title: Reproducibility of molecular phenotypes after long-term differentiation to Human iPSC-Derived Neurons: a multi-site omics study [bulk]

status: Public on Aug 20 2018

city: Cardiff

first: Viola

last: Volpato

address: Maindy Rd

department: UK DRI

institute: Cardiff University

summary: Reproducibility in molecular and cellular studies is fundamental to scientific discovery. To establish the reproducibility of a well-defined long term neuronal differentiation protocol, we repeated the cellular and molecular comparison of the same two iPSC lines across five distinct laboratories. Despite uncovering acceptable variability within individual laboratories, we detect poor cross-site reproducibility of the differential gene expression signature between these two lines. Factor analysis identifies the laboratory as the largest source of variation along with several variation-inflating confounds such as passaging effects and progenitor storage. Single cell transcriptomics shows substantial cellular heterogeneity underlying inter-laboratory variability and being responsible for biases in differential gene expression inference. Factor analysis-based normalization of the combined dataset can remove the nuisance technical effects, enabling the execution of robust hypothesis generating studies. Our study shows that multi-center collaborations can expose systematic biases and identify critical factors to be standardized when publishing novel protocols, contributing to increased cross-site reproducibility.

overall_design: RNAseq profiles of 57 bulk Human iPSC-Derived Neurons differentiated across five laboratories were generated in triplicates at two different time points and sequenced on 1 lane of HiSeq4000 at 75bp paired end. RNAseq profiles of .... single cells extracted from 2 of the 5 laboratories at the later time point were isolated by FACS onto 96-well plates and sequenced on 1 lane of HiSeq4000 at 75bp paired end.

last: Smith

first: Cynthia

last: Sandor

first: Janina

last: Ried

first: Anna

last: Baud

first: Adam

last: Handel

first: Sarah

last: Newey

first: Frank

last: Wessely

first: Moustafa

last: Attar

first: Emma

last: Whiteley

first: Satyan

last: Chintawar

first: An

last: Verheyen

first: Thomas

last: Barta

first: Majlinda

last: Lako

first: Lyle

last: Armstrong

first: Caroline

last: Muschet

last: Artati

first: Carlo

last: Cusulin

first: Klaus

last: Christensen

first: Christopher

last: Patsch

first: Eshita

last: Sharma

first: Jerome

last: Nicod

first: Philip

last: Brownjohn

first: Victoria

last: Stubbs

first: Wendy

last: Heywood

first: Paul

last: Gissen

first: Roberta

last: De Filippis

first: Katharina

last: Janssen

first: Peter

last: Reinhardt

first: Jerzy

last: Adamski

first: Ines

last: Royaux

first: Pieter

last: Peeters

first: Georg

middle: C

last: Terstappen

first: Martin

last: Graf

first: Frederick

last: Livesey

first: Colin

last: Akerman

first: Kevin

last: Mills

first: Rory

last: Bowden

first: George

last: Nicholson

first: Caleb

last: Webber

first: M

middle: Z

last: Cader

first: Viktor

last: Lakics


title: Functional interplay between c-Myc and Max in B lymphocyte differentiation

city: MADRID

first: Alfonsina

last: Trento

state: Madrid

address: Darwin 3

department: DIO

country: Spain

institute: Centro Nacional de Microbiologia - CSIC

type: Expression profiling by high throughput sequencing

summary: The Myc proteins (N-, L- and c-Myc) are transcription factors involved in many biological functions such as regulation of cell proliferation, differentiation, metabolism and apoptosis.  A large number of human cancers show enhanced expression of myc family proto-oncogenes as one of their hallmarks.  These proteins contain a basic region/helix-loop-helix/leucine zipper (bHLHZip) domain that mediates DNA binding and heterodimerization with its partner Max (Myc/Max heterodimer).  Among Myc proteins, c-Myc is the most widely expressed and relevant in primary B lymphocytes.  Some reports have implied that c-Myc can perform some functions without Max in different cell contexts.  However, the functional interplay in vivo between c-Myc and Max during B lymphocyte differentiation is not well-known.  Here we show that c-Myc requires Max.  However, key biological processes such as cell differentiation and DNA replication can initially progress without c-Myc/Max heterodimer in primary B lymphocytes.  We found that B lymphocytes lacking Myc, Max or both showed upregulation of signalling pathways associated with the B cell receptor.  Our data suggest that c-Myc/Max heterodimers are not essential for the initiation of certain biological processes in B lymphocytes.  Rather, c-Myc/Max are necessary for fine-tuning the initial response in these cells after activation.

overall_design: B cell mRNA profiles of 8-week old control (HET) Myc deficient (MycKO), Max deficient (MaxKO) and double deficient (DKO) mice were generated by deep sequencing, in duplicate, using a HiSeq2500 (Illumina.

first: Mercedes

last: Pérez-Olivares

first: Sara

last: Rodriguez-Acebes

first: Daniel

last: González-Acosta

first: David

last: Fernández-Antorán

last: Román-García

first: Dolores

last: Martinez

first: Tania

last: López-Briones

first: Carlos

last: Torroja

first: Yolanda

middle: R

last: Carrasco

first: Juan

last: Méndez

first: Ignacio

last: Moreno de Alborán


title: Cobomarsen, an Oligonucleotide Inhibitor of miR-155, Coordinately Regulates Multiple Survival Pathways to Reduce Cellular Proliferation and Survival in Cutaneous T-Cell Lymphoma

city: Boulder

first: Aimee

last: Jackson

state: CO

address: 6200 Lookout Rd.

country: USA

institute: miRagen Therapeutics

summary:
miR-155 is a microRNA associated with poor prognosis in lymphoma and leukemia and has been implicated in the progression of Mycosis Fungoides (MF), the most common form of cutaneous T-cell lymphoma (CTCL). In this study, we developed and tested Cobomarsen (MRG-106), a locked nucleic acid-modified oligonucleotide inhibitor of miR-155. In MF cell lines in vitro, inhibition of miR-155 with Cobomarsen de-repressed direct miR-155 targets, decreased expression of multiple gene pathways associated with cell survival, reduced survival signaling, decreased cell proliferation, and activated apoptosis.
We identified a set of genes that are significantly regulated by Cobomarsen, which includes direct and downstream targets of miR-155.

overall_design: CTCL cell lines were cultured with Cobomarsen (MRG-106) for 4 days (to identify direct targets) or 8 days (to identify downstream targets).

first: Anita

middle: G

last: Seto

middle: L

first: Joshua

middle: M

last: Lynch


title: Efficient C-to-T base editing in methylated region with human APOBEC3A

city: Shanghai

first: Li

last: Yang

address: 131 Dong-An Road

department: Institutes of Biological Sciences

country: China

institute: Fudan University

summary: Base editors (BEs) shed new light on correcting disease-related T-to-C mutations. However, current rat APOBEC1-based BEs are less efficient in editing cytosines in highly-methylated regions or in GpC context. By screening a variety of APOBEC/AID deaminases, we showed that human APOBEC3A-conjugated BE and its engineered forms can mediate efficient C-to-T base editing in all examined contexts, including regions with high-methylation levels and GpC dinucleotides, which extends base editing scope.

overall_design: Examination of indels and base substitutions induced by hA3A-BE3

first: Jia

last: Chen

first: Xingxu

last: Huang

first: Bei

first: Ying

last: Wang

first: Xiao


title: RNA-seq analysis of gene expression patterns of embryos in the post-implantation stages E5.25, E5.5 and E5.75

city: Cambridge

first: Christos

last: Kyprianou

address: Downing Street

department: Physiology Development and Neuroscience

country: United Kingdom

institute: University of Cambridge

summary: We analyzed the expression pattern of mouse embryos along the proximal-distal axis including both epiblast and extraembryonic ectoderm. The stages we investigated are E5.25, E5.5 and E5.75 to identify genes involved in formation of the pro-amniotic cavity.

overall_design: Examination of gene expression patterns in embryos in the post-implantation stages E5.25, E5.5 and E5.75 along the proximal-distal axis.

first: Ran

first: Neophytos

last: Christodoulou

first: Magdalena

last: Zernicka-Goetz


title: Next Generation Sequencing Facilitates Quantitative Analysis of Transcriptomes of  Colon Epithelial Cell from Wild Type and Pkm2-/- mice

city: Nanjing

first: HONGWEI

last: LIANG

state: Jiangsu Province

address: Qixia District, Xianlin Road No.163

institute: Nanjing University

summary:
Purpose: PKM2-mediated metabolic switch to aerobic glycolysis plays a critical role in promoting cell survival and proliferation. However, little is known about the function of intestinal epithelial PKM2 in intestinal homeostasis. Here we investigated whether and how intestinal epithelial PKM2 modulates the morphology and function of the adult intestine in experimental murine colitis.

Methods: Colonoscopic biopsies from the Crohn’s disease (CD) and ulcerative colitis (UC) patients (10 each) were analyzed for PKM2 expression. We also generated intestinal epithelial-specific Pkm2 knockout mice and employed them to examine PKM2 function. Mouse intestinal inflammation was induced with dextran sulfate sodium (DSS) in drinking water. Disease phenotypes were investigated by mouse survival, body weight, colon length and analysis of immune cell infiltration in colon, intestinal epithelial cell gene profiling and signal pathway.

Results: Intestinal epithelial PKM2 level in UC and CD patients was significantly decreased compared to that from non-inflamed intestinal epithelium. Similar reduction of intestinal epithelial PKM2 was observed in mice with experimental colitis. Supporting the notion that PKM2 serves as a safeguard against colitis, intestinal epithelial-specific Pkm2-deficient (Pkm2-/-) mice displayed a severer intestinal inflammation, companying with shortened colon, disruption of epithelial tight junction, higher permeability, elevation of inflammatory cytokines and immune cell infiltration, compared with wild-type mice. Gene profiling and western blot analysis indicated that cell survival signaling, particularly the Akt/β-catenin pathways, were downregulated in Pkm2-/- mice. Functional assay using mouse primary colonic epithelial cells confirmed that Pkm2 reduction decreased proliferation and migration of epithelial cells, while enhanced expression of Pkm2 was associated with increased transcriptional activity of β-catenin. Moreover, increasing mouse intestinal epithelial Pkm2 expression via delivery of Pkm2-expressing plasmid attenuated DSS-induced experimental colitis.

