title: Transcription dependent generation of a specialized chromatin structure at the TCRb locus

city: Marseille

first: Mohamed

last: BELHOCINE

address: 163, Avenue de Luminy

institute: INSERM

summary: V(D)J recombination assembles antigen receptor (AR) genes during lymphocyte development. Enhancers at AR loci are known to control V(D)J recombination at associated alleles, in part, by increasing chromatin accessibility of the locus, in order to allow the recombination machinery to gain access to its chromosomal substrates. However, whether there is a specific mechanism to induce chromatin accessibility at AR loci is still unclear. Here, we highlight a specialized epigenetic marking characterized by high and extended H3K4me3 levels throughout the Db-Jb-Cb gene segments. We show that extended H3K4 trimethylation at the Tcrb locus depends on RNA-Polymerase II (Pol II)-mediated transcription. Furthermore, we found that the genomic regions encompassing the two DJCb clusters are highly enriched for Ser5-phosphorylated Pol II and short-RNA transcripts, two hallmarks of transcription initiation and early transcription. Interestingly, these features are shared with few other tissue specific genes. We propose that the entire DJCb regions behave as transcription “initiation” platforms, therefore linking a specialized mechanism of Pol II transcription with extended H3K4 trimethylation and highly accessible Db and Jb gene segments.

overall_design: Genome-wide analysis via ChIP-Seq for RNA Pol II Ser5-phosphorylated binding in RAG2 -/-  thymocytes.

first: Joaquin

last: Zacarias-Cabeza

last: Belhocine

first: Pierre

last: Cauchy

first: Frederic

last: Koch

first: Aurélie

last: Bergon

first: Aleksandra

last: Pekowska

first: Laurent

last: Vanhille

first: Romain

last: Fenouil

first: Marta

last: Gut

first: Ivo

first: Dirk

last: Eick

first: Jean

last: Imbert

last: Ferrier

first: Jean-Christophe

last: Andrau

first: Salvatore

last: Spicuglia


title: PATZ1 is a DNA damage responsive transcription factor that inhibits p53 function

status: Public on Mar 27 2015

city: Istanbul

first: Batu

last: Erman

address: Orhanli, Tuzla

department: Biological Sciences and Bioengineering Program

country: Turkey

institute: Sabanci University

type: Expression profiling by high throughput sequencing

summary: Insults to cellular health cause p53 protein accumulation and loss of p53 function leads to tumorigenesis. Thus, p53 has to be tightly controlled. Here we report that the BTB/POZ domain transcription factor PATZ1 (MAZR), previously known for its transcriptional suppressor functions in T lymphocytes, is a crucial regulator of p53. The novel inhibitory role of PATZ1 on the p53 protein marks it as a proto-oncogene. PATZ1 deficient cells have reduced proliferative capacity which we assess by RNASeq and real time cell growth rate analysis. PATZ1 modifies the expression of p53 target genes associated with cell proliferation gene ontology terms. Moreover, PATZ1 regulates several genes involved in cellular adhesion and morphogenesis. Significantly, treatment with the DNA damage inducing drug doxorubicin results in the loss of the PATZ1 transcription factor, as p53 accumulates. We find that PATZ1 binds to p53 and inhibits p53 dependent transcription activation. We examine the mechanism of this functional inhibitory interaction and demonstrate that PATZ1 excludes p53 from DNA binding. This study documents PATZ1 as a novel player in the p53 pathway.

overall_design: RNA-seq was used to define differentially expressed genes in wild-type and PATZ1-/- MEFs. Each sample was represented in triplicate.

first: Nazli

last: Keskin

first: Emre

last: Deniz


title: Differential features of lamina-associated domains mapped by ChIP-sequencing from sonicated or micrococcal nuclease-digested chromatin

status: Public on Mar 24 2015

city: Oslo

first: Philippe

last: Collas

address: PO Box 1112 Blindern

department: Institute of Basic Medical Sciences

country: Norway

institute: University of Oslo

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The nuclear lamina interacts with the genome through megabase-size lamina-associated domains (LADs). LADs have been identified in proximity labeling assays and recently by chromatin immunoprecipitation-sequencing (ChIP-seq) of A- and B-type lamins. LADs localize mainly to the nuclear periphery, they are gene-poor and largely heterochromatic. Here, we show that the mode of chromatin fragmentation for ChIP, namely either bath sonication (used to date for ChIP of nuclear lamins) or digestion with micrococcal nuclease (MNase) leads to the discovery of distinct sets of lamin-interacting domains (which we refer to as LiDs) with distinct gene content, histone composition enrichment and relationship to lamin B1-interacting domains. We find that total genome coverage by lamin A/C ('LMNA') LiDs identified in sonicated or MNase-digested chromatin is similar (~730 megabases). Over half of these domains, however, are uniquely detected in sonicated or MNase-digested chromatin. Whereas sonication-specific LMNA LiDs are gene-poor and devoid of a broad panel of histone modifications, MNase-specific LMNA LiDs are of higher gene density and are enriched in H3K9me3, H3K27me3 and in histone variant H2A.Z. Analysis of published LMNB1 LADs and of LMNB1 LiDs identified by ChIP-seq further shows that LMNA can associate with 'open' chromatin domains displaying euchromatin features which are not associated with LMNB1. The differential genetic and epigenetic properties of lamin-interacting chromatin domains indicate the existence of distinct LiD populations identifiable in different chromatin contexts, including nuclease-accessible 'open' regions presumably localized in the nuclear interior.

