuroprotective effect of miR-204 OE in mouse models of Inherited Retinal Disease (IRD)

city: Pozzuoli

first: Rossella

last: De Cegli

address: Via Campi Flegrei 34

institute: Tigem

summary:
Transcriptome profile of retinas from wild-type (c57BL/6) and RHO-P347S mice following AAV-mediated subretinal delivery of pre-miR-204 to study the molecular changes underlying the neuroprotective effect of miR-204 OE in mouse models of Inherited Retinal Disease (IRD)
To determine the molecular changes associated with AAV-mediated delivery of miR-204 in the subretinal space of wild-type and IRD mouse models, we performed RNASeq analysis of retinas from c57BL/6 and RHO-P347S mice injected with the AAV2/8.CMV.miR204 and AAV2/8.CMV.miR204MUT vector in a paired manner.

overall_design: Total RNA was extracted at postnatal day (P) 12 from retinas injected at P4 with the AAV2/8.CMV.miR204 vector; RNA extracted from retinas of controlateral eyes (injected with the control). Designation of contralateral eyes i.e. OS=Left eye, OD=Right eye of the same animal AAV2/8.CMV.miR204MUT vector) was used as control.

first: Sandro

last: Banfi

first: Marianthi

last: Karali


title: Imprinted Cdkn1c Genomic Locus Cell-Autonomously Promotes Cell Survival in Cerebral Cortex Development [Cdkn1c_allelic expression]

first: Florian

last: Pauler

summary: The cyclin-dependent kinase inhibitor p57KIP2 is encoded by the imprinted Cdkn1c locus, exhibits maternal expression, and is essential for cerebral cortex development. How Cdkn1c regulates corticogenesis is however not clear. To this end we employ Mosaic Analysis with Double Markers (MADM) technology to genetically dissect Cdkn1c gene function in corticogenesis at single cell resolution. We find that the previously described growth-inhibitory Cdkn1c function is a non-cell-autonomous one, acting on the whole organism. In contrast we reveal a growth-promoting cell-autonomous Cdkn1c function which at the mechanistic level mediates radial glial progenitor and nascent projection neuron survival. Strikingly, the growth-promoting function of Cdkn1c is highly dosage sensitive but not subject to genomic imprinting. Collectively, our results suggest that the Cdkn1c locus regulates cortical development through distinct cell-autonomous and non-cell-autonomous mechanisms. More generally, our study highlights the importance to probe the relative contributions of cell intrinsic gene function and tissue-wide mechanisms to the overall phenotype.

overall_design: Targeted sequencing of cDNA to quantify allelic expression

first: Susanne

last: Laukoter

first: Robert

last: Beattie

first: Simon

last: Hippenmeyer


title: JMJD3 and UTX Determine Fidelity and Lineage Specification of Human Neural Progenitor Cells [RNA-seq]

first: qianyu

last: chen

address: 190 Kai Yuan Avenue, Science Park

institute: Guangzhou Institutes of Biomedicine and Health,Chinese Academy of Sciences

overall_design: We compared the transcriptome of  NPCs and differentiated NPC at day 28 of KDM6-deficient cells and their wildtype counterparts by RNA-Seq.

first: Yongli

last: Shan

first: Guangjin

last: Pan

first: Qianyu

last: Chen

first: Juan

last: Du

first: Zhongzhou

last: Yang

first: Andrew

last: Hutchins


title: JMJD3 and UTX Determine Fidelity and Lineage Specification of Human Neural Progenitor Cells [UTX ChIP]

first: Tian

last: Zhang

overall_design: We compared the UTX binding sites of  NPCs by ChIP-seq.


title: Transcriptomic profiling of blood and skin T cells.

status: Public on Dec 01 2019

city: Oxford

first: Ling-Pei

last: Ho

address: Headley Way

department: MRC Human Immunology Unit

country: United Kingdom

institute: University of Oxford

overall_design: Suction blister– and blood-derived peripheral blood mononuclear cells (PBMCs) were isolated by flow cytometry and collected gating on subsets of T cells.

first: Graham

last: Ogg


title: JMJD3 and UTX Determine Fidelity and Lineage Specification of Human Neural Progenitor Cells [ChIP-seq]

overall_design: We compared the H3K27me3 binding sites of  NPCs and differentiated NPC at day 28 of KDM6-deficient cells and their wildtype counterparts by ChIP-seq.


title: JMJD3 and UTX Determine Fidelity and Lineage Specification of Human Neural Progenitor Cells [ATAC-seq]

overall_design: We compared the chromatin accessibility of  NPCs by ATAC-seq.


overall_design: We compared the chromatin accessibility of  NPCs and differentiated NPC at day 28 of KDM6-deficient cells and their wildtype counterparts by ATAC-seq.


title: Re-evaluation of human BDCA-2+ DC during acute sterile skin inflammation

summary: Transcriptomic profiling of skin wounds in time-course manner

overall_design: Baseline full thickness punch biopsies were performed on the flank of healthy donors to create skin wounds. This was followed by larger biopsies, to encompass the original wounds, taken between one to four days later. The skin biopsies were subjected to targeted RNA sequencing analysis.

first: Yi-Ling

first: Danuta

last: Gutowska-Owsiak

first: Nicki

last: Gray


title: Effect of submergence on wild-type (Col-0) Arabidopsis plants

city: San Giuliano Terme

first: Pierdomenico

last: Perata

state: Pisa

address: Via Guidiccioni 8-10

department: Institute of Life Sciences

institute: Sant'Anna School of Advanced Studies

summary: We examined the expression of miRNAs in submerged plants and identified a set of submergence-regulated miRNAs.

overall_design: Each treatment is represented by two biologica replicates (Control) and three biological replicates (submerged plants). Each single biological replicate was obtained by pooling 4 plants. Genotypes of Arabidopsis thaliana used included Col-0 ecotype (wild-type) and the hypomorphic ARGONAUTE1 mutant ago1-27. Plants were grown in pots for three to four weeks at 23°C with a 12/12 h photoperiod at 120 μmol photons m–2 s–1 before being utilized for experiments. Plants were submerged in tanks with a water level of 10 cm above leaf level. The submergence treatment (12h) was carried out in the dark.

first: Elena

last: Loreti


title: The nucleosome core particle remembers its position through DNA replication and transcription

city: Berkeley

first: Gavin

middle: Schwebs

last: Schlissel

state: CA

address: 440 Barker Hall

department: Molecular Cell Biology

institute: UC Berkeley

summary: We tracked the fate of labelled nucleosomes through DNA replication, and established that nucleosomes present at a locus remembered their position during DNA replication. The replication-associated histone chaperones DPB3 and MCM2 were essential for nucleosome position memory, and in the absence of both DPB3 and MCM2 histone chaperone activity nucleosomes did not remember their position. Using the same approach, we tested the model that transcription results in retrograde transposition of nucleosomes along a transcription unit. We found no evidence of retrograde transposition. Our results suggest that nucleosomes have the capacity to transmit epigenetic memory across mitotic generations with exquisite spatial fidelity.

overall_design: Nucleosomes were tagged with the 15aa AviTag, then the AviTag was biotinylated using various versions of TetR-BirA (including a split TetR-BirA obligate heterodimer). Site-specific biotinyaltion was stopped by adding doxycycline, and the labeled nucleosomes were detected before and after replication using streptavidin affinity purification and sequencing. In the same samples, we directly detected the localization of the TetR-BirA enzyme by using a V5-tagged allele of TetR-BirA, and precipitating the TetR-BirA-bound chromatin using anti-V5 agarose resin.

first: Jasper

last: Rine


title: Campylobacter concisus Impairs sodium absorption in colonic epithelium via ENaC dysfunction and claudin-8 disruption

city: Berlin

first: Oliver

last: Nagel

address: Hindenburgdamm 30

department: Department of Gastroenterology, Rheumatology and Infectious Diseases

country: Germany

institute: Charité - Universitätsmedizin Berlin

summary: The study aimed to characterize C. concisus effects on ENaC using the HT-29/B6-GR/MR cell model.