Conclusions: Intestinal epithelial PKM2, through activating Wnt/β-catenin signaling, induces a strong proliferative and migratory response that increases cell survival and wound healing under colitic condition.

overall_design: mRNA profiles of colon epithelial cell from Wild Type and Pkm2-/- mice treated with  2.5% DSS for 3 days were generated by Illumina Hiseq X Ten platform

first: Hongwei

last: Liang

first: Ke

last: Zen


title: Reproducibility of molecular phenotypes after long-term differentiation to Human iPSC-Derived Neurons: a multi-site omics study [single-cell]

first: Viola

last: Volpato

overall_design: RNAseq profiles of 1440 single cells were extracted from 2 of the 5 laboratories (from bulk RNAseq experiment) at the later time point. Single cells were isolated by FACS onto 96-well plates and sequenced on 1 lane of HiSeq4000 at 75bp paired end.

first: James

last: Smith

first: Cynthia

last: Sandor

first: Janina

middle: S

last: Ried

first: Anna

last: Baud

first: Adam

last: Handel

first: Sarah

middle: E

last: Newey

first: Frank

last: Wessely

first: Moustafa

last: Attar

first: Emma

last: Whiteley

first: Satyan

last: Chintawar

first: An

last: Verheyen

first: Thomas

last: Barta

first: Majlinda

last: Lako

first: Lyle

last: Armstrong

first: Caroline

last: Muschet

last: Artati

first: Carlo

last: Cusulin

first: Klaus

last: Christensen

first: Christopher

last: Patsch

first: Eshita

last: Sharma

first: Jerome

last: Nicod

first: Philip

last: Brownjohn

first: Victoria

last: Stubbs

first: Wendy

last: Heywood

first: Paul

last: Gissen

first: Roberta

last: De Filippis

first: Katharina

last: Janssen

first: Peter

last: Reinhardt

first: Jerzy

last: Adamski

first: Ines

last: Royaux

first: Pieter

middle: J

last: Peeters

first: Georg

last: Terstappen

first: Martin

last: Graf

first: Frederick

last: Livesey

first: Colin

last: Akerman

first: Kevin

last: Mills

first: Rory

last: Bowden

first: George

last: Nicholson

first: Caleb

last: Webber

first: M

last: Cader

first: Viktor

last: Lakics


title: Comparison of transcriptional changes after CD28/CD3z and 4-1BB/CD3z chimeric antigen receptor ligation

status: Public on Aug 19 2018

city: Seattle

first: Alexander

middle: Isaac

last: Salter

state: WA

address: 1100 Fairview Avenue North, D3-100

department: Clinical Research Division

institute: Fred Hutchinson Cancer Research Center

summary: The adoptive transfer of chimeric antigen receptor- (CAR) modified T cells is revolutionizing the treatment of B cell malignancies and has the potential to be applied to other diseases. CARs redirect T cell specificity by linking an antigen recognition domain to T cell signaling modules comprised of CD3z to provide signal 1, and CD28 or 4-1BB to provide costimulation. CD28/CD3z and 4-1BB/CD3z CARs confer differences in effector function and cell fate that affect clinical efficacy and toxicity. These differences may result from activation of divergent transcriptional programs. To gain this insight, we analyzed changes in gene expression in stimulated and resting CD28/CD3z or 4-1BB/CD3z CAR T cells. CD28/CD3z CAR stimulation initiated more marked early transcriptional changes with greater fold increases in the expression of effector molecules including GZMB, IFNG, IL2, TNF, and IL6. Direct comparison of CD28/CD3z and 4-1BB/CD3z samples stimulated for 6 hours identified 1,673 differentially expressed genes. Of these, the memory T cell-associated genes KLF2, IL7R, and FAM65B were expressed at lower levels in CD28/CD3z CAR T cells. KLF2 and IL7R are FOXO transcription factor family targets and we found that FOXO4 expression was similarly reduced in CD28/CD3z CAR T cells. CD28/CD3z CAR stimulation induces an effector T cell-like transcriptional profile that may underlie the decreased persistence and increased risks of toxicities observed with CD28/CD3z CAR T cells in early clinical trials.

overall_design: Purified CD28/CD3z and 4-1BB/CD3z CAR T cells were prepared from healthy donors and stimulated by incubation with anti-CAR beads, or left unstimulated by incubation with control beads. Total RNA was harvested 6 or 24 hours after treatment. Three biological replicates for each treatment condition were prepared, yielding 24 total samples for analysis. A42 and A44 denote 4-1BB/CD3z CARs, A43 and A45 denote CD28/CD3z CARs.

first: Stanley

last: Riddell

middle: I

first: Valentin

last: Voillet


title: LXR-dependent gene expression in mouse bone marrow derived macrophages

status: Public on Aug 17 2018

city: CHARLOTTESVILLE

first: Ira

middle: Glenn

last: Schulman

state: Virginia

address: 1340 Jefferson Park Ave, Jordan Hall, room 5218

department: Pharmacology

institute: University of Virginia

summary: Bone marrow derived macrophages were isolated from C57BL/6J (LXR positive) and LXRalpha +LXRbeta double knockout mice (DKO) that had been treated with vehicle, Kado2-lipid A (KLA, and inflammatory stimulus), acetylated LDL (AcLDL, a mimic of pro-atherogenic lipoprotein particles) and GW3965 (GW, a synthetic LXR agonist) for 17 hours. Following treatment RNA was isolated and RNA-Seq was performed.

overall_design: For each genotype (LXR positive and DKO) 3 mice/treatment were used for a total of 24 samples (6 mice x 4 treatment groups). Single-end 50 basepair sequencing was carried out at HudsonAlpha using a HiSeq 2500.

first: Sophie

last: Liebergall


title: Expression data for WDFY4 het vs KO cDC1 from tumor bearing mice

city: St. Louis

first: Kenneth

last: Murphy

address: 660 S. Euclid

department: Pathology and Immunology

institute: Wustl/HHMI

summary:
WDFY4 is a gene essential for in vivo cross-presentation. The goal of this study was to find if there were transcriptional differences between WT and WDFY4 KO cDC1 under either steady state conditions
or under conditions of activation in order to give us clues as to the mechanism of action.

overall_design: WDFY4 het and KO mice were injected with regressor tumors and cDC1 from either the draining or non-draining skin-draining lymph nodes were harvested after 6 days of tumor growth

first: D

last: Theisen

first: K


title: A distinct isoform of ZNF207 controls self-renewal and pluripotency of human embryonic stem cells

city: Bozeman

first: Fang

last: Fang

state: Montana

address: CBB Room 103

department: Cell Biology

institute: Montana State University

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: A distinct isoform of ZNF207 controls self-renewal and pluripotency of human embryonic stem cells [ChIP-Seq]

summary: Self-renewal and pluripotency in human embryonic stem cells (hESCs) depends upon the function of a remarkably small number of master transcription factors (TFs) that include OCT4, SOX2, and NANOG. Endogenous factors that regulate and maintain the expression of master TFs in hESCs remain largely unknown and/or uncharacterized. We use a genome-wide, proteomics approach to identify proteins associated with the OCT4 enhancer. We identify known OCT4 regulators, plus a subset of potential regulators including a zinc finger protein, ZNF207, that plays diverse roles during development. In hESCs, ZNF207 partners with master pluripotency TFs to govern self-renewal and pluripotency while simultaneously controlling commitment of cells towards ectoderm through direct regulation of neuronal TFs, including OTX2. The distinct roles of ZNF207 during differentiation occur via isoform switching. Thus, a distinct isoform of ZNF207 functions in hESCs at the nexus that balances pluripotency and differentiation to ectoderm.

overall_design: examine ZNF207 binding sites in hESCs

first: Ninuo

last: Xia


title: A distinct isoform of ZNF207 controls self-renewal and pluripotency of human embryonic stem cells [RNA-Seq]

overall_design: examine gene expression changes in ZNF207 knock down hESCs


title: Reproducibility of molecular phenotypes after long-term differentiation to Human iPSC-Derived Neurons: a multi-site omics study

first: Viola

last: Volpato


title: A Virus-Packageable CRISPR Screen Identifies Host Factors Mediating Interferon Inhibition of HIV

first: Molly

last: Ohainle

address: 1100 Fairview Ave N, C2-023

summary: Interferon (IFN) inhibits HIV replication by inducing an array of antiviral effectors. Here we describe a novel CRISPR knockout screening approach to identify the ensemble of these HIV restriction factors. We assembled a CRISPR sgRNA library specific for Interferon Stimulated Genes (ISGs) into a modified lentiviral vector that allows for packaging of sgRNA-encoding genomes in trans into budding HIV-1 particles. We observed that knockout of Zinc Antiviral Protein (ZAP) improved the performance of the screen due to ZAP-mediated inhibition of the vector. We identify a small panel of IFN-induced HIV restriction factors, including MxB, IFITM1, Tetherin/BST2 and TRIM5 which together explain the inhibitory effects of IFN on the HIV-1 LAI strain in THP-1 cells. Further, we identify novel HIV dependency factors, including SEC62 and TLR2. The ability of IFN-induced restriction factors to inhibit an HIV strain to replicate in human cells suggests that these human restriction factors are incompletely antagonized.

sample_organism:
Homo sapiens
Human immunodeficiency virus

overall_design: A CRISPR screen targeting Interferon-Stimulated Genes in which enrichment of sgRNA sequences is compared in viral RNA to the library representation in genomic DNA. 2 biological replicates were performed in 3 indpendent cell lines (one wild type THP-1 and two independent ZAP Knockout THP-1 clonal cell lines). Cells were infected following treatment overnight with IFN and viral supernatants and cell pellets collected 3 days post-infection. sgRNA representation in viral RNA is compared to genomic DNA to determine which sgRNAs are over- or under-represented in the viral population.

middle: O

first: Louisa

last: Pendergast

first: Jolien

last: Vermeire

first: Ferdinand

last: Roesch

first: Daryl

last: Humes

first: Ryan

last: Basom

first: Jeffrey

last: Delrow

first: Julie

last: Overbaugh

first: Michael

last: Emerman


title: Mapping targets for small nucleolar RNAs in yeast.

city: Edinburgh

first: Hywel

last: Dunn-Davies

address: Max Born Crescent

department: Wellcome Trust Centre for Cell Biology

institute: University of Edinburgh

summary: We report the effects of depleting U3 and snR4 in yeast BY4741 cells on the abundance of different types of RNA within the cell.

overall_design: U3 and snR4 deletion mutants were sequenced in triplicate, and differential expression analysis was performed in comparison to wild type controls.

first: Tatiana

last: Dudnakova

first: Rosie

last: Peters

first: David

last: Tollervey


title: Mapping targets for small nucleolar RNAs in yeast

summary: We report the identification of potential novel RNA targets for box C/D snoRNAs in budding yeast, using the approach of UV crosslinking and sequencing of hybrids (CLASH).

overall_design: 28 different CLASH experiments were carried out on BY4741 cells, with NOP1, NOP56, NOP58, and MTR4 used as bait proteins. Cells were either grown solely in glucose, or grown in glucose then transferred to ethanol / glycerol for 20 minutes.


title: Genome-wide Expression Profiling in pancreatic cancer cells

city: LOS ANGELES

first: MARINA

last: KOUTSIOUMPA

state: CA

address: 650 Charles E. Young Dr South, CHS 33-315

department: MEDICINE

institute: UCLA

summary: Differential gene expression profiling in KMT2D-depleted MIA PaCa-2 cells was performed using Human Genome U133 Plus 2.0 Array

overall_design: Expression profiles of MIA PaCa-2 cells from technical duplicates of control and KMT2D-transiently silenced cells by siRNA.

first: Dimitrios

last: Iliopoulos

first: Marina

last: Koutsioumpa


title: Simplified ChIP-exo assays II

city: University Park

first: Matthew

middle: John

last: Rossi

state: Pennsylvania

address: 453 N Frear Bldg

department: Biochemistry & Molecular Biology

institute: Penn State University

summary: Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and its ultra-high resolution cousin ChIP-exo are methods that identify where proteins bind along any genome in vivo. ChIP-exo achieves near-base pair resolution by creating exonuclease stop sites just 5’ to where formaldehyde-induced protein-DNA cross-links occur. Whereas construction of ChIP genomic libraries is straightforward and widely adopted for ChIP-seq, ChIP-exo is technically more involved which has resulted in limited adoption. Here we describe multiple ChIP-exo protocols, each with use-specific advantages and limitations. The new versions are greatly simplified through removal of multiple enzymatic steps. This is achieved in part through the use of Tn5 tagmentation and/or single-stranded DNA ligation. The result is greater library yields, lower processing time, and lower cost. A similar streamlined approach was developed for ChIP-seq, called ChIP-seq 1-step, where library construction is achieved in one-step.