overall_design: There are two experiments of two samples each for LMNA ChIPs (ChIP and Input), and one LMNB1 ChIP sample, so 5 samples in total. The difference between the experiments are the chromatin fragmentation method (see extract protocols 1 and 2). One uses sonication while the other uses micrococcal nuclease (MNase).

first: Eivind

last: Lund

first: Isabelle

last: Duband-Goulet

first: Brigitte

last: Buendia


title: RNA expression profiling for Paf1 complex mutant strains [miRNA]

status: Public on Mar 25 2015

city: Basel

first: Tim

last: Roloff

address: Maulbeerstrasse 66

department: Functional Genomics

country: Switzerland

institute: FMI

type: Non-coding RNA profiling by high throughput sequencing

summary: Small RNA molecules are well known for silencing gene expression post-transcriptionally, but they are also implicated in epigenetic regulation across kingdoms. However, the mechanisms of epigenetic gene silencing by small RNAs are mostly unknown. In addition, stably silencing genes de novo using synthetic small RNAs to trigger locus-independent heterochromatin formation is impossible. Using fission yeast, we show that RNA-directed heterochromatin formation is negatively controlled by the highly conserved RNA polymerase-associated factor 1 (Paf1) complex. Temporary expression of a synthetic hairpin RNA in Paf1 mutants triggers stable heterochromatin formation at homologous loci, effectively silencing genes in trans. This repressed state is propagated over many generations by continual production of secondary siRNAs, independently of the synthetic hairpin RNA. Our work uncovers a novel mechanism for small RNA-mediated epigenome regulation and highlights fundamental roles for the Paf1 complex and the RNA interference machinery in building epigenetic memory.

overall_design: Small RNA was extracted from wild-type and mutant strains to compare miRNA gene expression.

first: Katarzyna

middle: M

last: Kowalik

first: Yukiko

last: Shimada

first: Michael

last: Stadler

first: Batki

last: Julia

first: Marc

last: Bühler


title: Genome-wide analysis of the endoplasmic reticulum stress response during lignocellulase synthesis in Neurospora crassa

city: Tianjin

first: feiyu

last: fan

state: Tianjin

address: 32 West 7th Avenue, Tianjin Airport Economic Area

department: Key Laboratory of Systems Microbial Biotechnology

country: China

institute: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences.

summary:
Purpose: To explore the pathway cross-talk of endoplasmic reticulum (ER) stress response and cellulases synthesis in Neurospora crassa.

Methods: To identify ER stress response targets(ESRTs), conidial suspensions of WT were obtained and 1 x 10^6 cells/ml incubated in cellulases induction medium (1x vogel’s salts , 1% w/v crystalline cellulose (Avicel PH-101; Sigma-Aldrich), 0.2% w/v NH4NO3 ,pH 7.0 )for 36 hours (25ºC, 200 rpm, constant light) followed by the addition of dithiothreitol (DTT, final concentration of 0.1mM for mild stress level while 5mM for acute stress level) or tunicamycin (TM, final concentration of 5ug/mL for mild stress level while 40ug/mL for acute stress level) and growth for 1 more hour.To identify which ESRTs regulated by IRE-1/Hac-1 mediated UPR pathway,conidial suspensions of the Ire-1 and Hac-1 KO mutants as well as their parental strains (FGSC#2489 and FGSC#9720, respectively) were obtained and 1 x 10^6 cells/ml incubated in minimal medium containing 2% sucrose for 16 hours, then young hyphae was harvested and transferred into cellulases induction medium as mentioned above culture for 4 hours followed by the addition of DTT (final concentration of 5mM)(or H2O as mock)and growth for 1 more hour.For Hac-1 KO mutant and FGSC#9720,cultures were plus with final concentration of 100ug/ml L-histidine.To explore the function of transcription factors RES-1, RES-2 and RRG-2 in response to ER stress and cellulases synthesis, each conidial suspension of the three strains was obtained and 1 x 10^6 cells/ml incubated in cellulases induction medium for 36 hours followed by the addition of dithiothreitol (DTT, final concentration of  5mM ) (or H2O as mock)and growth for 1 more hour.Cells were harvested by vacuum filtration with Whatman filter paper, frozen immediately in liquid nitrogen. Total RNA was isolated by the Trizol-Phenol-Chloroform method. An additional clean-up including the on-column DNase I treatment was performed by using the RNeasy mini kit (Qiagen).The thirteen 49-nt single-end RNA-seq libraries were generated commercially at Beijing Genomics Institute (BGI, Shenzhen, China) while eight 90-nt paired-end RNA-seq libraries were generated at Genewiz Co. Ltd (Suzhou) by using the HiSeq™ platform.Sequencing data was handled essentially with Bowtie2, Tophat2, NOIseq. Expression levels are presented as Reads Per Kilobase of transcript per Million mapped reads (RPKM).