overall_design: Examination of differential expression between colonic epithelium cells treated with DBS/treated with DBS and infected with C. consisius

first: Praveen

last: Nattramilarasu

first: Roland

last: Bücker

first: Fábia

middle: D

last: Lobo de Sá

first: Anja

last: Fromm

first: In-Fah

last: Lee

first: Eduard

last: Butkevych

first: Markus

last: Heimesaat

first: Stefan

last: Bereswill

first: Michal

middle: R

last: Schweiger

first: Hans

last: Nielsen

first: Hanno

last: Troeger

first: Jörg-Dieter

last: Schulzke


title: Effect of submergence on wild-type (Col-0) and ago1-27 mutant

first: Pierdomenico

last: Perata

summary: Hypoxia exerts major effects on gene expression, which is reconfigured to meet the needs of the stressed plant. In the recent years the mechanisms of oxygen sensing and transcriptional regulation of a cluster of anaerobic genes was described. We utilized a hypomorphic, post-transcriptional gene silencing defective ARGONAUTE1 (AGO1) mutant, ago1-27, to evaluate the contribution of AGO1 to plant tolerance to submergence and its effects on gene expression.

overall_design: Each treatment is represented by three biological replicates. Each single biologica replicate was obtained by pooling 4 plants. Genotypes of Arabidopsis thaliana used included Col-0 ecotype (wild-type) and the hypomorphic ARGONAUTE1 mutant ago1-27. Plants were grown in pots for three to four weeks at 23°C with a 12/12 h photoperiod at 120 μmol photons m–2 s–1 before being utilized for experiments. Plants were submerged in tanks with a water level of 10 cm above leaf level. The submergence treatment (12h) was carried out in the dark.


title: IL-33 signaling alters regulatory T cell diversity in support of tumor development

city: Cambridge

first: Rebecca

middle: H

last: Herbst

state: MA

address: 415 Main Street

department: Klarman Cell Observatory

institute: Broad Institute of MIT and Harvard

summary: Regulatory T cells (Tregs) can impair anti-tumor immune responses and are associated with poor prognosis in multiple cancer types. Tregs in human tumors span diverse transcriptional states distinct from those of peripheral Tregs, but their contribution to tumor development remains unknown. Here, we use single cell RNA-Seq to longitudinally profile dynamic shifts in the distribution of Tregs in a genetically-engineered mouse model of lung adenocarcinoma. In this model, interferon-responsive Tregs are more prevalent early in tumor development, while a specialized effector phenotype characterized by enhanced expression of the interleukin 33 receptor ST2 is predominant in advanced disease. Treg-specific deletion of ST2 alters the evolution of effector Treg diversity, increases infiltration of CD8+ T cells into tumors, and decreases tumor burden. Our study shows that ST2 plays a critical role in Treg-mediated immunosuppression in cancer, highlighting potential paths for therapeutic intervention.

overall_design: Tconv (DAPIneg, i.v.neg, Thy1.2+CD4+Foxp3-GFPneg) and Treg (DAPIneg, i.v.neg, Thy1.2+CD4+Foxp3-GFPpos) cells were single-cell sorted into Buffer TCL (Qiagen) plus 1% β-mercaptoethanol in 96-well plates using a MoFlo Astrios cell sorter. Each plate had 30-100 cell population well and an empty well as controls. Following sorting, plates were spun down for 1” at 2000 RPM and frozen immediately at -80C. Plates were thawed and RNA was purified using 2.2X RNAclean SPRI beads (Beckman Coulter) without final elution (Shalek et al., 2013). SMART-seq2 and Nextera library preparation was performed as previously described (Picelli et al., 2013), with some modifications as described in a previous study (Singer et al., 2017). Plates were pooled into 384 single-cell libraries, and sequenced 50 x 25 paired end reads using a single kit on the NextSeq500 5 instrument.

first: Amy

last: Li

first: David

last: Canner

first: Jason

last: Schenkel

first: Olivia

middle: C

last: Smith

first: Jonathan

last: Kim

first: Michelle

last: Hillman

first: Arjun

last: Bhutkar

first: Michael

middle: S

last: Cuoco

first: C

middle: G

last: Rappazzo

first: Patricia

last: Rogers

first: Celeste

last: Dang

first: Livnat

last: Jerby-Arnon

first: Orit

last: Rozenblatt-Rosen

first: Le

last: Cong

last: Birnbaum

first: Aviv

last: Regev

first: Tyler

last: Jacks


title: Genome-wide CRISPR screen Identifies Host Dependency Factors for Influenza A Virus Infection

status: Public on Nov 30 2019

first: Ang

last: Cui

address: 415 Main St.

institute: Broad Institute

summary: In this study, we performed a genome-wide CRISPR screen in A549 cells to identify host genes that are required forInfluenza A virus (IAV) infection.Amongst the identified genes, WDR7, CCDC115, TMEM199 and CMTR1 conferred strong protection against IAV infection when they were CRISPR-deleted without impacting host cell fitness. To investigate the roles of these genes on the transciptomic states of the cell, we performed bulk RNA-sequencing of WDR7, CCDC115, TMEM199 and CMTR1 polyclonal knockout cells. We found that cells lacking WDR7, CCDC115 or TMEM199 up-regulates expression of lysosomal genes due to increased nuclear translocation of Transcription factor EB (TFEB), which hampers IAV entry. In addition,we report that CRISPR deletion of CMTR1 results in increased expression of Interferon-stimulated genes (ISGs) that also plays a role inblocking IAV infection.

overall_design: WDR7, CCDC115, TMEM199 and CMTR1 polyclonal knockout cells were generated by CRISPR deletion and bulk RNA was sequenced for transcriptomic analysis.

first: Bo

last: Li

first: Nir

last: Hacohen


title: Next Generation Sequencing Facilitates Quantitative Analysis of EG7-CAFs and MSCs Transcriptomes

city: AUGUSTA

first: YAN

last: CUI

address: 1410 Laney Walker Blvd, CN 4120

department: Georgia Cancer Center

institute: Augusta University

summary:
Purpose: The goals of this study are to compare EG7 - CAFs (cancer-associated fibroblasts) and MSCs (mesenchymal stem cells) transcriptome profiling (RNA-seq) generated from Next-generation Sequencing (NGS) to perform optimal high-throughput data analysis
Methods: mRNA profiles of CAFs were purified from EG7 tumor bearing mice and early passage (p6) murine MSCs purchased from Cyagen were cultured in DMEM for no more than 2 passages were generated by deep sequencing, in triplicate, using Illumina HiSeq 3000. The sequence reads that passed quality filters were analyzed at the transcript isoform level TopHat followed by Cufflinks
Results: Comparative high throughput RNA-sequencing (seq) analysis of purified EG7-CAFs and the early passage murine MSCs, which are thought to be precursors for some CAFs and used as the control for normal mesenchymal cells, by using an optimized data analysis workflow, we mapped about 50 million sequence reads per sample to the mouse genome (build mm10) and identified 11,839 transcripts with TopHat workflow. Approximately 1584 of the transcripts showed differential expression between the EG7 - CAFs and MSCs, with a fold change ≥ 2 and p value <0.01
Conclusions: Our results clearly support the notion that CAFs are transcriptionally different from MSCs in various pathways and biological processes associated with immune modulation, inflammation, hypoxia, and activation of oncogenic pathways

overall_design: mRNA profiles of purified EG7-CAFs and the early passage murine MSCs were generated by deep sequencing, in triplicate, using Illumina HiSeq 3000.