overall_design: Several versions of optimized ChIP-exo and ChIP-seq protocols are described

first: William

last: Lai

first: B

middle: F

last: Pugh


title: Mechanistic insights into plant SUVH family H3K9 methyltransferases and their binding to context-biased non-CG DNA methylation

city: Los Angeles

first: Zhenhui

last: Zhong

address: 610 Charles E Young Dr East

department: Department of Molecular, Cell and Developmental Biology

institute: University of California, Los Angeles

summary:
DNA methylation functions in gene silencing and the maintenance
of genome integrity. In plants, non-CG DNA methylation is linked
through a self-reinforcing loop with histone 3 lysine 9 dimethylation (H3K9me2). The plant-specific SUPPRESSOR OF VARIEGATION
3–9 HOMOLOG (SUVH) family H3K9 methyltransferases (MTases)
bind to DNA methylation marks and catalyze H3K9 methylation.
Here, we analyzed the structure and function of Arabidopsis thaliana SUVH6 to understand how this class of enzyme maintains
methylation patterns in the genome. We reveal that SUVH6 has
a distinct 5mC base-flipping mechanism involving a thumb loop
element. Autoinhibition of H3 substrate entry is regulated by a
SET domain loop, and a conformational transition in the postSET domain upon cofactor binding may control catalysis. In vitro
DNA binding and in vivo ChIP-seq data reveal that the different
SUVH family H3K9 MTases have distinct DNA binding preferences,
targeting H3K9 methylation to sites with different methylated
DNA sequences, explaining the context biased non-CG DNA methylation in plants.

overall_design:
To determine the in vivo relevance of the in vitro binding preferences, we profiled levels of H3K9me2 by chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq)
in wild type (WT) and kyp, suvh5, suvh6, and kyp/suvh5/6 mutant
backgrounds.

first: Zhong

last: Zhenhui

first: Harris

last: Jake


title: Regulation of Gene Expression by Thyroid Hormone in Cultured Astrocytes: Transcriptional Targets and Factors Influencing the Genomic Response

status: Public on Aug 21 2018

city: Madrid

first: Enrique

last: Vazquez de Luis

address: Melchor Fernandez Almagro, 3

institute: CNIC

summary: Astrocytes mediate the action of thyroid hormone in the brain on other neural cells through the production of the active hormone triiodothyronine (T3) from its precursor thyroxine (T4). T3 has also many effects on the astrocytes in vivo and in culture, but whether these actions are directly mediated by transcriptional regulation is not clear. In this work, we have analyzed the genomic response to T3 of cultured astrocytes isolated from the postnatal mouse cerebral cortex, using RNA sequencing. Cultured astrocytes express relevant genes of thyroid hormone metabolism and action encoding type 2 deiodinase (Dio2), Mct8 transporter (Slc16a2), T3 receptors (Thra1 and Thrb), and nuclear corepressor (Ncor1) and coactivator (Ncoa1). T3 changed the expression of 668 genes (4.5% of expressed genes), of which 117 genes (0.8% of expressed genes) were primary, transcriptional responses. The Wnt and Notch pathways were down-regulated at the post-transcriptional level. Comparison with the effect of T3 on astrocyte-enriched genes in mixed cerebrocortical cultures isolated from fetal cortex revealed that the response to T3 is influenced by the degree of astrocyte maturation, and that in agreement with its physiological effects, T3 promotes the transition between the fetal and adult patterns of gene expression.

overall_design: To analyze the genomic response to T3 of cultured astrocytes, isolated from the postnatal mouse cerebral cortex, using RNA sequencing.

first: Beatriz

middle: B

last: Morte

first: Pilar

middle: P

last: Gil-Ibañez

first: Juan

last: Bernal


title: Transcriptome of Klf9-OE E13.5 PGCs cultured for 1 day in the conditions for PGC reprogramming

status: Public on Aug 16 2018

city: Sendai

first: Kei

last: Otsuka

state: miyagi

address: 4-1 Seiryomachi Aoba-ku

department: Institute of Development, Aging and Cancer

country: Japan

institute: Tohoku university

summary: We report transcriptome of Klf9-OE PGC by RNA-seq

overall_design: RNA-seq of  Klf9-OE clutured for one day in bFGF containing GS medium by using Illumina HiSeq2500.

first: Matsui

last: Yasuhisa


title: ATAC-seq with and without SET8 in U2OS cells

city: Copenhagen N

first: Mads

last: Lerdrup

address: Blegdamsvej 3C

department: Department of Cellular and Molecular Medicine

country: Denmark

institute: University of Copenhagen

summary: We report that loss of SET8 from cells leads to a global decrease in chromatin compaction status in G1 phase.

overall_design: Double thymidine block synchronized U2OS cells were depleted with SET8, released from G1/S and harvested in subsequent G1 phase. 4 biological replicates of each control siRNA and SET8 siRNA were processed for ATAC-seq as described previously by Buenrostro J et al; 2015.

first: Muhammad

last: Shoaib

last: Walter

first: Peter

last: Gillespie

first: Fanny

last: Izard

first: Birthe

last: Fahrenkrog

last: Lleres

first: Jens

middle: V

last: Johansen

first: Klaus

last: Hansen

first: Eric

last: Julien

first: J

last: Blow

first: Claus

last: Sørensen


title: Transcriptome of nascent teratoma cell-derived pluripotent stem cells, Dnd1-KD ESCs and control ESCs.

summary: We report transcriptome of nascent teratoma cell-derived pluripotent stem cells, Dnd1-KD ESCs and control ESC by RNA-seq.

overall_design: RNA-seq of two independent pluripotent stem cell lines derived form nascent teratoma cell in new-born testis of Dnd1-deficient mice, control E14tg2a ESCs and Dnd1-KD ESCs in dupulicate by using Illumina HiSeq2500.

first: Yuri

last: An

first: Yasuhisa

last: Matsui


title: Identification of NLRP3 inflammasome binding non-coding RNAs in activated iBMDMs

status: Public on Aug 18 2018

city: Hefei

first: Pengfei

last: Zhang

address: 443 Huang-Shan Road

department: School of Life Sciences

institute: University of Science and Technology of China

summary: High-throughput RNA-CLIP sequencing was employed to identify the NLRP3 inflammasome binding non-coding RNAs in iBMDMs.

overall_design: In order to identify the NLRP3 inflammasome binding ncRNAs and their binding sites, we performed NLRP3 CLIP and high-throughput sequencing.

first: Mian

last: Wu

first: Limian

last: Cao


title: T-cell DNA methylation in addicts treated with DHEA

city: Ariel

first: Elad

last: Lax

address: 65 Ramat HaGolan st.

department: Molecular Biology

country: Israel

institute: Ariel University

type: Methylation profiling by genome tiling array

summary: Previous studies in animal models of cocaine craving have delineated broad changes in DNA methylation profiles in the nucleus accumbens.  A crucial factor for progress in behavioral and mental health epigenetics is the discovery of epigenetic markers in peripheral tissues. Several studies in primates and humans have associated differences in behavioral phenotypes with changes in DNA methylation in T cells and brain. Herein, we present a pilot study (n=27) showing that the T cell DNA methylation profile differentiates persons with a substance use disorder from controls. Intervention with dehydroepiandrosterone (DHEA), previously shown to have a long-term therapeutic effect on human addicts herein resulted in reversal of DNA methylation changes in behavioral pathways associated with the addictive state.

overall_design: Examination of DNA methylation signatures in T cells from drug addicts before and after DHEA treatment and control patients

first: Gal

last: Warhaftig

first: David

last: Ohana

first: Rachel

last: Maayan

first: Yael

last: Delayahu

first: Paola

last: Roska

first: Alexander

last: Ponizovsky

first: Abraham

last: Weizman

last: Yadid

first: Moshe

last: Szyf


title: Multimodal enhancement of remyelination by exercise with a pivotal role for oligodendroglial PGC1a

city: Lethbridge

first: Yaroslav

last: Ilnytskyy

state: AB

address: 4401, University Drive W

department: Biology

country: Canada

institute: University of Lethbridge

summary: Remyelination is a multistep regenerative process that results in the reformation of myelin sheaths around demyelinated axons and is a critical therapeutic target. Here we show that immediate access to a running wheel following toxin-induced demyelination in mice enhances oligodendrogenesis, myelin thickness, and the proportion of remyelinated axons. RNA-sequencing suggests broad activation of pro-remyelination pathways including phagocytosis by exercise and highlights peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1a) activation. Our study demonstrates that physical activity is an integrative means to enhance remyelination and details a multimodal mechanism including the pivotal PGC1a-dependent enhancement of myelin thickness.

overall_design: We investigated the effect of physical exercise on remyelination using lysolecithin model of toxin induced demyelination. Voluntary exercise was introduced via unrestricted access to monitored running wheel. Control animals were subjected to the same conditions except the running wheel was locked. Generation of new olygodendrocytes was evaluated immunohistochemically using pan-olygodendrocyte lineage marker Olig2. The level remyelination was directly examined by immunohistochemical labeling of myelin basic protein (MBP) and additionally by electron microscopy. Transcriptome profiling of lesion sites was conducted using RNA-seq. The lesion tissue samples were obtained using laser micro dissections.

first: Samuel

middle: K

last: Jensen

first: Matthew

last: Michaels

first: Michael

last: Keough

first: Olga

last: Kovalchuk

first: V

middle: W

last: Yong


title: MCM2 promotes symmetric inheritance of modified histones during DNA replication

city: Copenhagen

first: Nataliya

last: Petryk

address: Ole Maaloees Vej 5

department: BRIC - Biotech Research and Innovation Centre

summary: During genome replication, parental histones are recycled to newly replicated DNA with their post-translational modifications (PTMs). It remains unknown whether sister chromatids inherit modified histones evenly. Here, we measured histone PTM partition to sister chromatids in embryonic stem cells. We found that parental histones H3-H4 segregate to both daughter DNA strands with a weak leading strand bias, skewing partition at topologically associating domain (TAD) borders and enhancers proximal to replication initiation zones. Segregation of parental histones to the leading strand increased markedly in cells with histone-binding mutations in MCM2, part of the replicative helicase, exacerbating histone PTM sister chromatid asymmetry. This work reveals how histones are inherited to sister chromatids and identifies a mechanism for how symmetric cell division is ensured by the replication machinery.

overall_design: Measurement of partition of new histone H4 (K5ac) and old histones H4 (K20me2) and H3 (K36me3) to Sister Chromatids After Replication in wild type and MCM2_2A histone binding mutants in mouse embryonic stem cells.

first: Maria

last: Dalby

first: Alice

last: Wenger

first: Caroline

last: Stromme

first: Anne

last: Strandsby

first: Robin

last: Andersson

first: Anja

last: Groth


title: Simplified ChIP-exo assays

last: Rossi

sample_organism:
Saccharomyces cerevisiae
Homo sapiens

platform_organism:
Saccharomyces cerevisiae
Homo sapiens

overall_design:
Several versions of optimized ChIP-exo and ChIP-seq protocols are described below:

ChIP-exo 1.1 libraries were prepared for sequencing using standard Illumina protocols with a minor modification; Reference paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813302/

ChIP-exo 1.1 Low Chromatin libraries were prepared for sequencing using standard Illumina protocols with a minor modification; Reference paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813302/, but used 5 times less starting material was used.

ChIP-exo 3.0 libraries were prepared for sequencing using standard Illumina protocols, Reference paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813302/, with modifications to include a Tn5 transposase to add adapters via tagmentation. Following tagmentation, the ChIP-exo 1.1 protocol was followed.