Results: Transcriptional profiling of ER stress during cellulase synthesis in N.crassa uncovered a set of 766 genes was identified as the ER stress response target genes (ESRTs). Of these 766 genes, canonical UPR components IRE-1 and HAC-1 regulated 223 and 186 genes, respectively. In screening 527 available ESRT mutants, we found 249 that exhibited DTT sensibility, including 100 targets with unknown function. Disruption of ire-1 or hac-1 renders N. crassa cells seriously deficient in cellulase secretion. Further investigation of the 39 transcription factors (TFs) that showed differential expression under ER stress uncovered three novel TFs (RES-1, RES-2, RRG-2) that markedly affected cellulase secretion.

Conclusions: Here, we determined the transcriptional scope of the ER stress response during lignocellulase synthesis in the model cellulolytic fungus N. crassa. Through genome-wide mutant screening and analysis, dozens of novel genes were discovered to be involved in the process. The findings of this work will be useful for strain improvement to facilitate lignocellulase and biomass-based chemical production.

overall_design: mRNA profiles of N.crassa FGSC#2489 (WT), FGSC#9720, Δire-1, Δhac-1, Δres-1, Δres-2 and Δrrg-2 under ER stress and cellulases induction condition were generated by deep sequencing, using Illumina HiSeq™ platform.

first: Feiyu

last: Fan

first: Guoli

last: Ma

first: Jingen

last: Li

first: Qian

last: Liu

first: J. Philipp

last: Benz

first: Chaoguang

last: Tian

first: Yanhe


title: Host-based markers for the early stage of infection by the grapevine trunk pathogen Neofusicoccum parvum

city: Tuebingen

first: Stefan

last: Czemmel

address: Auf der Morgenstelle 10

department: University of Tuebingen

country: Germany

institute: Quantitative Biology Center (QBiC)

summary:
In the vineyard, symptoms of the grapevine trunk disease Botryosphaeria dieback do not appear until 1 or 2 years after the causal fungus Neofusicoccum parvum establishes a necrotic canker in the permanent woody structure of the vine.  There are preventative management practices, but growers tend to wait until symptoms are widespread, at which point prevention has limited efficacy.
Toward development of an early detection tool, we examined the leaves of inoculated vs. non-inoculated plants for differential gene expression via RNASeq.  Stems were examined to monitor spread of the infection, and its spatial and temporal relationship to anatomical changes via light microscopy and high resolution computed tomography (HRCT).
The early stage of infection occurred prior to 2 months post-inoculation (MPI), at which point spread of the pathogen beyond the inoculation site was greatest.  This incubation period was also characterized by the largest stem lesions, the highest levels of fungal colonization and xylem vessels fully-occluded by gels, and the lowest starch content of xylem fibers and rays.
Prior to 2 MPI, RNASeq and validative qPCR analysis identified eight candidate markers, which are transcriptionally activated by infection, but not by wounding alone. Our best marker genes, a dehydrin, a BURP domain containing protein and an abscissic acid-induced wheat plasma membrane polypeptide 19 (AWPM-19), identified the pathogen’s presence with high specificity.  Screening of genome-wide expression data revealed that this signature is not affected by many abiotic and biotic stresses.

overall_design: Asymptomatic leaf mRNA profiles of Neofusicoccum parvum infected wounded plants (IW) and non-infected wounded plants (NIW) at two different time points post inoculation (0 MPI, 0.5-1.5 MPI) were generated by deep sequencing in biological triplicates using Illumina HiSeq2500 technology.

first: Erin

middle: R

last: Galarneau

first: Renaud

last: Travadon

first: Andrew

middle: J

last: McElrone

first: Grant

last: Cramer

first: Kendra

last: Baumgartner


title: Spatially heterogeneous choroid plexus transcriptomes encode positional identity and contribute to regional cerebrospinal fluid production

city: Boston

first: Matthew

middle: Bonser

last: Johnson

state: Massachusetts

address: 300 Longwood Ave.

department: Genetics and Genomics

country: USA

institute: Boston Children's Hospital

summary: A sheet of choroid plexus epithelial cells extends into each cerebral ventricle and secretes signaling factors into the cerebrospinal fluid (CSF). To evaluate whether differences in the CSF proteome across ventricles arise, in part, from regional differences in choroid plexus gene expression, we defined the transcriptome of lateral ventricle (telencephalic) vs. fourth ventricle (hindbrain) choroid plexus. We find that positional identities of mouse, macaque, and human choroid plexi derive from gene expression domains that parallel their axial tissues of origin. We then show that molecular heterogeneity between telencephalic and hindbrain choroid plexi contributes to region-specific, age-dependent protein secretion in vitro. Transcriptome analysis of FACS-purified choroid plexus epithelial cells also predicts their cell type-specific secretome. Spatial domains with distinct protein expression profiles were observed within each choroid plexus. We propose that regional differences between choroid plexi contribute to dynamic signaling gradients across the mammalian cerebroventricular system.