first: Miao

last: Yu

first: Gang

last: Guo

first: Yan


title: Plasmodium yoelii Erythrocyte Binding-like Protein Modulates Host Cell Membrane Structure, Immunity, and Disease Severity

status: Public on Nov 29 2019

city: Bethesda

first: Timothy

last: Myers

address: 50 South Drive, Room 5509

department: Research Technologies Branch

institute: National Institute of Allergy and Infectious Diseases

summary: Erythrocyte binding-like (EBL) proteins are known to play an important role in malaria parasite invasion; however, any roles in regulating host immune responses remain unknown. Here we show that an amino acid substitution (C741Y) in the protein trafficking domain of Plasmodium yoelii EBL (PyEBL) changes host immune response and disease severity, but not invasion of red blood cells (RBCs) as suggested previously. The C741Y substitution alters protein processing and trafficking in merozoites and in the cytoplasm of iRBCs, reduces its binding to Band 3, increases phosphatidylserine (PS) surface exposure, and elevates osmotic fragility of iRBCs. The modified iRBC surface triggers PS-CD36 mediated phagocytosis of iRBCs, host type I interferon (IFN-I) signaling, and T cell differentiation, leading to improved host survival. This study reveals a previously unknown role of PyEBL in regulating the innate immune response and host-pathogen interaction, which may be explored for developing disease control strategies.

overall_design: RNA was isolated from red blood cell lysates obtained from mice infected with Plasmodium y. yoelii nigeriensis N67, N67C and C or Y replaced four alleles. The same RNA samples were analyzed for parasite and host contributions. One to three mice per Py strain.

first: Yu-chih

last: Peng

first: Yanwei

last: Qi

first: Cui

last: Zhang

first: Xiangyu

last: Yao

first: Jian

last: Wu

first: Sittiporn

last: Pattaradilokrat

first: Lu

last: Xia

first: Keyla

last: Tumas

first: Xiao

last: He

first: Takahiro

last: Ishizaki

first: Chen-Feng

first: Anthony

last: Holder

first: Carole

last: Long

first: Osamu

last: Kaneko

first: Xin-zhuan

last: Su


title: Hyperactivation of Sympathetic Nerves Drives Melanocyte Stem Cell Depletion

status: Public on Nov 28 2019

city: Hangzhou

first: Bing

state: Zhejiang

address: 600 Dunyu Road, Xihu District

department: School of Life Sciences

institute: Westlake University

summary: Empirical and anecdotal evidence have associated stress with accelerated hair greying (formation of unpigmented hairs), but the scientific evidence linking the two is scant. Here, we report that acute stress leads to hair greying through fast depletion of melanocyte stem cells (MeSCs). Combining adrenalectomy, denervation, chemogenetics, cell ablation, and MeSC-specific adrenergic receptor knockout, we found that stress-induced MeSC loss is independent of immune attack or adrenal stress hormones. Rather, hair greying results from activation of the sympathetic nerves that innervate the MeSC niche. Upon stress, sympathetic nerve activation leads to burst release of the neurotransmitter norepinephrine, which drives quiescent MeSCs into rapid proliferation, followed by differentiation, migration, and permanent depletion from the niche. Transient suppression of MeSC proliferation prevents stress-induced hair greying. Our studies demonstrate that acute stress-induced neuronal activity can drive rapid and permanent loss of somatic stem cells, and illustrate an example in which somatic stem cell maintenance is directly influenced by the overall physiological state of the organism.

overall_design: Cell type: FACS purified melanocyte stem cells (MeSCs) from mouse skin; 2 conditions: stressed vs control; 2 replicate for each condition.

first: Sai

last: Ma

first: Ya-Chieh

last: Hsu


title: Large 1p36 deletions affecting Arid1a locus facilitate Mycn-driven oncogenesis in a new neuroblastoma model

city: Memphis

first: Yong-Dong

last: Wang

state: TN

address: 262 Danny Thomas Pl, MS1135

institute: St. Jude Children's Research Hospital

type: Genome variation profiling by genome tiling array

summary: Loss of heterozygosity (LOH) at 1p36 is a frequent chromosomal event in a broad range of human cancers with increased incidence observed in nervous system malignancies, including neuroblastoma (NBL). MYCN amplification and 1p36 deletions are highly correlated markers of tumor aggressiveness in NBL. While short distal 1p36 losses are associated with single copy MYCN tumors, larger hemizygous 1p deletions correlate with MYCN amplification, suggesting that two tumor suppressor regions exist in 1p36, a distal and proximal region, which have MYCN-independent and dependent roles. To interrogate this, we genome-edited a proximal candidate, Arid1a, a distal candidate, Chd5, and engineered a syntenic 1p36 LOH deletion in primary mouse NCCs, the likely cell of origin for NBL. Chd5 deletions were found to confer most of the pro-tumorigenic functions of 1p36 LOH in cells expressing endogenous Mycn levels. In contrast, reduced tumor latency in Mycn-driven oncogenesis significantly correlated with the loss of Arid1a. Gene expression analysis found that tumors presenting early onset display a Polycomb Repressive Complex 2 (PRC2) signature and an impaired Trp53 signaling pathway signature. Our findings delineate the tumor suppressor functions of 1p36 LOH in NBL and potentially provide critical insights for the constellation of cancers with 1p36 deletion.

overall_design: Profiling the copy number changes in parental cell lines and tumor samples of neuroblastoma model

first: J

last: García-López

first: K

last: Wallace

last: Otero

first: R

last: Olsen

first: Y

first: D

last: Finkelstein

last: Rehg

first: A

last: Davidoff

last: Freeman


title: Gene expression comparison of resting human peripheral-blood NK cells and activated counterparts

city: Montreal

first: Panojot

last: Bifsha

state: QC

address: 3175 Chemin de la Côte Ste-Catherine

department: Pediatrics

country: Canada

institute: CHU Sainte-Justine

summary: Resting NK cells are notoriously difficult to transduce, especially using the VSVG lentivirus pseudotype. When NK cells are expanded and activated using the NKAES system, there is a modest improvement of transduction rates compared to their resting NK counterparts. Still, the transduction efficiency remains poor for robust development of NK cell-based cancer immunotherapy. We envisaged to have a genome-wide expression profile of resting and activated NK (NKAES) cells in order to improve currrent transduction protocols .

overall_design: Blood samples were obtained from 4 healthy adult volunteers. NK-cells that were enriched from PBMC using a CD56-positive selection kit were defined as NK resting. A subset of NK resting cells (from 4 donors) were expanded using the Amplification and Expansion System (NKAES) with irradiated K562mbIL21 feeders. Cells were cultured in RPMI1640 supplemented with 10% FCS and penicillin/streptomycin and 200 UI/mL of human recombinant IL-2.

first: Simon

last: Nicoletti

first: Jean-Christophe

last: Grenier

first: Luis

last: Barreiro

first: Elie

last: Haddad


title: CHUK/IKKalpha loss in lung epithelial cells enhances NSCLC growth associated with HIF up-regulation

status: Public on Nov 26 2019

city: Belmont

first: David

last: Habiel

address: 375 Acorn Park Drive,, Apt. 1416

institute: Cedars-Sinai Medical Center

summary:
During the natural progression of Non-small-cell lung cancer (NSCLC), tumor cells evolve progressively through the accumulation of mutations, some of which involve oncogenic (K-RAS, EGFR) or tumor suppressor (P53) genes.  These mutations alter cell signaling pathways to promote tumor growth and survival in the tumor microenvironment.  Herein we show that CHUK (IKK-alpha) acts as a prominent tumor suppressor in two independent NSCLC models.
Using a novel transgenic mouse strain, where IKK-alpha gene is ablated using tamoxifen in alveolar type II epithelial cells, loss of IKK-alpha increased the number and size of lung tumors in response to the chemical carcinogen urethane. Furthermore, IKK-alpha knock-down in three human NSCLC lines (showing independent K-Ras or p53 mutations and status) promoted their growth as xenografts in immunocompromised mice.  Transcriptomic and functional studies of IKK-alpha knock-down tumors, relative to their wild type counterparts, suggested that the loss of IKK-alpha promoted the activation of HIF-1 alpha and higher tumor cell growth and survival under hypoxic conditions.
Together, these results suggest that IKK-alpha acts as a tumor suppressor by suppressing the activity of HIF-1 alpha and tumor cell growth/survival under hypoxic conditions.