ChIP-exo 3.0 Nextera libraries were prepared as ChIP-exo 3.0, but the in-house Tn5 was replaced by the protein that is commercially available from Illumina.

ChIP-exo 4.0 libraries were prepared for sequencing using standard Illumina protocols, Reference paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813302/, with modifications to the method of adapter ligation.

ChIP-exo 4.1 libraries were prepared for sequencing using standard Illumina protocols, Reference paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813302/, with modifications to the method of adapter ligation. that were opposite polarity of those used in ChIP-exo 4.0.

ChIP-exo 5.0 libraries were prepared for sequencing using standard Illumina protocols, Reference paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813302/ with modifications throughout the protocol that reduced the number of steps and incubation times compared to ChIP-exo 1.1.

ChIP-exo 5.0 Low Chromatin Low Chromatin libraries prepared for sequencing as for ChIP-exo 5.0, but used 5 times less starting material was used.

ChIP-nexus (ChIP-exo 2.0) libraries were prepared for sequencing using standard Illumina protocols. Reference paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4390430/

ChIP-seq 1-step (altChIP-seq) libraries were prepared for sequencing using standard Illumina protocols with modifications that removed standard end repair steps and ligated the adapters in a single step.

ChIPmentation (altChIP-seq) libraries were prepared for sequencing using standard Illumina protocols; Reference paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4589892/

first: William

last: Lai

first: B

last: Pugh


title: VEGF-D deficiency causes hyperlipidemia and delayed clearance of chylomicron remnants

city: Kuopio

first: Henri

last: Niskanen

department: A.I. Virtanen Institute

country: Finland

institute: University of Eastern Finland

summary:
Objective: Dyslipidemia is one of the key factors behind coronary heart disease. Blood and lymphatic vessels play pivotal roles in both lipoprotein metabolism and development of atherosclerotic plaques. Recent studies have linked members of Vascular Endothelial Growth Factor (VEGF) family to lipid metabolism but the function of VEGF-D has remained unexplored. Here we investigated how the deletion of VEGF-D affects lipid and lipoprotein metabolism in atherogenic LDLR-/-ApoB100/100 mice.

Approach and Results: Deletion of VEGF-D (Vegfd-/-LDLR-/-ApoB100/100) led to markedly elevated plasma cholesterol and triglyceride levels without an increase in atherogenesis. Size distribution and hepatic lipid uptake studies confirmed a delayed clearance of large chylomicron remnant particles that cannot easily penetrate through the vascular endothelium. Mechanistically, the inhibition of VEGF-D signaling significantly decreased the hepatic expression of syndecan 1 (SDC1), which is one of the main receptors for chylomicron remnant uptake when LDLR is absent. Immunohistochemical staining confirmed reduced expression of SDC1 in the sinusoidal surface of hepatocytes in VEGF-D deficient mice. Furthermore, hepatic RNA sequencing revealed that VEGF-D is also an important regulator of genes related to lipid metabolism and inflammation. The lack of VEGF-D signaling via VEGF receptor 3 led to lowered expression of genes regulating triglyceride and cholesterol production as well as downregulation of peroxisomal β-oxidation pathway.

Conclusions: These results demonstrate that VEGF-D, a powerful lymphangiogenic and angiogenic growth factor, is also a major regulator of chylomicron metabolism in mice.

overall_design: Gene expression profiling of mouse liver tissue from control and VEGF-D knock-out mice. Control and VEGF-D KO mice were both in C57Bl/6 and atherosclerotic background, i.e., deficient of LDLR and expressing only apolipoprotein B100.

first: Minna

middle: U

last: Kaikkonen

first: Annakaisa

last: Tirronen

first: Taina

last: Vuorio


title: Systematic perturbation of retroviral LTRs reveals widespread long-range effects on human gene regulation [RNA-seq]

city: Stanford

first: Daniel

middle: R

last: Fuentes

address: 265 Campus Drive, G3065

department: Chemical and Systems Biology

institute: Stanford University

summary: Recent work suggests extensive adaptation of transposable elements (TEs) for host gene regulation. However, high numbers of integrations typical of TEs, coupled with sequence divergence within families, have made systematic interrogation of the regulatory contributions of TEs challenging. Here, we employ CARGO, our recent method for CRISPR gRNA multiplexing, to facilitate targeting of LTR5HS, a higher primate-specific class of HERVK (HML-2) LTRs that is active during early development and present in ~700 copies throughout the human genome. We combine CARGO with CRISPR activation or interference to, respectively, induce or silence LTR5HS en masse, and demonstrate that this system robustly targets the vast majority of LTR5HS insertions. Remarkably, activation/silencing of LTR5HS is associated with reciprocal up- and down-regulation of hundreds of human genes. These effects require presence of retroviral sequences, but occur over long genomic distances, consistent with a pervasive function of LTR5HS elements as early embryonic enhancers in higher primates.

overall_design:
Identify transcriptional changes of CARGO-CRISPRa/CRISPRi by RNA-seq
CRISPR dCas9 fusions to target transposable elements in the genome;
'VPR' and 'KRAB' refer to the name of the dCas9 fusion protein expressed in the given sample. 'Sp' and 'Sa' refer to the gRNA scaffold species for the CRISPR targeting construct, and 'LTR5HS' and 'nontarget' refer to the class of transposable element (or none at all) that the CRISPR targeting construct aims to target.

first: Joanna

last: Wysocka


title: Systematic perturbation of retroviral LTRs reveals widespread long-range effects on human gene regulation [ChIP-seq]

overall_design:
Identify genome-wide binding sites of dCas9 with CARGO by ChIP-seq
CRISPR dCas9 fusions to target transposable elements in the genome;
'VPR' and 'KRAB' refer to the name of the dCas9 fusion protein expressed in the given sample. 'Sp' and 'Sa' refer to the gRNA scaffold species for the CRISPR targeting construct, and 'LTR5HS' and 'nontarget' refer to the class of transposable element (or none at all) that the CRISPR targeting construct aims to target.


title: miRNAs expression in G0  phase of mouse melanoma B16 cell lines in comparison with G1 phase by use of small RNA sequence

status: Public on Aug 15 2018

city: Callaghan

first: Ting

last: La

state: NSW

address: University Drive

department: School of Biomedical Sciences & Pharmacy

country: Australia

institute: The University of Newcastle

type: Non-coding RNA profiling by high throughput sequencing

summary: To investigate miRNAs in quiescent cancer cell

overall_design: Examination of miRNAs specificlly expressing in G0 and G1 phases.

first: Xu

middle: D

last: Zhang


title: Systematic perturbation of retroviral LTRs reveals widespread long-range effects on human gene regulation

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

overall_design: Refer to individual Series


title: Single-cell chromatin conformation capture of diploid cells by Dip-C

city: Cambridge

first: Longzhi

last: Tan

state: MA

address: 12 Oxford St

department: Chemistry and Chemical Biology

institute: Harvard University


title: Single-cell chromatin conformation capture of diploid cells by Dip-C (analyzed with the hickit package)

summary: To reconstruct the 3D genomes of single diploid human and mouse cells, we performed single-cell chromatin conformation capture by a novel method, Dip-C, on human cells. This submission is similar to our submission GSE117109, but analyzed the data with the hickit package instead of the dip-c package. The raw data for the samples in this series are identical to the related samples in GSE117109. There are links between the original and re-analyzed data on the sample pages.

overall_design: We performed Dip-C on 17 GM12878 human lymphoblastoid cells and 18 peripheral blood mononuclear cells (PBMCs) from a male human donor, and analyzed the data with the hickit package. BioProject for the data is PRJNA473369.

first: Heng

last: Li


title: CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops

first: Yu

middle: (Phoebe)

last: Guo

state: California

address: 1450 Biggy Street, NRT 6514

institute: Univ. of Southern California

summary: Background: Recent genome-wide association studies (GWAS) have identified more than 100 loci associated with increased risk of prostate cancer, most of which are in non-coding regions of the genome. Understanding the function of these non-coding risk loci is critical to elucidate the genetic susceptibility to prostate cancer. Results: We generated genome-wide regulatory element maps and performed genome-wide chromosome confirmation capture assays (in situ Hi-C) in normal and tumorigenic prostate cells. Using this information, we annotated the regulatory potential of 2,181 fine-mapped PCa risk-associated SNPs and predicted a set of target genes that are regulated by PCa risk-related H3K27Ac-mediated loops. We next identified PCa risk-associated CTCF sites involved in long-range chromatin loops. We used CRISPR-mediated deletion to remove PCa risk-associated CTCF anchor regions and the CTCF anchor regions looped to the PCa risk-associated CTCF sites; we observed up to 100 fold increases in expression of genes within the loops when the PCa risk-associated CTCF anchor regions were deleted.  Conclusions: We have identified GWAS risk loci involved in long-range loops that function to repress gene expression within chromatin loops. Our studies provide new insights into the genetic susceptibility to prostate cancer.

overall_design: ChIP-seq, RNA-seq, Hi-C in prostate cells.

first: Andrew

last: Perez

first: Fidas

last: Lay

first: Suhn

last: Rhie

first: Peggy

last: Farnham


title: A single cell transcriptional portrait of embryoid body differentiation and comparison to progenitors of the developing embryo

city: Baltimore

first: Patrick

last: Cahan

state: MD

address: 733 North Broadway, MRB 653

department: ICE and Biomedical Engineering

institute: Johns Hopkins University School of Medicine


title: A single cell transcriptional portrait of embryoid body differentiation and comparison to progenitors of the developing embryo [scRNA-seq]

summary: We report the application of single-cell sequencing technology for high-throughput transcriptional profiling of differentiating mouse embryoid bodies. We make comparisons of the these transcriptional profiles to the transcriptional profiles of progenitors of a developing embryo.

overall_design: We examine two time points of the embryoid body directed differentiation protocol: day 4 and day 6. 5,062 day 4 cells and 4,842 day 6 cells were sequenced.

first: Abby

last: Spangler


title: Regulation of genes by HDAC2 via enzyme activity-independent manner during HL-60 cell differentiation by TPA

city: Seoul

first: Jiyoung

last: KIM

address: 84 Heuk Seok-Dong, Dong jak -Gu

department: Life Science

country: South Korea

institute: Chung-Ang University

summary: Human myeloid leukemia cell line, HL-60 cells, have a potential to differentiate into monocytes with treatment of 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the underlying mechanism of differentiation by TPA is not fully elucidated yet. Here, we performed ChIP-seq profiling using RNA Pol II, HDAC2, AcH3 and H3K27me3, and analyzed the differential chromatin state changes during HL-60 differentiation by TPA. In this study, we focused on the atypical active genes, which showed enhanced H3 acetylation occupancies despite increased HDAC2 recruitment. We found that HDAC2 regulates the expression of these genes positively via histone deacetylase activity-independent manner. HDAC2 interacted and recruited Paired Box 5 (PAX5) to promoter of the target genes and regulated PAX5-mediated gene activation. Taken together, these data demonstrated the profiling of differentiation specific-chromatin status during the differentiation in HL-60 cells by TPA and suggested that HDAC2 activates certain genes through the interaction with PAX5 in enzyme activity independent pathway.