overall_design: Two-factor design with two levels per factor and n=2 biological replicates. Lateral (telencephalic) and fourth (hindbrain) choroid plexus samples are paired in that they are isolated from the same brains.

middle: B

first: Melody

middle: P

last: Lun

first: Maria

middle: K

last: Lehtinen


title: 4C-seq: Clustering of mammalian Hox genes with other H3K27me3 targets within an active nuclear domain

status: Public on Mar 23 2015

city: Lausanne

first: Marion

last: LELEU

address: EPFL-SV-ISREC-UPDUB- Station 19

department: School of Life Sciences

institute: EPFL

summary: Embryogenesis requires the precise activation and repression of many transcriptional regulators. The Polycomb group proteins and the associated H3K27me3 histone mark are essential to maintain the inactive state of many of these genes. Mammalian Hox genes are targets of Polycomb proteins and form local 3D clusters centered on the H3K27me3 mark. More distal contacts have also been described, yet their selectivity, dynamics and relation to other layers of chromatin organization remained elusive. We report that repressed Hox genes form mutual intra- and inter-chromosomal interactions with other genes located in strong domains labeled by H3K27me3. These interactions occur in a central and active nuclear environment that consists of the HiC compartment A, away from peripheral Lamina Associated Domains. Interactions are independent of nearby H3K27me3-marked loci and determined by chromosomal distance and cell-type specific scaling factors, thus inducing a moderate reorganization during embryogenesis. These results provide a simplified view of nuclear organization whereby Polycomb proteins may have evolved to repress genes located in gene-dense regions whose position is restricted to central, active nuclear environments.

overall_design: Circular Chromosome Conformation Capture (4C-seq) samples from mouse ES cells and mouse embryonic samples at different anatomical positions. Data based on 34 biological samples.

first: D

last: Noordermeer

first: M

last: Vieux-Rochas

last: Leleu

last: Duboule


title: Transcriptome Characterization of developing bean (Phaseolus vulgaris L.) pods from two genotypes with contrasting seed zinc concentrations.

city: East Lansing,

first: Carolina

last: Astudillo

state: MI

address: A494 PSSB 1066 Bogue St.

institute: Michigan State University

summary: Dry bean (Phaseolus vulgaris L.) seeds are a rich source of dietary zinc, especially for people consuming plant-based diets.  Within P. vulgaris there is at least two-fold variation in seed Zn concentration.  Genetic studies have revealed seed Zn differences to be controlled by a single gene in two closely related navy bean genotypes, Albion and Voyager.  In this study, these two genotypes were grown under controlled fertilization conditions and the Zn concentration of various plant parts were determined.  The two genotypes had similar levels of Zn in their leaves and pods but Voyager had 52% more Zn in its seeds than Albion. RNA was sequence from developing pods of both genotypes.  Transcriptome analysis of these genotypes identified 27,198 genes in the developing bean pods, representing 86% of the genes in the P. vulgaris genome (v 1.0 DOE-JGI and USDA-NIFA).  Expression was detected in 18,438 genes.  A relatively small number of genes (381) were differentially expressed between Albion and Voyager.  Differentially expressed genes included three genes potentially involved in Zn transport, including zinc-regulated transporter, iron regulated transporter like (ZIP), zinc-induced facilitator (ZIF) and heavy metal associated (HMA) family genes.  In addition 12,118 SNPs were identified between the two genotypes.  Of the gene families related to Zn and/or Fe transport, eleven genes were found to contain SNPs between Albion and Voyager.

overall_design: Transcriptome of developing pods of two contrasting common bean genotypes in zinc concentration  generated by deep sequencing using Illumina Genome Analyzer II

first: Karen

middle: A

last: Cichy

last: Astudillo-Reyes


title: Clustering of mammalian Hox genes with other H3K27me3 targets within an active nuclear domain

summary:
[This SuperSeries is composed of the SubSeries listed below.]
Embryogenesis requires the precise activation and repression of many transcriptional regulators. The Polycomb group proteins and the associated H3K27me3 histone mark are essential to maintain the inactive state of many of these genes. Mammalian Hox genes are targets of Polycomb proteins and form local 3D clusters centered on the H3K27me3 mark. More distal contacts have also been described, yet their selectivity, dynamics and relation to other layers of chromatin organization remained elusive. We report that repressed Hox genes form mutual intra- and inter-chromosomal interactions with other genes located in strong domains labeled by H3K27me3. These interactions occur in a central and active nuclear environment that consists of the HiC compartment A, away from peripheral Lamina Associated Domains. Interactions are independent of nearby H3K27me3-marked loci and determined by chromosomal distance and cell-type specific scaling factors, thus inducing a moderate reorganization during embryogenesis. These results provide a simplified view of nuclear organization whereby Polycomb proteins may have evolved to repress genes located in gene-dense regions whose position is restricted to central, active nuclear environments.