sample_organism:
Homo sapiens
Mus musculus

overall_design:
Tumor RNA samples were sequenced in quadruplicate
RNAseq was performed in Human and mouse WT and CHUK-Knockdown NSCLC tumors or tumor xenografts.  For human tumor xenografts, three independent human Wt or CHUK-knockdown NSCLC lines (H1437, A549 and H1299), which differ either in their p53 or K-Ras functional status were obtained from the Sanger (UK) cell line database, which provides the status of their K-Ras and p53, EGFR, ARF and p16 alleles. These three human NSCLC lines (IKK WT and IKK KD) and H1299 and A549  (p52WT and p52KD) were grown as tumor xenografts by subcutaneous transplantation into either side (Left side for WT and right side for KD cells) of immune compromised 5 week old NSG (NOD-SCID-IL2Rgamma) mice (2 x 10^6 cells per injection in 200 μl of PBS). Mice were sacrificed 3 weeks and part of the dissected tumors was immediately snap frozen in liquid nitrogen prior to RNA extraction with the TRI-Reagent (Merck) protocol, according to the manufacturer's instructions.  For mouse tumors, Mice with IKKa allele containing LoxP recombination sites and a ROSA-fLacz Cre-inducible LacZ reporter gene were crossed with Sftpc-CreERT2 mice (Rock et al., 2011), which harbor a tamoxifen-inducible CreERT2 recombinase under the control of the Sftpc gene promoter that is only active in alveolar type II (AT-II) lung epithelial cells. To induce either IKK or IKK deletion in AT-II lung epithelial cells six week-old male or female mice were injected intraperitoneally (i.p.) for 5 consecutive days with 2mg tamoxifen (Sigma) dissolved in corn oil. To induce NSCLCs, one week after tamoxifen administration mice received weekly i.p. urethane injections (1g/kg) (Sigma) dissolved in PBS for 12 consecutive weeks, and were sacrificed 6 months after the first urethane injection.


title: Splicing and gene expression changes in human mammary epithelial MCF-10A cells expressing SR or SR-like proteins

city: Farmington

first: Marina

last: Yurieva

address: 10 Discovery dr

department: Computational Sciences

institute: Jackson Laboratory

summary: RNA-seq data from human mammary epithelial MCF-10A cells expressing the coding sequence of SRSF2, SRSF4, SRSF6, SRSF9, TRA2B, or a control empty vector plasmid grown for 8 days in 3D culture in matrigel

overall_design: We examined the differential gene expression and splicing changes in MCF-10A cells overexpressing SRSF2, SRSF4, SRSF6, SRSF9 or TRA2B compared to control cells.

first: Olga

last: Anczukow

first: Adrian

last: Krainer


title: Transcriptomic Profiles of Sepsis in the Human Brain

city: Ann Arbor

first: Benjamin

middle: H.

last: Singer

state: Mi

address: 109 Zina Pitcher Place

department: Internal Medicine Division of Pulmonary and Critical Care Medicine

institute: University of Michigan

summary: The purpose of this study is to examine the transcriptomic profiles (RNAseq) of post-mortem brain tissue samples from patients who have died of sepsis compared to non-sepsis controls using two analytic approaches. Tissue samples originated from the Adult Changes in Thought study (ACT) brain bank. In order to determine cause of death, hospital charts for 89 ACT subjects who died while hospitalized were reviewed using a structured instrument for diagnosis of sepsis.  RNA was extracted from 24 post-mortem parietal cortex tissue samples. RNA sequencing was performed on the 24 samples using Illumina's Hi-Seq platform. Raw data was exported, pre-processed, and analyzed by two methods, differential expression and weighted gene co-expression network analysis (WGCNA). 176 genes were differentially expressed with fold change of > 1.5 and adjusted p < 0.5. The top differentially expressed genes were immune-related. WGCNA reveled 6 modules were significantly correlated with sepsis. Significant nodules were enriched in terms associated with innate immunity, cytokines, DAMPs, synaptic function, ion channel function, neuronal growth, and T-cell signalling among others. These data suggest sepsis is associated with specific transcriptional responses in the human brain. These results provide support for previously identified targets as well as provide evidence to suggest investigation into new targets for mechanistic exploration of sepsis-associated brain injury.

overall_design: RNAseq data was generated from post-mortem human brain tissue samples from Sepsis cases and non-sepsis controls.

first: Angela

last: Bustamante

first: Kristopher

last: Opron

first: William

middle: J

last: Ehlenbach

first: Eric

middle: B

last: Larson

first: Paul

last: Crane

first: C

last: Keene

first: Theodore

last: Standiford


title: Siah2 control of T-regulatory cells limits anti-tumor immunity

status: Public on Nov 27 2019

city: New York

first: Igor

last: Dolgalev

state: New York

address: 550 1st Ave

institute: NYU Grossman School of Medicine

summary: Understanding mechanisms underlying anti-tumor immunity is pivotal for improving immune-based cancer therapies. Here we demonstrate that growth of inoculated BRAF-mutant melanoma cells was inhibited, up to complete loss, in Siah2-/- mice.

overall_design: CD45+ cells from YUMMER1.7 tumors grown 11 days in WT or Siah2-/- mice, including one thousand CD45- cells for every 20,000 CD45+ cells.

first: Marzia

last: Scortegagna

first: Ze’ev

last: Ronai


title: JMJD3 and UTX Determine Fidelity and Lineage Specification of Human Neural Progenitor Cells

city: Guangzhou

first: qianyu

last: chen

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

overall_design: Refer to individual Series


title: Transcriptomes of pssu(z)12 mutant from phytophthora sojae P6497 at mycelium stage

status: Public on Nov 25 2019

city: Nanjing

first: Haidong

last: Shu

state: Jiangsu

address: Weigang NO.1

department: Department of Plant Pathology

institute: Nanjing Agricultural University

summary: Purpose and methods:Transcriptome profiling of Phytophthora sojae pssu(z)12 mutant T34 (lost 561bp by CRISPR/Cas9) mycelium (3-days old) were generated to find out the relationship between H3K27me3 and gene expression on Avr1b locus. Wild-type P. sojae P6497 was took as a comparative control. RNA-seq data was mapped using Tophat2, and gene expression data was generated by Cufflinks. Transcriptome profiles were displayed using IGV browser.

overall_design: mRNA profiles of Phytophthora sojae pssu(z)12 mycelium(mu,3-days old)was generated by deep sequencing, in two replicates. Wild-type P.sojae P6497 RNA-seq data was accessed from NCBI (GSE116089).

first: Suomeng

last: Dong

first: Han

last: Chen

first: Liyuan

last: Wang


title: ADAR1 mediated regulation of neural crest-derived melanocytes and Schwann cell development.

city: Paris

first: Pierre

last: de la Grange

address: 29, rue du Faubourg Saint-Jacques

department: Paris Biotech Santé

institute: GenoSplice technology

summary: We studied the physiological requirement of A-to-I RNA editing in neural crest cells. To this end, we generated mouse lines that undergo neural crest cell-specific deletion of Adar1 making use of the HtPAcre reporter. The mutants show global depigmentation and absence of myelin within the peripheral nerves. To assess global changes in gene regulation caused by conditional Adar1 deficiency in Schwann cells, a we performed transcriptomic analysis (RNA-seq) of sciatic nerves from independent samples from three control and three mutant animals at P4. The steady-state level of 3,009 mRNAs differed by at least two-fold in abundance between the two genotypes. Analysis of the 3,009 differentially-expressed mRNAs using the interferome database showed 75% of them to be within or associated with IFN type 1 or 2 signaling pathway. Interestingly, 34% of the RNA transcripts deregulated upon Adar1 invalidation are also deregulated upon nerve injury. Overall, our results suggest that Adar1 safeguards neural crest-derived cells from unwanted MDA5-mediated interferon production and chronic Interferon stimulated genes upregulation, with implications for the human diseases caused by ADAR1 mutations and neural crest development and linked-disorders.

overall_design: we performed transcriptomic analysis (RNA-seq) of sciatic nerves from independent samples from three control and three mutant animals at Post-natal day 4.

first: Nadege

last: Bondurand

first: Nadjet

last: Gacem


title: Splicing and gene expression changes in human MDAM-MB231 breast cancer cells with TRA2B knockdown

first: Marina

last: Yurieva

summary: RNA-seq data from human MDA-MB231 breast cancer cells expressing control or TRA2B-targeting shRNAs grown for 8 days in 3D culture in matrigel

overall_design: We examined the differential gene expression and splicing changes in MDA-MB231 cells expressing a DOX-inducible non targeting (shCTL) or TRA2B-targeting shRNA (TRA2Bsh1 and TRA2B sh2). For each shRNA, we compared  -DOX vs +DOX.