overall_design: Genome binding/occupancy profiling of HDAC2, RNA PolII, acetyl-H3, H3K27me3 in differentiated-HL-60 cells

first: Hyeonsoo

last: Jung

first: Ji-Young

last: Kim

first: Sang-Beom

last: Seo


title: Gene expression in cultured mouse neural progenitor cells

city: Pasadena

first: Nagarajan

last: Nandagopal

address: 1200 E. California Blvd, M/C 114-96

institute: California Institute of Technology

summary: Bulk RNA sequencing data from neural progenitor cells under conditions of low or high growth factor and Notch pathway activation

overall_design: Cells were treated with high (20 ng/ml EGF and FGF) or low (0.5 ng/ml EGF) recombinant growth factors, with or without Notch pathway inhibitor (DAPT, 10 uM) for 12h.

first: Michael

last: Elowitz


title: RNA Sequencing in CD11b cells for Quantitative Analysis of WT and myeloid specific NRP2 Knock out transcriptomes in subcutaneous pancreatic tumors

status: Public on Aug 14 2018

city: Omaha

first: Sohini

last: Roy

state: Nebraska

address: 985870 nebraska medical center, DRC1

department: Biochemistry and molecular biology

institute: University of nebraska medical center

summary: The purpose of the study is to identify the alterations in transcriptome (RNA Seq) in CD11b+ myeloid cells isolated from WT and monocytes/macrophage specific Neuropilin2 (NRP2) Knock out mice harboring subcutaneous pancreatic cancer tumors. The main goal is to identify the intrinsic changes in gene signature occuring in myeloid cells in NRP2 deleted conditions in tumor that indicates the role of NRP2 in regulating phagocytosis/efferocytosis and immune responses in myeloid cells in tumors.

overall_design: Mouse pancreatic cancer cell lines were subcutaneously implanted into NRP2f/f i-Cre mice. Once tumors became palpable, the test group animals were injected with Tamoxifen to specifically knock out NRP2 from monocytes/macrophages. Tumors were harvested at 15days and CD11b cells were isolated using magnetic beads. RNA was isolated using trizol and RNA Seq performed for analysis of differentially expressed transcriptomes in WT and in myeloid specific NRP2 KO conditions. We had n=3 animals in each control and test groups. Since the RNA isolated from each individual animal was less, we pooled RNA from n=3 animals. We validated expression of some of the differentially altered genes in seperate biological replicates by RT-PCR analysis.

first: Karen

last: Yip


title: m6A-facilitated hippocampal learning and memory through Ythdf1

city: Chicago

first: Chuan

last: He

state: ILLINOIS

address: 929 E 57th St., GCIS E319

department: Department of Chemistry

institute: The University of Chicago

summary: N6-methyladenosine (m6A) is the most prevalent internal RNA modification in mammalian messenger RNAs (mRNAs). While m6A has been shown to mark groups of mRNAs for coordinated degradation in various physiological processes, the relevance of m6A in affecting translation remains to be determined in intact biological systems in vivo. Here we show that, through its reader protein Ythdf1, m6A promotes a pulse of protein synthesis of target transcripts in response to neuronal stimuli in the adult mouse hippocampus, thereby facilitating learning and memory processes. Mice with genetic deletion of the Ythdf1 gene (Ythdf1-/-) exhibit learning and memory defects, as well as impaired hippocampal synaptic transmission and long-term potentiation (LTP). Selective re-expression of Ythdf1 in the hippocampus of adult Ythdf1-/- mice fully rescues the behavioral and synaptic defects, while hippocampus-specific knockdown of Ythdf1 or Mettl3, the catalytic component of m6A methyltransferase complex, recapitulates the hippocampal deficiency in adult mice. At the molecular level, transcriptome-wide mapping of m6A sites and RNA binding sites of Ythdf1 on hippocampal mRNAs using crosslinking immunoprecipitation followed by high-throughput sequencing (CLIP-seq) uncovered key neuronal genes, including those involved in synaptic transmission and long-term potentiation. Nascent protein labelling and tether reporter assays in cultured hippocampal neurons revealed that Ythdf1 is critical for initiating a pulse of protein synthesis of target transcripts in a neuronal-stimulus-dependent manner. Collectively, our results uncover a pathway of mRNA m6A methylation in learning and memory, which is mediated through Ythdf1 in response to stimuli.

overall_design: (1) The RNA binding targets of Ythdf1 were determined by anti-Ythdf1 antibody CLIP-seq in the adult mouse hippocampal tissue. (2) m6A-modified transcripts were mapped by anti-m6A antibody CLIP-seq using polyA RNA purified from the adult mouse hippocampus. (3) mRNA abundance in wild-type and Ythdf1-KO mouse hippocampus was determined by mRNA-seq. (4) Abundance of m6A-modified transcripts in mouse dentate gyrus before and after electroconvulsive treatment was determined by full length m6A-RIP-seq.

first: Hailing

last: Shi

first: Zhike

last: Lu

first: Yi-Lan

last: Weng

first: Guo-Li

last: Ming

first: Hongjun

last: Song

first: Tao

last: Zhou


title: Adipose tissue of overfed animals during and after overfeeding

city: New York

first: Ethan

last: Edwin

state: NY

address: 1150 St. Nicholas Ave

institute: Columbia University Medical Center

summary: We report the transcriptomes (polyadenylated mRNAs over 200bp) of animals overfed through intragastric tubes either in the overfed state (OIO) or 24 hours after cessation of overfeeding (OIOneg). We also report transcriptomes of weight-matched mice either fed (ALO, or fasted 24 hours (ALOneg).

overall_design: Examination of transcriptomes in two models of weight gain at either peak weight or after 24 hours without feeding.

first: Yann

last: Ravussin

first: Anthony

last: Ferrante Jr


title: Differential open chromatin profile and transcriptomic signature define depot-specific human subcutaneous preadipocytes [RNA-seq]

city: orlando

first: Adeline

last: Divoux

state: FL

address: 301 E. Princeton St

department: Translational Research Institute

institute: Adventhealth

summary: RNA-seq experiments revealed an A-fat and GF-fat selective gene expression signature, with 126 genes up-regulated in abdominal preadipocytes and 90 genes up-regulated in GF cells. Assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) identified almost 10-times more A-specific chromatin-accessible regions. Using a combined analysis of ATAC-seq and global gene expression data, we identified 74 of the 126 abdominal-specific genes (59%) with A-specific accessible chromatin sites within 200kb of the transcription start site (TSS). Interestingly, only 14 of the 90 GF-specific genes (15%) had GF-specific accessible chromatin sites within 200kb of the corresponding TSS, whereas 25 of them (28%) had abdominal-specific accessible chromatin sites.

overall_design: Preadipocytes cultured from A and GF adipose tissue obtained from premenopausal apple-shaped women were used to perform transcriptome analysis by RNA-seq and assess accessible chromatin regions by ATAC-seq.

first: Steven

last: Smith

first: Timothy

last: Osborne


title: Alignment of different types of resting or murine cytomegalovirus-activated mononuclear phagocytes across different datasets

status: Public on Aug 23 2018

city: marseille

first: Thien-Phong

last: Vu Manh

address: 163 avenue de Luminy

country: France

institute: CIML

type:
Expression profiling by array
Third-party reanalysis

summary:
The goal of this experiment was to use global gene expression profiling to assess the global genetic reprogramming of different types of splenic mononuclear phagocytes early after MCMV infection in vivo. This study includes new samples (GSM3178486-GSM3178497; available below) profiling splenic CD11b+ conventional dendritic cells (cDC2), classical monocytes (cMo) and red pulp macrophages (RPM) from untreated or day 1.5 MCMV-infected mice together with re-analysis of previously published data in order to examine the similarities in the pDC gene expression profiles across datasets.

The complete processed data for all analyzed samples, with and without batch correction, are available at the foot of this series. Included is a text file with a list of all re-analyzed samples and data processing protocols for the re-analysis.

overall_design: The new samples in this series are for a comparison of the gene expression programs of different types of resting or MCMV-activated mononuclear phagocytes isolated from the spleen of untreated versus d1.5 infected wild-type CD45.2 C57BL/6 mice.

first: Anissa

last: Fries

first: Rabie

last: Chelbi

first: Emeline

last: Pollet

first: Marc

last: Dalod

first: Elena

last: Tomasello


title: High definition analysis of host protein stability during human cytomegalovirus infection reveals antiviral factors and viral evasion mechanisms

first: Michael

middle: Paul

last: Weekes

address: Hills Road

department: Cambridge Institute for Medical Research

institute: University of Cambridge

summary: Human cytomegalovirus (HCMV) is an important pathogen with multiple immune evasion strategies, including virally facilitated degradation of host antiviral restriction factors. Here, we describe a multiplexed approach to discover proteins with innate immune function on the basis of active degradation by the proteasome or lysosome during early phase HCMV infection. Using three orthogonal proteomic/transcriptomic screens to quantify protein degradation, with high confidence we identified 35 proteins enriched in antiviral restriction factors. A final screen employed a comprehensive panel of viral mutants to predict viral genes that target >250 human proteins. This approach revealed Helicase-like Transcription Factor (HLTF), a DNA helicase important in DNA repair, potently inhibits early viral gene expression but is rapidly degraded during infection. The functionally unknown HCMV protein UL145 facilitates HLTF degradation by recruiting the Cullin4 E3 ligase complex. Our approach and data will enable further identifications of innate pathways targeted by HCMV and other viruses.

overall_design: 9 samples comprising three sets of three replicates (0h, 24h, 72h)


title: Differential open chromatin profile and transcriptomic signature define depot-specific human subcutaneous preadipocytes [ATAC-Seq]


title: Inhibition of aberrant DNA re-methylation improves development of cloned embryos (RNA-Seq data set 2)

city: Shanghai

first: Shaorong

last: Gao

state: Shanghai

address: 1239 Siping Road, Yangpu District

department: School of life science and technology

institute: Tongji University

summary: Somatic cell nuclear transfer (SCNT) enables the genome of a differentiated somatic cell to be reprogrammed to totipotency. However, this process is extremely inefficient, and the underlying mechanism of the epigenetic rearrangements following SCNT remains largely unknown. Here, we generated a genome-wide DNA methylome of mouse SCNT preimplantation embryos. Surprisingly, we identified widespread re-methylated regions (rDMRs) in 2- to 4-cell stage cloned embryos, which caused mis-expression of genes and retrotransposons important for zygotic genome activation and embryo development. Knocking-down DNA methyltransferases can specifically rescue the re-methylation defects of SCNT embryos and evidently improve the poor developmental capacity of cloned embryos. In addition, inactivation of DNA methyltransferases combined with overexpression of histone demethylases led to a more significant reduction in DNA methylation as well as exhibited a synergistic enhancement effect on the full-term development of nuclear transfer embryos. Our study therefore reveals that aberrant re-methylation functions as an unavoidable barrier for SCNT embryo development, and that the removal of multiple epigenetic barriers would be a promising approach to achieve the highest cloning efficiency.

overall_design: For SCNT embryos or injected SCNT embryos 2-3 replicates were performed for each stage.

first: Chenfei

last: Wang

first: Rui

first: Wenchao

last: Xiu

first: Wenqiang

last: Liu

first: Yawei

first: Xiaochen

last: Kou

first: Yanhong

last: Zhao

first: Dandan

last: Bai

first: Yong

last: Zhang


title: Inhibition of aberrant DNA re-methylation improves development of cloned embryos (WGBS-Seq)

type: Methylation profiling by high throughput sequencing

overall_design: For SCNT embryos, 1-2 replicates were performed for each stage.


title: OCaR1, a gatekeeper of lysosomal and granular Ca2+ release and regulated exocytosis

city: Phoenix

first: Tobias

last: Jakobi

state: Arizona

address: 475 N. 5th Street

department: Internal Medicine

institute: University of Arizona

summary: During exocytosis, a Ca2+ increase arises in close vicinity of secretory granules which is strictly prevented in resting cells. Granular Ca2+ homeostasis and its role in granular exocytosis are not well understood.

overall_design: Comparison of male wild type (C57Bl6/N) and OCaR1–/– mice. RNA was isolated from the pancreas of three 3 weeks old male wild type (C57Bl6/N) and OCaR1–/– mice, respectively, using TRIZOL reagent (Thermo Fisher Scientific, Germany).

first: Christoph

last: Dieterich

last: Freichel


title: Migration through a small pore disrupts inactive chromatin organisation in neutrophil-like cells [Hi-C]

city: Auckland

first: Justin

middle: M.

last: O'Sullivan

address: 85 Park Road, Grafton

department: Liggins Institute

country: New Zealand

institute: University of Auckland

summary:
Background. Mammalian cells are flexible and can rapidly change shape when they contract, adhere, or migrate. Their nucleus must be stiff enough to withstand cytoskeletal forces, but flexible enough to remodel as the cell changes shape. This is particularly important for cells migrating through constricted space, where the nuclear shape must change in order to fit through. This happens many times in the life cycle of a neutrophil, which must protect its chromatin from damage and disruption associated with migration.