overall_design: Refer to individual Series


title: RNA expression profiling for Paf1 complex mutant strains

first: Tim

last: Roloff

type: Expression profiling by genome tiling array

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


title: ADAR1 is required for differentiation and neural induction by regulating microRNA processing in a catalytically independent manner (iCLIP)

city: Shanghai

first: Li

last: Yang

address: 131 Dong-An Road

department: Institutes of Biological Sciences

institute: Fudan University

summary: We used iCLIP to identify ADAR1 binding sites in embryonic stem cells.

overall_design: iCLIP in ADAR1 KD H9 cells infected with Flag tagged ADAR1 or ADAR1-E912A.

first: Ling-Ling

last: Chen

first: Tian

first: Jian-Feng

last: Xiang

first: Qing-Fei

last: Yin

first: Shanshan

last: Zhu

first: Xiao-Ou

last: Zhang


title: Inhibition of transcriptional elongation in the P5424 T cell line

first: Mohamed

last: BELHOCINE

country: France

summary: We study the impact of inhibiting Pol II-Se2 phosphorylation on Pol II binding and histone modifications in the mouse T cell line P5424.

overall_design: Genome-wide analysis via ChIP-Seq for total RNA Pol II binding, H3K4me1, H3K4me2 and H3K4me3 in murine P5424 cells with tow conditions: KM05283 treatment (Inhibitor of Pol II Ser2 phosphorylation) or control DMSO.

first: Joaquin

last: Zacarias-Cabeza

last: Belhocine

first: Aurélien

last: Griffon

first: Laurent

last: Vanhille

first: Romain

last: Fenouil

first: Marta

last: Gut

first: Ivo

first: Jean-Christophe

last: Andrau

first: Salvatore

last: Spicuglia


title: The IDH2 mutation cooperates with the NPM1 mutation to activate Hoxa9/Meis1 and hypoxia pathways in acute myeloid leukemia

city: Cho-ku

first: yoko

last: ogawara

state: Tokyo

address: 5-1-1, Tsukiji

country: Japan

institute: National Cancer Center Research Institute

type: Methylation profiling by high throughput sequencing


title: LED, a long non-coding RNA activator of enhancer RNAs, is hypermethylated in human cancers

city: Amsterdam

first: Koos

last: Rooijers

address: Plesmanlaan 121

department: Gene Regulation

country: Netherlands

institute: Netherlands Cancer Institute

summary: LED, a long non-coding RNA induced by Nutlin-3a, was found to activate enhancer RNAs, one of which was found in an intron of CDKN1A (p21).

overall_design: Impact of expression of LED was assessed in MCF7 cells using RNAseq and GROseq. Chromatin binding of LED was assessed using ChIRPseq.

first: Nicolas

last: Léveille

first: Carlos

last: Melo

first: Angel

last: Díaz

first: Sonia

first: Patrick

last: Wijchers

first: Geert

last: Geeven

first: Gözde

last: Korkmaz

first: Rui

last: Lopes

first: Farhad

last: Moqadam

first: Monique

middle: L

last: den Boer

first: Raghu

last: Kalluri

first: Wouter

last: de Laat

first: Manel

last: Esteller

first: Reuven

last: Agami


title: TRIM24 suppresses development of spontaneous hepatic lipid accumulation and hepatocellular carcinoma in mice

city: HOUSTON

first: Michelle

middle: C.

last: Barton

state: TEXAS

address: 6767 Bertner St. Unit Number: 1000 Room Number: S9.8116B

department: Department of Epigenetics and Molecular Carcinogenesis,

institute: The University of Texas MD Anderson Cancer Center

summary:
Purpose: berrantly high expression of TRIM24 occurs in human cancers, including hepatocellular carcinoma. In contrast, TRIM24 in the mouse is reportedly a liver-specific tumor suppressor. To address this dichotomy and uncover direct regulatory functions of TRIM24 in vivo, we developed a new mouse model that lacks expression of all Trim24 isoforms, as the previous model expresses normal levels of Trim24 lacking only exon 4.
Methods: To produce germline-deleted Trim24dlE1 mice, deletion of the promoter and exon 1 of Trim24 was induced in Trim24LoxP mice by crossing with a zona pellucida 3-Cre line for global deletion. Liver-specific deletion (Trim24hep) was achieved by crossing with an Albumin-Cre line. Phenotypic analyses were complemented by protein, gene-specific and global RNA expression analyses and quantitative chromatin immunoprecipitation.
Results:Global loss of Trim24 disrupted hepatic homeostasis in 100% of mice with highly significant, decreased expression of oxidation/reduction, steroid, fatty acid and lipid metabolism genes, as well as increased expression of genes in unfolded protein, endoplasmic reticulum stress and cell cycle pathways. Trim24dlE1/dlE1 mice have markedly depleted visceral fat and, like Trim24hep/hep mice, spontaneously develop hepatic lipid-filled lesions, steatosis, hepatic injury, fibrosis and hepatocellular carcinoma.
Conclusions: TRIM24, an epigenetic co-regulator of transcription, directly and indirectly represses hepatic lipid accumulation, inflammation, fibrosis and damage in the murine liver. Complete loss of Trim24 offers a model of human nonalcoholic fatty liver disease, steatosis, fibrosis and development of hepatocellular carcinoma in the absence of high-fat diet or obesity.