first: Olga

last: Anczukow

first: Adrian

last: Krainer


title: RNA-seq from archival FFPE Breast Cancer samples: Molecular Pathway Fidelity and Novel Discovery

city: Portland

first: Zheng

last: Xia

state: Oregon

address: 3181 SW Sam Jackson Park Rd

department: Department of Biomedical Engineering

institute: Oregon Health & Science University

summary: Archival Formalin-Fixed Paraffin Embedded (FFPE) tissue represents an abundant and more easily transferable source of patient tissue than live specimens. Furthermore cancer FFPE tissue deposited at major cancer research institutes is often paired with survival data increasing the value of biomarker and prediction based research from these samples. However the known fragility of RNA and artifacts introduced through the processes of tissue preservation and storage introduce the possibility of unfaithful RNA expression signatures from these sources. To evaluate this we established an RNA isolation, library prepration and data analysis pipeline at Oregon Health and Science University to evaluate the fidelity of RNA expression profiles from FFPE tissues utilizing gene and pathway expression comparisons between ER positive and ER negative samples  in archival FFPE specimens compared to those obtained from public available data sets of fresh specimens.

overall_design: Two Breast Cancer ER+ FFPE samples  prepared by  two different RNA expression library methodologies (Illumina-Access, and Nugen-Ovation) and sequenced in triplicate.  3 ER positive and 3 ER negative FFPE derived Breast Cancer specimens prepared by Illumina Access RNA Expression library and sequenced in singlet on HiSeq2000, with four samples per lane permitting approximately 70 million reads per sample.

first: Nathan

last: Pennock

first: Wes

last: Horton

first: Sonali

last: Jindal

first: Pepper

last: Schedin


title: Genome-wide maps of chromatin accessibility governing the transition from founder cells to distinct fate-restricted cardiopharyngeal precursors

first: Lionel

last: Christiaen

state: NY

address: 100 Washington Square East 1009 Silver Center

department: Biology

institute: New York University

summary: We used the assay for transposon-accessible chromatin using sequencing (ATAC-seq) on FACS-purified cells to profile chromatin accessibility during early cardiopharyngeal fate choices in Ciona.  We obtained ~500 million unique reads from samples comprising cardiopharyngeal mesoderm and mesenchymal cells, as well as whole Ciona embryos and genomic DNA control. We combined ATAC-seq peaks from all the replicates to generate an atlas of 56,090 unique and non-overlapping accessible regions (accessome) covering 9.25% of the C. robusta genome. We used the accessome to analyze differential accessibility and integrated expression data to compare the chromatin and gene expression dynamics underlying cardiopharyngeal fate specification. In summary, we revealed that most changes in accessibility occur upon induction of multipotent cardiopharyngeal progenitors from the founder cells. Comparing differential expression to differential accessibility shows that genes activated in the multipotent progenitors tend to have regions that specifically open nearby them. We found that the elements accessible specifically in multipotent cardiopharyngeal progenitors were enriched in Fox, GATA and nuclear receptor binding motifs. CRISPR-mediated loss of Foxf function followed by FACS, ATAC-seq and RNA-seq showed that Foxf is required to open ~22% of the cardiopharyngeal-specific elements. Notably, elements associated with de novo expressed genes, which turn on either in heart or pharyngeal muscle progenitors, were also opening in multipotent progenitors, whereas only ~10% of differentially expressed genes had differentially accessible elements. Finally, we propose a general model for chromatin dynamics whereby most lineage-specific elements open in multipotent progenitors, and control both early pan-cardiopharyngeal and late cell-type-specific expression.

overall_design:
Examination of 52 ATAC-seq samples: we collected ~4,000 cells per independent replicate for samples from embryos dissociated at five time points encompassing cardiopharyngeal development: naive Mesp+ mesoderm (aka founder cells) at 6 hours post-fertilization, hpf), anterior tail muscle (aka ATMs), first and second multipotent cardiopharyngeal progenitors (aka trunk ventral cells, TVCs) at 10hpf,  fate-restricted progenitors obtained from larvae dissociated at three time points (15, 18 and 20hpf) encompassing second multipotent cardiopharyngeal progenitors (and aka STVCs), as well as fate-restricted first and second heart precursors (FHPs and SHPs), and pharyngeal muscle precursors (aka atrial siphon muscle cells -ASMs). From the same dissociated embryos embryonic cell populations, we isolated B-line mesenchymal cells at 10, 15, 18, and 20hpf. We also collected samples from whole embryo at 10hpf and genomic DNA as input control. We obtained FACS-purified cells at 10hpf after inducing loss of Foxf function using CRISPR/Cas9-mediated mutagenesis (FoxfCRISPR – 10hpf). We examined 6 RNA-seq samples from FoxfCRISPR and ControlCRISPR at 10hpf.

B7.5 = cardiopharyngeal mesoderm precursors

The "new" ATAC-seq method includes addition of EDTA quenching to block the tagmentation reaction (i.e. 1.1  l of 500mM EDTA (final concentration approx. 50mM, incubating at 50  C for 30mins). No EDTA quenching step was used in the "old" method.

first: Claudia

last: Racioppi

first: Keira

last: Wiechecki


title: Expression data from CD4+IL-4gfp+ Th2 cells isolated from BALB/c IL-4gfp 4get mice infected with the filarial nematode Litomosoides sigmodontis.

city: Edinburgh

first: Alasdair

last: Ivens

address: Kings Buildings

institute: Centre for Immunity, Infection and Evolution

summary:
Litomosoides sigmodontis is parasitic filarial nematode that can infect mice, and is used as a murine model for human filariasis. Type 2 immune responses are protective, but infection is typically chronic due to a combination of immune suppression by the parasite and immune regulation by the host dampening protective Type 2 immunity.
We have shown that the CD4+ Th2 cells develop an intrinsically hyporesponsive or dysfunctional phenotype between day 20 and 60 of infection, denoted by an impaired ability to proliferate and produce Th2 cytokines. The hyporesponsive phenotype is PD-1/PD-L2 dependent, and impairs parasite killing (van der Werf et al. 2013. PloS Path. e1003215). This study aimed to investigate the mRNA expression of the intrinsically-hyporesponsive Th2 cells to determine the mechanisms by which they become dysfunctional, and to test whether Th2 cell intrinsic hyporesponsiveness has similarities with other forms of T cell-intrinsic regulation such as exhaustion, tolerance or anergy.

overall_design: The study included 6 groups with 4 – 5 biological replicates per group. For four of the groups, BALB/c IL-4gfp 4get reporter mice were infected with L. sigmodontis. CD4+IL-4gfp+ Th2 cells were purified from the infection site (pleural cavity, PleC), and CD4+IL-4gfp+CXCR5- Th2 cells purified from the lymph nodes within the thoracic cavity (tLN). Th2 cells were sampled pre-onset of hyporesponsiveness (day 20 post-infection) and post-onset of hyporesponsiveness (day 60 post-infection). To generate a reference Th2 population, CD4+IL-4gfp+CXCR5- were purified from the tLN of mice infected with the parasitic nematode Nippostrongylus brasiliensis for 6 days. N. brasiliensis stimulates a prototypical acute Th2 response, and the tLN drain its lung migration stage. Naïve CD4+IL-4gfp-CXCR5-CD25-CD44-CD62L+CD45RB+ T cells were purified from the spleens of uninfected mice as a control. Due to limiting cell numbers, each biological replicate was created by pooling cells from 20 – 31 mice for day 20 L. sigmodontis infection, 16 – 21 mice for day 60 L. sigmodontis infection, 20 – 23 for N. brasiliensis infection, and 3 naïve mice.