Results. Total RNA-sequencing identified that neutrophil migration through 5 or 14 µm pores was associated with changes in the transcript levels of inflammation and chemotaxis-related genes, when compared to unmigrated cells. Differentially expressed transcripts specific to migration with constriction were enriched for groups of genes associated with cytoskeleton remodelling. Hi-C was used to capture the genome organisation in control and migrated cells. Chromatin did not switch between the active (A) and inactive (B) compartments after migration. However, global depletion of mid- to long-range contacts was observed following active migration through the 5 µm pores. Chromatin contacts that decreased in frequency were enriched in inactive chromatin.

Conclusion. Mid- to long-range contacts are preferentially lost within inactive chromatin, thus protecting transcriptionally active contacts from the disruptive effects of migration with constriction. This is consistent with current hypotheses implicating heterochromatin as the mechanoresponsive form of chromatin. Further investigation concerning the contribution of heterochromatin to stiffness, flexibility, and protection of nuclear function will be important for understanding cell migration in human health and disease.

overall_design: Hi-C sequencing of neutrophil-like cells migrated through large (14µm) or small (5µm) pores, with an unmigrated control population.

first: Elsie

middle: C

last: Jacobson

middle: M


title: Loss of E3 ubiquitin protein ligase Cbl-b provides protection against UVB-induced cytotoxicity and inflammation.

city: Graz

first: Karin

last: Wagner

state: Styria

address: Stiftingtalstrasse 24

country: Austria

institute: Medical University Graz

summary: The exposure of skin to ultraviolet (UV) radiation results in sunburn cell (SBC) and cyclobutane pyrimidine dimer (CPD) formation and local immune suppression. However, the pathways that regulate these events are poorly understood. Here, we exposed mice to UVB to study the early cytotoxic effects in absence of E3 ubiquitin protein ligase Cbl-b. We observed that Cbl-b-/- mice developed significantly less SBCs and CPDs compared to WT mice. In addition, microarray analysis revealed that loss of Cbl-b-induced cell tolerance genes rather than DNA repair genes. Taken together, our results suggest a novel role of Cbl-b in regulating UV cytotoxicity.

overall_design: Samples of dorsal skin were collected from UV-exposed clb-b -/- (group 1) and wild type (group 2) mice 24 hours after exposure. The number of animals was 3 per experimental group.

first: Tej

last: Singh

first: Peter

last: Wolf


title: Combinatorial Smad2/3 Activities Downstream of Nodal Signaling Maintain Embryonic/Extra-Embryonic Cell Identities during Lineage Priming [RNA-Seq]

city: Oxford

first: Anna

middle: Dorothea

last: Senft

address: South Parks Road

department: Sir William Dunn School of Pathology

institute: University of Oxford

summary: Epiblast cells in the early post-implantation stage mammalian embryo undergo a transition described as lineage priming before cell fate allocation, but signaling pathways acting upstream remain ill defined. Genetic studies demonstrate that Smad2/3 double-mutant mouse embryos die shortly after implantation. To learn more about the molecular disturbances underlying this abrupt failure, here we characterised Smad2/3-deificient embryonic stem cells (ESCs). We found that Smad2/3 double-knockout ESCs induced to form epiblast-like cells (EpiLCs) display changes in naïve and primed pluripotency marker gene expression, associated with the disruption of Oct4-bound distal regulatory element. In the absence of Smad2/3, we observed enhanced Bmp target gene expression and de-repression of extra-embryonic gene expression. Cell fate allocation into all three embryonic germ lakers is disrupted. Collectively, these experiments demonstrate that combinatorial Smad2/3 functional activities are required to maintain distinct embryonic and/or extra-embryonic cell identity during lineage priming in the epiblast before gastrulation.

overall_design: mRNA profiles of wild type and Smad2/3 DKO day 2 epiblast-like cells (EpiLCs) were generated by deep sequencing in biological triplicate using Illumina HiSeq4000.

first: Ita

last: Costello

first: Hamish

last: King

first: Arne

last: Mould

first: Elizabeth

last: Bikoff

middle: J

last: Robertson


title: Differential open chromatin profile and transcriptomic signature define depot-specific human subcutaneous preadipocytes

first: Adeline

last: Divoux

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

overall_design: Refer to individual Series


title: Combinatorial Smad2/3 Activities Downstream of Nodal Signaling Maintain Embryonic/Extra-Embryonic Cell Identities during Lineage Priming [ATAC-Seq]

overall_design: Tn5-accessible chromatin profiles of wild type and Smad2/3 DKO day 2 epiblast-like cells (EpiLCs) were generated by deep sequencing in biological triplicate using Illumina HiSeq4000.


title: Inhibition of aberrant DNA re-methylation improves development of cloned embryos

first: Shaorong

last: Gao


title: CnAb1

city: Madrid

first: Jesus

middle: Maria

last: Gómez-Salnero

address: Melchor Fernández Almagro, 3

institute: CNIC


title: CnAb1 knock down in R1 mESCs culture in pluripotent conditions

summary:
R1 mESCs were trasnfected with siRNAs against CnAb1 or Control (Luciferase) using Lipofectamine. mRNA was extracted 48 hours using Qiagen RNAeasy Mini kit
Microarray was performed after QPCR validation of the downregulation of CnAb1.

overall_design: Cells were transfected with siRNAs: Control, CnAb1 (1) and CnAb1 (2). For the MicroArray analysis we used 3 control, 3 CnAb1 (1), and 2 CnAb1 (2) samples.

first: Jesús

last: Gómez-Salinero

first: Enrique

last: Lara-Pezzi


title: Hexokinase 2 is dispensable for T cell-dependent immunity

status: Public on Aug 13 2018

first: Panagiotis

last: Ntziachristos

state: IL

address: 320 E Superior

department: Biochemistry and Molecular Genetics

institute: Northwestern University

summary: Hexokinase 2 (HK2) is one the most highly upregulated enzymes in glycolysis in activated T cells and cancer cells. We genetically abolished HK2 expression in T cells in vivo and infected mice with LCMV to induce T cell proliferation. CD8+ T cells from were sorted from spleens of WT and HK2 null mice 8 days after infection.  Additionally, WT CD8+ T cells were compared to WT primary T cell acute lymphoblastic leukemia (T-ALL). T-ALL cells were sorted from spleens of mice 6 weeks after adoptive transfer of bone marrow that overexpresses mutant Notch1 (Notch1-deltaE).

overall_design: Whole RNA was extracted from 100k primary cells from sorted T cells and T-ALL using the RNAeasy kit (Qiagen) according to the manufacturer’s protocol. SMART-Seq v4 Ultra Low Input RNA Kit was used to generate full-length cDNA and Nextera XT DNA sample preparation kit was used to prepare final library. Libraries were sequenced on the NextSeq 500 with single-end sequencing (1x75bp). Differential gene expression analysis was performed between WT and and HK2 null (KO) T cells. Additionally, WT T cells were compared to WT T-ALL. Analysis was performed using edgeR package.

first: Carlos

middle: A

last: Martinez

first: Navdeep

last: Chandel

first: Manan

last: Mehta


title: SIRT1 Activation Disrupts Maintenance of Myelodysplastic Syndrome Stem and Progenitor Cells by Restoring TET2 Function [hMeDIP-Seq]

city: Duarte

first: Sun

last: Jie

address: 1500 East Duarte Road

institute: City of Hope

summary: Improved understanding of mechanisms regulating myelodysplastic syndrome (MDS) hematopoietic stem/progenitor cell (HSPC) growth and self-renewal is critical for developing MDS therapy. We revealed a novel regulatory axis that SIRT1-deficiency induced TET2 hyperacetylation promotes MDS HSPC functions, and provide an approach to target MDS HSPCs by activating SIRT1 deacetylase.

overall_design: 5-hydroxymethylcytosine (5hmC) modified DNA fragments from SIRT1 overexpressed or MOCK MDS-L cells were immunoprecipited by anti-5hmC antibody for Next-Generation Sequencing

first: Jie

last: Sun

first: Juan

last: Du

first: Xiwei

last: Wu

first: Ling

last: Li

first: Jinhui

last: Wang


title: AmpliSeq Transcriptome Analysis of Human Prostate Cancer Cells With or Without Overt SRRM4 Expression

city: Vancouver

first: Xuesen

last: Dong

state: British Columbia

address: 2660 Oak Street

department: Department of Urologic sciences, Vancouver Prostate Centre

institute: University of British Columbia

summary: Prostate adenocarcinoma (AdPC) cells can undergo lineage switching to neuroendocrine cells and develop into therapy-resistant neuroendocrine prostate cancer (NEPC). While genomic/epigenetic alterations are shown to induce neuroendocrine differentiation via an intermediate stem-like state, RNA splicing factor SRRM4 can transform AdPC cells into NEPC xenografts through a direct neuroendocrine transdifferentiation mechanism. Whether SRRM4 can also regulate a stem-cell gene network for NEPC development remains unclear. Here, we use the Ion AmpliSeqTM Transcriptome Human Gene Expression Kit (AmpliSeq) to analyze the transcriptome of human AdPC cell line DU145 overexpressing SRRM4 via lentiviral transduction compared to the control, empty vector-transduced DU145 cells. This study reveals that SRRM4 induces a pluripotency gene network consisting of the stem-cell differentiation gene, SOX2. In summary, we report a novel mechanism by which SRRM4 drives NEPC progression via a pluripotency gene network.

overall_design: Assessment of transcriptome profiles from DU145 cells with or without overt SRRM4 expression using AmpliSeq.

first: Ahn

last: Lee

first: Yu

last: Gan


title: Expression data from erythroblast (CD71 and GPA positive cells) derived from fetal liver and adult  blood

city: Khlong Nung

first: Pavita

last: Kaewprommal

state: Pathum Thani

department: NBT

country: Thailand

institute: NSTDA

summary:
The fetal hemoglobin (HbF) levels were 95.4 ± 1.5% and 4.4 ± 0.2% in fetal liver-derived and adult blood derived-cultured erythrocytes (n=5), respectively. Following RNA isolation from the CD71 high/GPA positive erythroblasts, gene expression analyses were performed using Affymetrix Human Gene 2.0 ST Array.
The Affymetrix raw data files of microarray were preprocessed using robust multi-array average method for background correction, log-transformation, and quantile normalization. Differential gene expression between fetal liver derived- and adult peripheral blood derived-erythroblasts was determined using moderated t-statistics implemented in the Limma package. We found the 1366 genes were upregulated by more than 1.5-fold change with adjusted p-value less than 0.05 in fetal liver derived-erythroblasts.