overall_design: mRNA profiles of 8 weeks wild type (WT) and Trim24-/- mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000

first: Zeynep

middle: H

last: Coban Akdemir


title: The role of Tet3 in regulating gene expression in response to global synaptic activity changes

city: Baltimore

first: Jaehoon

last: Shin

state: Maryland

address: 733 N Broadway

institute: Johns Hopkins University

summary: Counter to the long-held belief that DNA methylation of terminally differentiated cells is permanent and essentially immutable, post-mitotic neurons exhibit extensive DNA demethylation. The causal role of active DNA demethylation in neurons, however, is not known. Tet family proteins oxidize 5-methylcytosine to initiate active DNA demethylation through the base-excision repair pathway. Here, we show that synaptic activity bi-directionally regulates neuronal Tet3 expression. Functionally, knockdown of Tet or inhibition of base-excision repair in hippocampal neurons elevates excitatory glutamatergic synaptic transmission, whereas overexpressing Tet3 or Tet1 catalytic domain decreases it. Furthermore, dysregulation of Tet3 signalling prevents homeostatic synaptic plasticity. Mechanistically, Tet3 dictates neuronal surface GluR1 levels. RNA-seq analyses further revealed a pivotal role of Tet3 in regulating gene expression in response to global synaptic activity changes. Thus, Tet3 serves as a synaptic activity sensor to epigenetically regulate basic properties and meta-plasticity of neurons via active DNA demethylation.

overall_design: Bicuculin, Tetradotoxin, shTet3

first: Hongjun

last: Song

first: Guo-li

last: Ming

first: Giovanni

last: Coppola


title: Active repression of Sox9 by Jag1 is required for silencing the default chondrogenic fate of the vascular smooth muscle wall

city: Los Angeles

first: Anais

last: Briot

state: CA

address: 621 Charles E. Young Drive, South

department: Molecular, Cell and Developmental Biology

institute: University of California, Los Angeles


title: Active repression of Sox9 by Jag1 is required for silencing the default chondrogenic fate of the vascular smooth muscle wall [set 1]

summary: Acquisition and maintenance of vascular smooth muscle fate is essential for the morphogenesis and function of the circulatory system. Loss of contractile properties or changes in the identity of vascular smooth muscle cells (vSMC) can result in structural alterations associated with aneurysms and vascular wall calcifications. Here we report that maturation of sclerotome-derived vSMC is dependent on a transcriptional switch between mouse embryonic days 13 and 14.5. At this time point, Jag1-mediated repression of sclerotome transcription factors Pax1, scleraxis and Sox9 is necessary to fully enable vSMC maturation. Specifically, Notch signaling in vSMC antagonizes sclerotome and cartilage transcription factors, and promotes upregulation of contractile genes. In the absence of Jag1, vSMC acquire a chondrocytic transcriptional repertoire that can lead to ossification of the vascular wall. Importantly, our findings suggest that sustained Notch signaling is essential throughout vSMC life to maintain contractile function, prevent vSMC reprogramming and promote vascular wall integrity.

overall_design: mRNA profile of vascular Smooth Muscle Cells, isolated from the descending aorta of Immorto mouse, treated or not with gamma-secretase inhibitor  was generated by  deep sequencing, in triplicate.

first: Artur

last: Jaroszewicz

first: Jing

last: Lu

first: Matteo

last: Pellegrini

first: M

last: Iruela-Arispe


title: Rat pineal gland RNA-Seq PolyA project

city: Bethesda

first: Steven

last: Coon

state: MD

address: 49 Convent Drive Room 6A83

department: NICHD/PDEGEN

institute: NIH

summary: The rat pineal gland is a highly dynamic tissue with many hundreds of genes changing more than two-fold in a 24-hr daily rhythm as measured by Affymetrix GeneChip analysis. We sought to more completely understand this dynamic transcriptome using RNA-Seq in order to capture information regarding alternative splicing, novel exons, unannotated mRNAs, non-coding RNAs, etc. We also wished to identify transcripts that were selectively expressed in the pineal glands relative to other tissues. Toward this end we performed RNA-Seq on three types of samples; 1) a pool of pineal glands sampled at mid-day (ZT7); 2) a pool of pineal glands sampled at mid-night (ZT19); and a pool of 15 different tissues collected from 3 animals at mid-day (ZT7). Animals were housed in a 14:10 light:dark lighting cycle. PolyA-selected RNA was fragmented and sequenced on an Illumina GAII machine, yielding paired-end 51-mer reads.

overall_design: The rat pineal transcriptome was measured from mid day and midnight samples and compared to mixed non-pineal tissues.