first: Matthew

last: Taylor

first: Johanna

last: Knipper


title: Transcriptome-wide analysis of RNA structure in MCF7 cells with and without inhibition of eIF4A by hippuristanol treatment

city: Glasgow

first: Joseph

middle: Alexander

last: Waldron

address: Switchback road

institute: Beatson Institute for Cancer Research

summary: Translational dysregulation is an emerging hallmark of cancer, and increased activity of the mRNA helicase eIF4A is associated with poor survival in malignancies. This is believed to be due to the unwinding of secondary structures within the 5’UTRs of oncogenic mRNAs, with studies showing that in general eIF4A-dependent mRNAs have longer 5’UTRs with more stable secondary structures, yet our ability to predict eIF4A-dependency from 5’UTR properties alone remains poor. We therefore used Structure-seq 2 to measure transcriptome-wide changes in RNA structure in MCF7 cells, following eIF4A inhibition with hippuristanol. This technique measures the single-strandedness of RNA by specific and rapid methylation of single-stranded adenosines and cytosines with Dimethyl Sulphate (DMS). When paired with polysome profiling data to identify which mRNAs are most translationally repressed, we can identify the structural determinants of eIF4A-dependency. Upon eIF4A inhibition, both 5’UTRs and CDSs become generally more structured, while overall this was not observed for 3’UTRs. This was most pronounced in CDSs, supporting recent findings that the ribosome sculpts RNA structure in this region. 5’UTRs are generally more structured at their 5’ ends and highly translated mRNAs are less structured just upstream of the CDS. Following eIF4A inhibition, the 5’UTR is remodelled. eIF4A-dependent mRNAs have greater localised gains of structure. The degree of these structural changes is strongly correlated with 5’UTR length, explaining why eIF4A-dependent mRNAs have longer 5’UTRs. Crucially, in eIF4A-dependent mRNAs these highly-structured elements are located predominantly at the 3’ end of the 5’UTR, suggesting that increased structure just upstream of the CDS is most inhibitory to translation following eIF4A inhibition and is a key determinant of eIF4A-dependency.

overall_design: MCF7 cells were treated with and without dimethyl sulphate (DMS) following treatment with and without hippuristanol in triplicate. Poly(A) RNA was extracted and underwent Structure-seq 2 library preparation

first: David

last: Tack

first: Laura

middle: E

last: Ritchey

first: Sarah

middle: L

last: Gillen

first: Ania

last: Wilczynska

first: Ernest

last: Turro

first: Philip

last: Bevilacqua

last: Assmann

first: Martin

last: Bushell

first: John

last: Le Quesne


title: Angiogenic Transformation in Human Brain Micro Endothelial Cells: Whole Genome DNA Methylation and Transcriptomic Analysis

status: Public on Nov 23 2019

city: TUCSON

first: Ravi

last: Goyal

state: Arizona

address: 1501 N. Campbell Ave

institute: University of Arizona

summary: We tested the hypothesis that endothelial capillary tube formation in 3D cultures in basement membrane extract (BME) is secondary to the altered DNA promoter methylation and mRNA expression in Human Brain Micro Endothelial Cells (HBMEC). We conducted a whole-genome transcriptomic and methylation microarray and CRISPR/Cas9-mediated gene knockdown to test our hypothesis. The data demonstrated that with angiogenic transformation 1318 and 1490 genes were significantly (p<0.05) upregulated and downregulated, respectively. We compared our gene expression data with the published databases on GEO and found several genes in common. PTGS2, SELE, ID2, HSPA6, DLX2, HEY2, FOSB, SMAD6, SMAD7, and SMAD9 showed a very high level of expression during capillary tube formation. Among downregulated gene were ITGB4, TNNT1, PRSS35, TXNIP, IGFBP5. The most affected canonical pathways were ATM signaling and cell cycle G2/M DNA damage checkpoint regulation. The top upstream regulators of angiogenic transformation were identified to be VEGF, TP53, HGF, ESR1, and CDKN1A. We compared the changes in gene expression with the change in gene methylation and found hypomethylation of the CpG sites was associated with upregulation of 515 genes and hypermethylation was associated with the downregulation of 31 genes. Furthermore, the silencing of FOSB, FZD7, HEY2, HSPA6, NR4A3, SELE, PTGS2, SMAD6, SMAD7, and SMAD9 significantly inhibited angiogenic transformation as well as cell migration of HBMECs. We conclude that the angiogenic transformation is associated with altered DNA methylation and gene expression changes.

overall_design: We conducted the experiments on human primary brain microendothelial cells obtained from the Cell Systems Repository of Applied Cell Biology Research Institute (Kirkland, WA). We performed the experiments cells at passage 8. The microarray was conducted on the biological replicates of 6 each for control group and tube formation group (after 24 hours of plating of HBMEC cells on Basal matrix extract).

first: Dipali


title: miRNAs-seq of ovary at  postnatal 3 day PD3 after DEHP treatment with CD1 mouse

city: Qingdao

first: Xiaofeng

last: Sun

address: 700 changcheng road

institute: Qingdao Agricultural University

type: Non-coding RNA profiling by high throughput sequencing

summary: The the ovaries of new born mouse were collected after birth and cultured in vitro with DEHP at the concentration of 0μM (vehicle control DMSO), 10μM and100μM respectively for 72 hours.Then they were performed miRNAs-seq to analyze  the effects of DEHP on ovarian miRNA profile.

overall_design: Construction of miRNA landscape of ovary after different concentration of DEHP treatment.

first: Wei

last: Shen


title: Analysis of redundancy between RNAi and piRNA pathways in the mouse oocytes

status: Public on Nov 22 2019

city: Prague 4

first: Josef

last: Pasulka

address: Vídeňská 1083

country: Czech Republic

institute: Institute of Molecular Genetics of the ASCR, v. v. i.

summary: Suppression of retrotransposons is an integral part of germline genome defense. The PIWI-associated RNA (piRNA) pathway mediates post-transcriptional and transcriptional silencing of retrotransposons in vertebrates. The loss of the piRNA pathway in mice results in male sterility while females remain fertile. Unlike spermatogenic cells, mouse oocytes utilize also RNA interference (RNAi) pathway, another small RNA pathway capable of retrotransposon suppression. To examine redundancy of piRNA and RNAi pathway in the mouse female germline, we produced an RNAi pathway mutant DicerSOM and crossed it with a catalytically-dead mutant of Mili, an essential piRNA factor. Ovaries and follicular development in mice lacking both pathways appear normal suggesting that RNAi in oocytes does not rescue a piRNA pathway knock-out phenotype. We observed that one of the pathways dominates in suppression of different retrotransposons. Intracisternal A Particle retrotransposon is mainly targeted by the piRNA pathway, MT and RLTR10 retrotransposons were targeted mainly by RNAi. Importantly, both pathways redundantly suppress LINE-1 retrotransposons where the loss of both RNA silencing pathways yielded ~6-fold upregulation of negligible levels of LINE-1 transcripts. This implies that another transcriptional silencing mechanism must contribute to LINE-1 repression and could explain why both, RNAi and piRNA pathways, are non-essential defense pathway in the mouse female germline.

overall_design: We report transcriptome profiles of partial and complete knockout of Dicer1 specific isoform in oocytes.

first: Eliska

last: Svobodova

first: Radek

last: Malik

first: Filip

last: Horvat

first: Irena

last: Jenickova

first: Zoe

last: Matosevic

first: Petr

last: Svoboda


title: Mesostigma viride genome and transcriptome provide insights into the origin and evolution of Streptophyta

status: Public on Nov 21 2019

city: Beijing

last: Gu

address: Zhongguancun south street

institute: Chinese Academy of Agricultrual Sciences

type: Methylation profiling by high throughput sequencing


title: Clonal kinetics and single cell transcriptional profiling of adoptively transferred CD19-specific CD8+ CAR-T cells in adults with B cell malignancies

city: Cape Town

first: Valentin

last: Voillet

state: Western Cape

address: First Floor Wembley Square, 3 McKenzie St, Gardens

country: South Africa

institute: cape town hvtn immunology laboratory

summary: Lymphodepletion chemotherapy followed by infusion of T cells modified to express a CD19-targeting chimeric antigen receptor (CAR) has produced remarkable anti-tumor responses in patients with B cell malignancies.  However, little is known about the clonal composition and transcriptional heterogeneity of CAR-T cells in the infusion products (IP) and clonal kinetics after adoptive transfer.  We performed single-cell RNA sequencing (scRNA-seq) on CD8+ CAR-T cells isolated from the IP and the blood of patients treated on a phase 1 clinical trial (NCT01865617) with lymphodepletion chemotherapy and a defined formulation of CD4+ and CD8+ CD19-specific CAR-T cells.  Infused CD8+ CAR-T cells displayed transcriptional heterogeneity which declined after adoptive transfer, coincident with early expression of genes associated with activation.  We identified four transcriptionally distinct CAR-T cell subsets in the IP and found that these subsets differed in their contributions to the CAR-T cell population detected in blood after infusion.  Better understanding of the kinetics of clonal expansion of CAR-T cells after adoptive transfer may provide insight into strategies to improve CAR-T cell immunotherapy.