overall_design: The fetal liver-derived and adult blood derived-cultured erythrocytes (n=5), respectively. Gene expression analyses were performed using Affymetrix Human Gene 2.0 ST Array. The Affymetrix raw data files of microarray were preprocessed using robust multi-array average method for background correction, log-transformation, and quantile normalization. Differential gene expression between fetal liver derived- and adult peripheral blood derived-erythroblasts was determined using moderated t-statistics implemented in the Limma package.

first: Amornrat

last: Tangprasittipap

first: Orapan

last: Sripichai

first: Nuankanya

last: Sathirapongsasuti

first: Chonthicha

last: Satirapod

first: Philip

last: Shaw

first: Jittima

last: Piriyapongsa

first: Suradej

last: Hongeng


title: Single-cell RNA-seq reveals differentiation of bona fide human pDCs and cDC1s in cultures of cord blood CD34+ progenitors, and a newly identified terminal differentiation step of cDC1s

first: Thien-Phong

last: Vu Manh

summary: CD34+ cord blood hematopoietic progenitors were expanded in vitro as previously described (Balan et al., J Immunol, 2014) and then differentiated on a mixed feeder layer of OP9 cells expressing or not the Notch ligand Delta-like 1, with FLT3-L, TPO and IL-7. At the end of the cultures, single live Lin- HLA-DR+ cells were index sorted in 96-well plates containing lysis buffer, and snap frozen. Four putative cell types were sorted according to their expression patterns of key combinations of cell surface markers: putative pDCs, putative cDC1s, putative pre-cDC2s and putative cDC2s. Single cell RNA-sequencing libraries were subsequently generated for 90 single cells and 6 control wells using an adaptation of Smart-Seq2 (Villani et al., Science, 2017). Cells were sequenced at a depth of 1-3M reads/cell.

overall_design: A total of 90 single cells and 6 controls from one culture were processed using a protocol adapted from Smart-Seq2 protocol (Villani et al., Science, 2017), which allows for the generation of full-length single cell cDNA, and sequencing libraries were generated using Illumina Nextera XT DNA library preparation kit. A few samples (10) were profiled but excluded from the processed data since they were either bulk (5) or blank (1) control samples or excluded due to QC (4). Therefore, there are 86 samples included here.

first: Catharina

last: Arnold-Schrauf

first: Abdenour

last: Abbas

first: Norbert

last: Couespel

first: Alexandra

last: Villani

first: Thien

first: Marc

last: Dalod


title: Analysis of single-cell RNA-seq data from human PBMCs and from in vitro cultures of human cord blood CD34+ progenitors encompassing different DC types

summary: For both PBMC and cells from the in vitro cultures, RNA purification and library generation was performed using the Chromium Single Cell Controller apparatus and associated protocols (10X Genomics). Libraries were sequenced by 75-bp single-end reading on a NextSeq500 sequencer (Illumina). Reads were aligned on the GRCh38 human genome assembly. Data analysis was performed using the R software package Seurat (https://github.com/satijalab/seurat)

overall_design: Single cell RNA-seq data were generated on the 10X emulsion platform (10X Genomics, Pleasanton, CA) according to the manufacturer's instructions. NextSeq data from the Chromium platform were processed using CellRanger v1.3.1, and subsequent normalization, QC, filtering, and differential gene expression analysis was performed in R using Seurat v1.4.0.16.


title: RNA-seq and Hi-C sequencing of neutrophil-like cells migrated through large or small pores

first: Justin

last: O'Sullivan


title: Inhibition of aberrant DNA re-methylation improves development of cloned embryos (RRBS-Seq)

first: Shaorong

last: Gao

overall_design: For SCNT embryos 4-8 replicates were performed for each stage . As the control, 3 replicates were performed for each stage of wild type samples

first: Chenfei

last: Wang

first: Rui

first: Wenqiang

last: Liu

first: Yawei

first: Xiaochen

last: Kou

first: Yanhong

last: Zhao

first: Dandan

last: Bai

first: Yong

last: Zhang


title: Maf1 and repression of RNA polymerase III-mediated transcription drives adipocyte differentiation

city: Houston

first: Deborah

last: Johnson

state: Texas

address: One Baylor Plaza

department: Department of Molecular and Cellular Biology

institute: Baylor College of Medicine

summary: RNA polymerase (pol) III transcribes a variety of small untranslated RNAs that are involved in essential cellular processes that include transcription, RNA processing, and translation. RNA pol III and its components are altered in various human developmental disorders, yet their roles in cell fate determination and development are poorly understood. Here we demonstrate that Maf1, a transcriptional repressor, promotes induction of mouse embryonic stem cells into mesoderm and their terminal differentiation into adipocytes. Reduced Maf1 expression in preadipocytes impairs adipogenesis while ectopic Maf1 expression in Maf1-/- deficient cells enhances differentiation. RNA pol III repression by either chemical inhibition or knockdown of Brf1, promotes adipogenesis. Altered RNA pol III-dependent transcription produces select changes in RNA pol II-derived transcripts with a significant enrichment of adipogenic gene signatures. Furthermore, RNA pol III-mediated transcription positively regulates long non-coding RNA H19 and Wnt6 expression, established adipogenesis inhibitors. Together, these studies reveal an important and unexpected function for RNA pol III-mediated transcription and Maf1 in mesoderm induction and cellular differentiation.

overall_design: Gene expression profiling by RNA-seq at two time points (day 0: before differentiation, day 2: two days after differentiation) with knockdown by shRNA of Maf1 and Brf1, respectively, and RNA Pol III inhibitor, ML-60218, treatment compare to the control (empty shRNA vector) in 3T3-L1 cells.

first: Chun-Yuan

last: Chen

first: Rainer

middle: B

last: Lanz

middle: L


title: Therapeutic efficacy of BET bromodomain protein inhhibitor and PD-1 blockade in genetically engineered mouse model of non-small cell lung cancer

first: Igor

last: Dolgalev

state: New York

address: 550 1st Ave

institute: NYU Grossman School of Medicine

summary: KRAS mutation is present in about 30% of human lung adenocarcinomas. While recent advances in targeted therapy have shown great promise, KRAS remains undruggable and concurrent alterations in tumor suppressors render KRAS mutant tumors even more resistant to existing therapies. Contributing to the refractoriness of KRAS mutant tumors harboring these co-mutations are immunosuppressive mechanisms such as increased presence of suppressive Tregs in tumors and elevated expression of the inhibitory receptor PD-1 on tumor-infiltrating T cells. BET bromodomain inhibitors demonstrate clinical benefit in hematologic malignancies, and prior reports demonstrate their Treg-disruptive effects in a NSCLC model. Targeting PD-1 inhibitory signals through anti-PD-1 antibody blockade has also shown substantial therapeutic impact in lung cancer although these outcomes are still limited to a minor pool of patients. We therefore hypothesized that the BET bromodomain inhibitor JQ1 would synergize with PD-1 blockade to promote robust anti-tumor response in lung cancer. In the present study, using Kras+/LSL-G12D; Trp53L/L (KP) mouse models of non-small cell lung cancer, we identified cooperative effects between JQ1 and anti-PD-1 antibody that included reduced numbers of tumor-infiltrating Tregs and enhanced activation of tumor-infiltrating T cells, which exhibited a Th1 cytokine profile that favored their demonstrated improved effector function. Furthermore, lung-tumor-bearing mice under this combinatorial treatment regimen showed robust and long-lasting anti-tumor responses compared to either agent alone, culminating in substantial improvement in the survival of treated mice. Thus, combining BET bromodomain inhibition with immune checkpoint blockade offers a promising therapeutic approach for solid malignancies such as lung adenocarcinoma.

overall_design: Gene expression analyses of tumor nodules in lung tumor-bearing mice treated with Vehicle (control), JQ1 (Bromodomain inhibitor) and/or anti-PD-1 antibody

first: Dennis

last: Adeegbe

first: Kwok-Kin

last: Wong


title: FTO-Dependent m6A Regulates Cardiac Function During Remodeling and Repair

first: Susmita

last: Sahoo

address: One Gustave L. Levy Place, Box 1030

department: CVRC

institute: Icahn School of Medicine at Mount Sinai

summary:
Background: Despite its functional importance in various fundamental bioprocesses, the studies of N6-methyladenosine (m6A) in the heart are lacking.
Methods: We performed methylated (m6A) RNA immunoprecipitation sequencing (MeRIP-seq) to map transcriptome-wide m6A in healthy and failing hearts.
Results: Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is carried out by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts
Conclusion: Collectively, our study demonstrates the functional importance of FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.

overall_design: MeRIP-seq was performed on total RNA extracted from 1 week post-MI mouse left ventricles using the Illumina HiSeq 2500 platform as 50bp single reads

first: Sahoo

last: Susmita

first: Mathiyalagan

last: Prabhu

first: Mayourian

last: Joshua


title: Inhibition of aberrant DNA re-methylation improves development of cloned embryos (RNA-Seq data set 1)

first: Shaorong

last: Gao

overall_design: For SCNT embryos or injected SCNT embryos 3-8 replicates were performed for each stage . As the control, 3-6 replicates were performed for each stage of wild type samples

first: Chenfei

first: Rui


title: The Estrogen Receptor variants β2 and β5 Induce Stem Cell Characteristics and Chemotherapy Resistance in Prostate Cancer through activation of Hypoxic Signaling

first: Anders

middle: Mikael

last: Strom

address: 3517 Cullen Blvd Bldg 545 room 3018

department: Biology & Biochemistry

institute: University od Hpuston

summary: Chemotherapy resistant prostate cancer is a major clinical problem. When the prostate cancer has become androgen deprivation resistant, one of the few treatment regimens left is chemotherapy. There is a strong connection between a cancer’s stem cell like characteristics and drug resistance. By performing RNA-seq we observed several factors associated with stem cells being strongly up-regulated by the estrogen receptor β variants, β2 and β5. In addition, most of these factors were also up-regulated by hypoxia. One mechanism of chemotherapy resistance was expression of the hypoxia-regulated, drug transporter genes, where especially ABCG2 and MDR1 were shown to be expressed in recurrent prostate cancer and to cause chemotherapy resistance by efficiently transporting drugs like docetaxel out of the cells. Another mechanism was expression of the hypoxia-regulated notch3 gene, which causes chemotherapy resistance in urothelial carcinoma, although the mechanism is unknown. It is well known that hypoxic signaling is involved in increasing chemotherapy resistance. Regulation of the hypoxic factors, HIF-1α and HIF-2α is very complex and extends far beyond hypoxia itself. We have recently shown that two of the estrogen receptor β variants, estrogen receptor β2 and β5, bind to and stabilize both HIF-1α and HIF-2α proteins leading to expression of HIF target genes. This study suggests that increased expression of the estrogen receptor β variants, β2 and β5, could be involved in development of a cancer’s stem cell characteristics and chemotherapy resistance, indicating that targeting these factors could prevent or reverse chemotherapy resistance and cancer stem cell expansion.