first: Cong

last: Fu

first: Samuel

last: Clokie

first: Peter

last: Munson

first: Zoila

last: Rangel

first: David

middle: C

last: Klein


title: Active repression of Sox9 by Jag1 is required for silencing the default chondrogenic fate of the vascular smooth muscle wall [set 2]

overall_design: mRNA profile of vSMC from the descending aorta of  14.5 embryos Wild type (WT), SMC Jag1-heterozygous (HTZ) and SMC Jag1-null (KO) was generated by  deep sequencing, in duplicate.


title: RNA-seq and small RNA-seq from WT and ADAR1 knockdown H9 lines and their differentiation to specific types of neurons

first: Li

last: Yang

summary: Adenosine deaminases acting on RNA (ADARs) are involved in adenosine (A) to inosine (I) RNA editing and human ADARs are implicated in neurological diseases. Here we generated human embryonic stem cells (hESCs) lacking ADAR1 to investigate its role in neural development in a human context. We found that ADAR1 deficiency significantly retarded neural induction with widespread mRNA and miRNA expression changes. We have genome widely examined the changes of  A-to-I editing and miRNA expression after ADAR1 knockdown. Such aberrant mRNA and miRNA expression was not due to reduced A-to-I editing after ADAR1 knockdown. We further revealed that ADAR1 regulates miRNA biogenesis independent of its editing activity.

overall_design: In order to study the function role of ADAR1 in neural induction, we used shRNA to stably knocked down ADAR1 in human embryonic stem cell (hESC) h9 line and then differentiated ADAR1 deficiency hESC h9 cells to specific types of neurons. We then collect total RNAs at different time points at undifferentiated stage (day 0, d0 for short), day 10 (d10), day 17 (d17) and day 35 (d35), and obtained RNA-seq and small RNA-seq datasets for further analyses.

first: Tian

last: Chen

first: Shanshan

last: Zhu

first: Qing-Fei

last: Yin

first: Hua-Hong

last: Fang

first: Ling-Ling


title: Epigenome analysis of human epidermal samples with aging and sun exposure

first: Andrew

last: Feinberg

address: 855 N. Wolfe St

institute: Johns Hopkins University School of Medicine

summary: Genome wide DNA methylation profiling of epidermal samples obtained from sum-exposed and sun-protected body sites from younger (<35 years old) and older (>60 years old) individuals.

overall_design: Bisulphite converted DNA from 12 samples was sequenced using Illumina HiSeq.

first: Amy

last: Vandiver


title: Genome-wide maps of KDM5A/JARID1A/RBP2 and its isoforms in differentiated U937 cells

city: Chicago

first: Elizaveta

middle: V

last: Benevolenskaya

state: IL

address: 900 S. Ashland Ave.

department: Biochemistry and Molecular Genetics

institute: University of Illinois at Chicago

summary: We report the identification of genomic regions bound by RBP2 isoforms and define the functional categories that these regions represent. RBP2 modifies methylated lysine residues on histone tails. We found that RBP2 large isoform containing the recognition module for histone H3K4me3 and RBP2 small isoform lacking this module bind to to different regions in the human genome. Importantly, isoform-specific regions and overlapping regions belong to genes with different molecular functions. For example, chromatin binding and transcription factor binding functions can be ascribed to gene targets of RBP2 small isoform but not of RBP2 large isoform. By comparing gene sets generated for all isoforms and RBP2 large isoform, we can define if the small isoform is specifically recruited to genomic regions dispaying certain signatures, such as transcription start sites, CpG-rich regions, transcriptional activity and transcription factor binding.

overall_design: Examination of location of different isoforms

first: Abul

last: Islam


title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not DNA methylation

last: Oler

address: 31 Center Drive, Room 3B62E

department: Bioinformatics and Computational Biosciences Branch (BCBB)

institute: NIAID/NIH

summary: Nearly half the human genome is comprised of repetitive DNA, including short interspersed nuclear elements (SINEs) such as Alu.  SINEs spread by retrotransposition, which requires their transcripts to be copied into DNA and then inserted into new chromosomal sites.  This can lead to genetic damage through insertional mutagenesis and through chromosomal rearrangements between nonallelic SINEs at distinct loci.  SINE DNA is heavily methylated and this is thought to suppress its accessibility and transcription, thereby protecting against retrotransposition.  However, we provide several lines of evidence that methylated SINE DNA is occupied genome-wide by RNA polymerase III, including the use of high-throughput bisulphite sequencing of ChIP DNA (ChIP-BS-Seq).  Loss of DNA methylation has little effect on expression of SINEs or their accessibility to transcription machinery. We present evidence that methylation of histones rather than DNA plays a dominant role in suppressing SINE expression.

overall_design: RPC155 ChIP DNA was bisulfite-converted prior to sequencing (ChIP-Bis-seq).  A single library was prepared for the ChIP-Bis-seq and two libraries were prepared for Input DNA from the same sonicate.