overall_design: We performed scRNA-seq on CD8+ CAR-T cells isolated from the IP and blood at the early, late, and very late time points from four patients who received CD19-specific CAR-T cells for refractory B cell malignancies.  Single CD8+ CAR-T cells were sorted as CD8+/CD4-/EGFRt+ events and scRNA-seq was performed using the Chromium Single Cell 5’ library and V(D)J enrichment kit (10x Genomics).  Libraries were sequenced on HiSeq2500.

first: Alyssa

last: Sheih


title: Mesostigma viride genome and transcriptome provide insights into the origin and evolution of Streptophyta [Bisulfite-seq]

summary: The Streptophyta include unicellular and multicellular charophyte green algae and land plants. Colonization of the terrestrial habitat by land plants was a major evolutionary event that has transformed our planet. So far lack of genome information on unicellular charophyte algae hinders our understanding of the origin and the evolution from unicellular to multicellular life in Streptophyta. This work reports the high-quality reference genome and transcriptome of Mesostigma viride, a single-celled charophyte alga with a position at the base of Streptophyta. There are abundant segmental duplications and transposable elements in M. viride, which contribute to a relatively large genome with high gene content compared to other algae and early diverging land plants. This work identifies the origin of genetic tools that multicellular Streptophyta have inherited and key genetic innovations required for evolution of land plants from unicellular aquatic ancestors. The findings shed light on the age-old questions of the evolution of multicellularity and the origin of land plants.

overall_design: Bisulfite-seq with two replicates.

first: Zhe

last: Liang

first: Yuke

last: Geng

first: Hao

last: Yu


title: Mediator complex component MED13 regulates zygotic genome activation and is required for postimplantation development

city: Research Triangle Park

first: Carmen

last: Williams

state: NC

address: 111 T W ALEXANDER DR

institute: NIH/NIEHS

summary: Understanding the factors that regulate the transition from oocyte to embryo is critical for determining how cells are reprogrammed to become totipotent or pluripotent. Although factors that can reprogram somatic cells into induced pluripotent stem cells have been discovered, we have limited information regarding how this process occurs physiologically in vivo. Here we identified a specific mediator complex subunit, the kinase domain subunit MED13, as being recruited for translation during oocyte maturation and transcribed very early from the zygotic genome. Both knockdown and conditional knockout genetic approaches demonstrate that MED13 is absolutely essential for zygotic genome activation in the mouse in part through regulation of the embryo-specific chromatin remodeling complex, esBAF. This role is mediated by interactions with E2F family transcription factors. In addition to MED13, its paralog, MED13L, is also required for successful preimplantation embryo development. Although MED13L can partially compensate for loss of MED13 function, post-implantation embryo development is not rescued by MED13L because the pluripotency factor NANOG is not expressed at normal levels. Our data demonstrate for the first time an essential role for MED13 in supporting chromatin reprogramming and directed transcription of essential genes during zygotic genome activation

overall_design: Examination of gene expression differences in CF-1 mouse 2-cell stage embryos. Early 1-cell stage embryos were injected with either a scrambled control morpholino or MED13-targeting morpholino, and then allowed to develop to the late 2-cell stage. Four biological replicates were collected for each group.

first: Yi-Liang

last: Miao

first: Weichun

last: Huang

first: Elizabeth

last: Padilla-Banks

first: Andrés

last: Gambini

first: Yingpei

last: Zhang

first: Leping

last: Li

first: Miranda

last: Bernhardt

first: Wendy

middle: N

last: Jefferson


title: H3K27me3 in Phytophthora sojae P6497 and its pssu(z)12 mutant at mycelium stage

first: Haidong

last: Shu

summary: We report the H3K27me3 profile on Avr1b locus in two Phytophthora sojae strains P6497 and pssu(z)12 mutant. Nuclei of Phytophthora sojae P6497 and pssu(z)12 mutant T34 (lost 561bp by CRISPR/Cas9) mycelium (3-days old) was extracted and digested to 200-400bp using micrococcal nuclease (MNase: NEB M0247S). The antibody Millipore 07-449 was used to immunoprecipitation. We find significant accumulation of H3K27me3 at Avr1b locus in Avr1b silencing strain P6497 and clear H3K27me3 depletion at Avr1b locus in Avr1b unsilenced strain pssu(z)12 mutant T34.

overall_design: Examination of H3K27me3 on Avr1b locus in two phytophthora sojae strains

first: Han

last: Chen

first: Liyuan

last: Wang

first: Suomeng

last: Dong


title: STING-dependent paracriny shapes apoptotic priming of breast tumors in response to anti-mitotic treatment

city: Saint Herblain

first: Wilfried

last: Gouraud

address: bd Jacques Monod

department: Integrated Center of Oncology René Gauducheau

institute: ICO (Integrated Center of Oncology Nantes)

summary:
A fascinating but uncharacterized action of antimitotic chemotherapy is to collectively prime cancer cells to apoptotic mitochondrial outer membrane permeabilization (MOMP), while impacting only on cycling cell subsets.
Here, we show that a proapoptotic secretory phenotype is induced by activation of cGAS/STING in cancer cells that are hit by antimitotic treatment, accumulate micronuclei and maintain mitochondrial integrity despite intrinsic apoptotic pressure.
Organotypic cultures of primary human breast tumors and patient-derived xenografts sensitive to paclitaxel exhibit gene expression signatures typical of type I IFN and TNFalpha exposure.
These cytokines induced by cGAS/STING activation trigger NOXA expression in neighboring cells and render them acutely sensitive to BCL-xL inhibition.
cGAS/STING-dependent apoptotic effects are required for paclitaxel response in vivo, and they are amplified by sequential, but not synchronous, administration of BH3 mimetics.
Thus anti-mitotic agents propagate apoptotic priming across heterogeneously sensitive cancer cells through cytosolic DNA sensing pathway-dependent extracellular signals, exploitable by delayed MOMP targeting.

overall_design:
Patient-provided breast cancer cells treated by the antimitotic Paclitaxel produce inflammatory cytokines induced by the cGAS/STING pathway, that render neighboring cells sensitive to BCL-xL inhibition.