overall_design: Examination of the transcriptome changed by two estrogen reseptor beta variants (ERbeta2 and ERbeta5). Control (lacking expression) and  variant expressing cells in two repeats

first: Michelle

last: Faria


title: RNAseq analysis for Fast Generated Functional Subtype Astrocytes from Human Pluripotent Stem Cells

status: Public on Aug 11 2018

city: Madison

first: Xiang

last: Li

state: Wisconsin

address: University of Wisconsin

institute: University of Wisconsin-Madison

summary: Differentiation of astrocytes from human pluripotent stem cells (hPSCs) is a tedious and variable process. This hampers the study of hPSC-generated astrocytes in disease processes and drug development. By using CRISPR/Cas9-mediated inducible expression of NFIA or NFIA plus SOX9 in hPSCs, we developed a method to efficiently generate astrocytes in 4-7 weeks. The astrocytic identity of the induced cells was verified by their characteristic molecular and functional properties as well as after transplantation. Furthermore, we developed a strategy to generate region-specific astrocyte subtypes by combining differentiation of regional progenitors and transgenic induction of astrocytes. This simple and efficient method offers a new opportunity to study the fundamental biology of human astrocytes and their roles in disease processes.

overall_design: Total 5 samples analyzed, including two transgenic induced samples, two non-transgenic induced samples and primary astrocytes.

first: Yezheng

last: Tao

first: Robert

last: Bradley

first: Zhongwei

last: Du

first: Su-chun


title: RNA sequencing analysis of human embryonic stem cells and axial progenitors

city: Napoli

first: Ilaria

last: Granata

address: Via Pietro Catellino, 111

department: Institute for high performance computing and networking (ICAR)

country: Italy

institute: National Research Council of Italy (CNR)

summary: We analysed the transcriptomes of human embryonic stem cells (hESCs) and in vitro derived axial progenitors.

overall_design: RNA sequencing of triplicate samples of undifferentiated hESCs grown in Essential 8 medium and Geltrex and axial progenitors generated after a 3 day culture of hPSCs on fibronectin in the presence of bFGF (20 ng/ml) and the WNT agonist/GSK3 inhibitor CHIR99021

first: Anestis

last: Tsakiridis


title: Unravelling subclonal heterogeneity and aggressive disease states in TNBC through single-cell RNA-seq [WES]

city: Boston

first: Simona

last: Cristea

state: Massachusetts

address: 450 Brookline Ave.

department: Biostatistics and Computational Biology

institute: Dana-Farber Cancer Institute

summary: Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by extensive intratumoral heterogeneity. To investigate the underlying biology, we conduct single-cell RNA- sequencing (scRNA-seq) of >1500 cells from six primary TNBC, together with whole exome sequencing (WES) for four of the tumors. Intercellular heterogeneity of gene expression programs within each tumor was variable and largely correlated with clonality of inferred genomic copy number changes, suggesting that genotype drives the gene expression phenotype of individual subpopulations. Clustering of gene expression profiles identified distinct subgroups of malignant cells shared by multiple tumors, including a single subpopulation associated with multiple signatures of treatment resistance and metastasis, and characterized functionally by activation of glycosphingolipid metabolism and associated innate immunity pathways. A novel signature defining this subpopulation was predictive of long-term outcomes for TNBC patients in a large patient cohort. Collectively, this analysis reveals the functional heterogeneity and its association with genomic evolution in TNBC, and uncovers unanticipated biological principles dictating poor outcomes in this disease.

overall_design: Whole exome sequencing of 4 FFPE TNBC samples and 1 lymphoblastoid cell line


title: Combinatorial Smad2/3 Activities Downstream of Nodal Signaling Maintain Embryonic/Extra-Embryonic Cell Identities during Lineage Priming

first: Anna

last: Senft

overall_design: Refer to individual Series


title: Unravelling subclonal heterogeneity and aggressive disease states in TNBC through single-cell RNA-seq


title: SIRT1 Activation Disrupts Maintenance of Myelodysplastic Syndrome Stem and Progenitor Cells by Restoring TET2 Function

first: Sun

last: Jie

state: CA


title: TCRgd cell gene expression

status: Public on Aug 09 2018

city: Manchester

first: Joanne

middle: Elizabeth

last: Konkel

address: AV Hill Building, Oxford Road

department: DIIRM

institute: University of Manchester

summary: Comparision of gene expression profiles of TCRgd cells from spleen, gut and gingiva

overall_design: 9 samples total, 3 biological replicates of 3 different samples

middle: E


title: Genetic ablation of TonEBP/NFAT5 in smooth muscle cells inhibits arterial remodeling

status: Public on Aug 10 2018

city: Mannheim

first: Carsten

last: Sticht

address: Theodor-Kutzer-Ufer

department: ZMF

country: Germany

institute: University Heidelberg

summary: Chronic biomechanical stress elicits remodeling of the arterial wall and causes detrimental arterial stenosis and stiffening. In this context, molecular determinants controlling proliferation and stress responses of vascular smooth muscle cells (VSMCs) have been insufficiently studied. We identified the transcription factor ‘nuclear factor of activated T-cells 5’ (NFAT5) as crucial regulatory element of mechanical stress responses of VSMCs. The relevance of this observation for biomechanically induced arterial remodeling was investigated in mice upon SMC-specific knockdown of NFAT5. While blood pressure levels, vascular architecture and flow-induced collateral growth were not affected in these mice, both hypertension-mediated arterial thickening and muscularization of pulmonary arteries during pulmonary artery hypertension (PAH) were impaired. In all models, a decrease in VSMC proliferation was observed indicating that NFAT5 controls activation of VSMCs in the remodeling arterial wall. Mechanistically, mechanoactivation of VSMCs promotes nuclear translocation NFTA5c upon its phosphorylation at Y143 and dephosphorylation at S1197. As evidenced by transcriptome studies, loss of NFAT5 in mechanoactivated VSMCs impairs expression of gene products controlling cell cycle and transcription/translation. These findings identify NFAT5 as molecular determinant of VSMC responses to biomechanical stress and arterial thickening.

overall_design: Human arterial smooth muscle cells (HUASMCs) were freshly isolated and transfected with short interfering RNA directed against NFAT5 compared to HUASMCs transfected with siGENOME Non-Targeting siRNA as controls.

first: Maren

last: Zappe

first: Thomas

last: Korff


title: RNA-seq analysis of paired kidney-infiltrating and splenic T cells in the MRL/lpr murine model of systemic lupus erythematosus.

city: Pittsburgh

first: Mark

last: Shlomchik

state: PA

address: The Assembly, 5051 Centre Avenue

department: Department of Immunology

institute: University of Pittsburgh

summary:
Purpose: The goal of this study is to compare the transcriptional phenotype of lymphoid and kidney-infiltrating T cell populations in the setting of systemic inflammatory disease to determine how tissue location alters their phenotype.

Methods: mRNA profiles of T cells isolated from 23-week-old nephritic (protein score of 3+ on dipstick) mice were used in this study. T cells were isolated by flow cytometry gated on CD45+Thy1.1+CD44+ and either CD4 or CD8+ T cells. RNA was isolated using the RNeasy Plus Micro Kit (Qiagen). Samples were sequenced using Illumina NextSeq 500 with 75bp paired-end reads and aligned to the mm10 genome using the STAR aligner. The number of uniquely aligned reads ranged from 10 to 12 million. Using an optimized data analysis workflow, Gene-level counts were determined using featureCounts and raw counts were analyzed for differential expression using the “voom” method in the “limma” R package.

Results: After determining genes that were differentially expressed between splenic T cells and KIT, we performed gene set enrichment analysis (GSEA. Differentially expressed genes were compared to several previously defined gene signatures that are characteristic of CD8+ and CD4+ T cell exhaustion in the chronic LCMV infection model and tumor infiltrating lymphocytes. Genes from the CD8+ exhaustion cluster were significantly enriched among genes that were differentially expressed in CD8+ KITs vs CD8+ splenocytes.

overall_design: mRNA profiles of CD4 and CD8 T cells from spleen and kidney of 23 week old wild MRL/lpr mice were generated in triplicate by sequencing using Illumina NextSeq 500

first: Jeremy

middle: S

last: Tilstra

first: Shuchi

last: Smita

first: Maria

last: Chikina


title: Metabolic labeling of Hek293 cells using 4-thiouracil

city: Stockholm

first: Sten

last: Linnarsson

address: Scheeles väg 1

department: Medical Biochemistry and Biophysics

country: Sweden

institute: Karolinska Institutet

summary: Hek293 cells were metabolically labelled using 4-thiouracil as described in (Schwalb et al, Science. 2016 Jun 3;352(6290):1225-8) but without fragmentation, and then bulk RNA was prepared for sequencing using the STRT method (Islam et al, Genome Res. 2011 Jul;21(7):1160-7). Samples were incubated in duplicate for 5, 15 and 30 minutes and included an unlabeled control representing the steady-state expression state.

overall_design: 2 samples each of 4 incubation times, 2 cDNA preparations, 2 tagmentation replicates, and 2 biological replicates

first: Gioele

last: LaManno

first: Ruslan

last: Soldatov

first: Peter

middle: V

last: Karchenko


title: Transcriptional profiling of cochlear neurons

first: Gabriela

last: Pregernig

address: 1325 Boylston Street, Suite 500

country: USA

institute: Decibel Therapeutics

summary: In order to characterize the various subpopulations of neurons present in the inner ear, we dissected out the cochleas from wild-type mice, removing non-neuronal populations as possible, then dissociated the tissue to obtain a single cell suspension and profiled these by single cell RNA-Seq.

overall_design: This study's goal was to characterize the native populations of murine cochlear neurons. To ensure reproducibility of our findings, neurons from 4 separate mice were profiled.

first: Joseph

last: Burns

first: Kathy

last: So

first: Matthew

last: Nguyen

first: Adam

last: Palermo


title: Somatic piRNA pathway prevents transgenerational germline transposition [RNA-seq]

city: MONTPELLIER

first: SEVERINE

last: CHAMBEYRON

address: 141, rue de la Cardonille

department: Institute of Human Genetics

institute: CNRS

summary: Transposable elements (TEs) are genomic parasite that threat genome integrity. One major strategy organisms evolved to balance TE activity in the germline is the Piwi-interacting RNAs (piRNAs) pathway. It prevents transposition by repressing TE at transcriptional and/or post-transcriptional level. However, evidence that TE derepression caused by piRNA pathway impairment is actually followed by bursts of transposition is still lacking. Here we present a genome-wide TE-analyses of the replicative TE life cycle (their expression, their translation to their transposition in the germline) after  piRNA pathway impairment in the somatic follicle cells. We observed a high transposition-rate in the germline, leading to a 10-fold increase in genomic TE-load after 70 successive generations of piRNA pathway impairment. Moreover, we demonstrate that siRNAs co-regulate TE activity at post transcriptional level in follicle cells. These observations demonstrate that the small RNA-mediated TE repression in follicle cells contribute to genome homeostasis.

overall_design: Analysis of effects of conditional piwi knock down in somatic follicle cells on TE behaviour and progeny genome.

first: Bridlin

last: Barckmann

first: Marianne

last: El-Barouk

first: Alain

last: Pelisson

first: Blaise

last: Li

first: Bruno

last: Mugat

first: Anna-Sophie

last: Fiston Lavier

first: Marie

last: Mirouze

first: Klaus

last: Föstermann

last: Fablet

first: Severine

last: Chambeyron


title: Somatic piRNA pathway prevents transgenerational germline transposition [DNA-seq]


title: Somatic piRNA pathway prevents transgenerational germline transposition [smallRNA-seq]


title: Somatic piRNA pathway prevents transgenerational germline transposition

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