Protocol: THEREX: 2012-A00682-41

first: Steven

last: Lohard

first: Nathalie

last: Bourgeois

first: Laurent

last: Maillet

first: Fabien

last: Gautier

first: Aurélie

last: Fétiveau

first: Hamza

last: Lasla

first: Frédérique

last: Nguyen

first: Céline

last: Vuillier

first: Alison

last: Dumont

first: Agnès

last: Moreau-Aubry

first: Morgane

last: Frapin

last: David

first: Delphine

last: Loussouarn

first: Olivier

last: Kerdraon

first: Mario

last: Campone

first: Pascal

last: Jézéquel

first: Philippe

middle: P

last: Juin

first: Sophie

last: Barillé-Nion


title: Transcriptome wide analysis of translation efficiency in MCF7 cells using polysome profiling with and without eIF4A inhibition by hippuristanol treatment

first: Joseph

last: Waldron

summary: To identify mRNAs that are most translationally repressed following eIF4A inhibition and those that are relatively insensitive, polysome profiling was carried with and without eIF4A inhibition by hippuristanol treatment. Polysomal, sub-polysomal and total RNA was sequenced and we used a Bayesian model to identify mRNAs that with greatest confidence had shifted from the polysomal into the sub-polysomal fractions, from the sub-polysomal into the polysomal fractionsand those mRNAs that did not change in their polysomal to sub-polysomal ratio, following hipp treatment, which were termed eIF4A-dependent, eIF4A-antidependent and eIF4A-independent mRNAs respectively

overall_design: Polysomal, sub-polysomal and total RNA samples were sequenced from both control and hippuristanol treated cells in triplicate

middle: A

first: David

last: Tack

first: Laura

last: Ritchey

first: Sarah

last: Gillen

first: Ania

last: Wilczynska

first: Ernest

last: Turro

first: Philip

last: Bevilacqua

middle: M

last: Assmann

first: Martin

last: Bushell

first: John

last: Le Quesne


title: Mesostigma viride genome and transcriptome provide insights into the origin and evolution of Streptophyta [lncRNA]

first: Xiaofeng

last: Gu

overall_design: LncRNA sequencing with three replicates.

first: Zhe

last: Liang

first: Yuke

last: Geng

first: Hao

last: Yu


title: The G protein-coupled bile acid receptor TGR5 (Gpbar1) modulates endothelin-1 signalling in liver

status: Public on Nov 19 2019

city: Düsseldorf

first: Verena

last: Keitel

address: Moorenstr. 5

department: Department of Gastroenterology, Hepatology and Infectious Diseases

institute: University Hospital, Medical Faculty of Heinrich Heine University Düsseldorf

summary: TGR5 (Gpbar1) is a G protein-coupled receptor responsive to bile acids (BAs), which is expressed in different non-parenchymal cells of the liver, including biliary epithelial cells, liver-resident macrophages, sinusoidal endothelial cells (LSECs) and activated hepatic stellate cells (HSCs). Mice with targeted deletion of TGR5 are more susceptible towards cholestatic liver injury induced by cholic acid-feeding and bile duct ligation, resulting in a reduced proliferative response and increased liver injury. Conjugated lithocholic acid (LCA) represents the most potent TGR5 BA ligand and LCA-feeding has been used as a model to rapidly induce severe cholestatic liver injury in mice. Thus, TGR5 knockout (KO) mice and wildtype littermates were fed a diet supplemented with 1%LCA for 84 hours. Liver injury and gene expression changes induced by the LCA-diet revealed an enrichment of pathways associated with inflammation, proliferation and matrix remodelling. Knockout of TGR5 in mice caused upregulation of endothelin-1 (ET-1) expression in the livers. Analysis of TGR5-dependent ET-1 signalling in isolated LSECs and HSCs demonstrated that TGR5 activation reduces ET-1 expression and secretion from LSECs and triggers internalization of the ET-1 receptor in HSCs dampening ET-1 responsiveness. Thus, we identified two independent mechanisms by which TGR5 inhibits ET-1 signalling and modulates portal pressure.

overall_design: Ten male TGR5 wildtype mice were fed a diet containing 1% (w/w) lithocholic acid (LCA, six mice) or standard chow diet (four mice) ad libitum.

first: Caroline

last: Klindt

first: Maria

last: Reich

first: Birte

last: Hellwig

first: Jan

last: Stindt

first: Jörg

last: Rahnenführer

last: Hengstler

first: Karl

last: Köhrer

first: Kristina

last: Schoonjans

first: Dieter

last: Häussinger


title: Mesostigma viride genome and transcriptome provide insights into the origin and evolution of Streptophyta [microRNA]

overall_design: Small RNA sequencing with three replicates.


title: Mesostigma viride genome and transcriptome provide insights into the origin and evolution of Streptophyta [RNA-seq]

overall_design: RNA-seq in different temperature, light and pH conditions with two replicates.


title: The MicroRNAs target mitochondrial dynamics and metabolism to regulate mitochondrial-mediated antiviral innate immunity

city: Fukuoka

first: Kai

last: Yasukawa

state: Fukuoka

address: 744 Motooka, Nishi-ku

department: Department of Systems Life Sciences

country: Japan

institute: Kyusyu University

summary: MicroRNAs (miRNAs) are short non-coding RNAs act as a suppressor of multiple genes and it is known that miRNAs link to numerous biological functions. Mitochondria act as a platform for innate immune response against RNA viruses via a mitochondrial outer membrane protein, MAVS. Here we report miR-302b and miR-372 involve in mitochondrial-mediated antiviral response. These miRNAs were upregulated by viral infection and influenced not only the production of type I interferon and inflammatory cytokines, but also mitochondrial function. Furthermore, we revealed that these phenotypes were induced by changing several mitochondrial genes expression. miR-302b induced mitochondrial fragmentation by changing gene expression related to mitochondrial dynamics to regulate Drp1 activity. Indeed, downregulation of SLC25A12 by miR-302b suppressed antiviral responses via inhibiting the MAVS activation directly. We first propose that miR-302b and miR-372 act as negative regulator of antiviral response by affecting mitochondrial function to inhibit excessive innate immune response.

overall_design: Change of gene expression by miR-302b in HEK293 cells was measured. The gene expression in the cells transfected with miR-302b mimic was compared with the cells transfected with negative control miRNA mimic.

first: Takumi

last: Koshiba


title: Pathogenic pathways in early onset autosomal recessive Parkinson’s disease discovered using isogenic human dopaminergic neurons

city: Cambridge

first: Lee

last: Rubin

address: 7 Divinity Ave

department: Stem Cell and Regenerative Biology

institute: Harvard University

summary: Purpose of NGS transcriptomics: We generated RNA-sequencing transcriptomics datasets from isogenic Parkinson’s disease (PD) HUES1 cell lines (harboring loss of function mutations in either PARKIN (PRKN), DJ-1-/- (PARK7), or ATP13A2-/- (PARK9)) to identify common and distinct dysregulated genes and networks in our three isogenic PD lines in an unbiased way, with the goal of grouping similar and distinct forms of PD and identify common or divergent dysregulation.

overall_design: Transcriptional profiles of 12 samples (3 replicates of 4 isogenic cell lines) were generated after sorting hPSC derived dopaminergic neurons using Tyrosine Hydroxylase knock-in fluroescence reporter

first: Tim

last: Ahfeldt


title: RNA-seq profiling of BAG neurons in C. elegans npr-1 and npr-1;arcp-1 animals

first: Isabel

last: Beets

address: Francis Crick Avenue

department: Cell Biology

institute: MRC-LMB

summary: The extent to which behavior is shaped by experience varies between individuals. Genetic differences contribute to this variation but the neural mechanisms are not understood. In this study, we identify a natural genetic variant of the dendritic scaffolding protein ARCP-1 that modifies individual behavioral flexibility in C. elegans. Disrupting ARCP-1 alters how previous oxygen experience modulates escape behavior in response to carbon dioxide. ARCP-1 normally suppresses CO2 aversion by inhibiting signaling from the BAG CO2-sensing neurons. To further investigate how disrupting ARCP-1 alters BAG function, we specifically labeled these neurons with GFP, used FACS to isolate the fluorescent cells from acutely dissociated arcp-1 mutant and control animals, and profiled their gene expression using RNA-Seq. We find transcriptional changes for a broad range of gene classes and link altered neuropeptide expression to increased CO2 aversion in the arcp-1 mutant.

overall_design: Examination of gene expression in C. elegans BAG neurons for two different genotypes

first: Mario

last: de Bono

first: Gurpreet

last: Ghattaoraya


title: RNAseq transcriptome profiling of melanoma cell lines

status: Public on Nov 20 2019

city: Camperdown

first: Stuart

last: Gallagher

state: NSW

address: Building 93, RPA Hospital

department: Melanoma Oncology

institute: Centenary Institute

summary: The transcriptome of 55 cutaneous melanoma cell lines was assessed using RNAseq.

overall_design: RNAseq was performed on each cell line, using untreated cells and a single replicate

first: Nick

last: Hayward

first: Jessamy

last: Tiffen

first: Ken

last: Dutton-Regester