first: Dhaval

last: Varshney

first: Jana

last: Vavrova-Anderson

first: Bradley

last: Cairns

first: Robert

last: White


title: Transcriptome Differences between Alternative Sex Determining Genotypes in the House Fly, Musca domestica

status: Public on Mar 20 2015

city: Houston

first: Richard

last: Meisel

state: TX

address: 3455 Cullen Blvd

department: Biology and Biochemistry

institute: University of Houston

summary: Sex determination evolves rapidly, often because of turnover of the genes at the top of the pathway. The house fly, Musca domestica, has a multifactorial sex determination system, allowing us to identify the selective forces responsible for the evolutionary turnover of sex determination in action. There is a male determining factor, M, on the Y chromosome (Y^M), which is probably the ancestral state. An M factor on the third chromosome (III^M) has reached high frequencies in multiple populations across the world, but the evolutionary forces responsible for the invasion of III^M are not resolved. To test if the III^M chromosome invaded because of sex-specific selection pressures, we used mRNA sequencing to determine if isogenic males that differ only in the presence of the Y^M or III^M chromosome have different gene expression profiles. We find that more genes are differentially expressed between Y^M and III^M males in testis than head, and that genes with male-biased expression are most likely to be differentially expressed between Y^M and III^M males. This suggests that male phenotypes, especially those related to male fertility, are more likely to be affected by the male-determining chromosome, supporting the hypothesis that sex-specific selection acts on alleles linked to the male-determining locus driving evolutionary turnover in the sex determination pathway. We additionally find that III^M males have a "masculinization" gene expression profile, suggesting that the III^M chromosome has accumulated an excess of male-beneficial alleles because of its male-limited transmission.

overall_design: 3 replicates of testes and male heads from a Y^M and a III^M strain, and one sample of head and ovary from each of the strains

first: Jeffrey

middle: G

last: Scott

last: Clark


title: Cytosolic Accumulation of Small Nucleolar RNAs (snoRNAs) is Dynamically Regulated by NADPH Oxidase

city: Durham

first: Christopher

last: Holley

state: NC

address: 2301 Erwin Rd

institute: Duke University

summary: Small nucleolar RNAs (snoRNAs) guide nucleotide modifications of cellular RNAs in the nucleus. We have previously shown that box C/D snoRNAs from the Rpl13a locus are unexpected mediators of physiologic oxidative stress, independent of their predicted ribosomal RNA modifications. Here, we demonstrate that oxidative stress induced by doxorubicin causes rapid cytoplasmic accumulation of the Rpl13a snoRNAs through a mechanism that requires superoxide and a nuclear splice variant of NADPH oxidase. RNA-sequencing analysis reveals that box C/D snoRNAs as a class are present in the cytoplasm, where their levels are dynamically regulated by NADPH oxidase. These findings suggest that snoRNAs may orchestrate the response to environmental stress through molecular interactions outside of the nucleus.

overall_design: Cytosolic RNA was isolated from H9c2 cells by digitonin permeabilization of the plasma membrane and differential centrifugation. Cells were treated as follows: control, DPI (Nox inhibitor), doxorubicin (dox), or DPI+dox. N=3 independent experiments were analyzed for each treatment (12 samples total).

first: Scot

last: Matkovich

first: Jean

middle: E

last: Schaffer


title: Epigenome analysis of human epidermal and dermal samples with aging and sun exposure.

type: Methylation profiling by genome tiling array


title: G9a, ZNF644 and WIZ ChIP-seq results

city: Duarte

first: Xiaochun

last: Yu

address: 1500 E. Duarte Rd, Rm 202, Lippman-Graff Bldg

department: Internal Medicine

institute: city of hope

summary: The G9a mediates mono- and dimethylation of Lys9 of histone H3 at specific gene loci, which is associated with transcriptional repression. ZNF644 and WIZ contain multiple zinc finger motifs that recognize consensus DNA sequences.

overall_design:
The molecular mechanism by which the G9a is targeted to the specific gene loci for H3K9 methylation is unclear, as G9a can't specifically recognize genomic DNA. Here, with unbiased protein affinity purification, we found that ZNF644 and WIZ are two binding proteins of G9a. Both ZNF644 and WIZ contain multiple zinc finger motifs that recognize consensus DNA sequences. Thus, ZNF644 and WIZ target G9a to the chromatin, and mediate the G9/GLP complex-dependent H3K9 methylation as well as gene repression.

Please note that the data files (both raw and processed) have been updated on Sep 22, 2016.

first: Chunjing

last: Bian


title: METTL3 HITS-CLIP

city: New York

first: Claudio

last: Alarcon

state: New York

address: 1230 York Ave

department: Systems Cancer Biology

institute: The Rockefeller University

summary: To identify METTL3 binding sites on endogenous nuclear RNAs, we performed HITS-CLIP endogenous METTL3

overall_design: Wild type Hela Cells cells were subjected to the HITS-CLIP procedure (Licatalosi D, et al. 2008, Nature 456:464-U22)

first: Cludio

first: Hyeseung

last: Lee

first: Hani

last: Goodarzi

first: Sohail

middle: F

last: Tavazoie


title: H3K9me3 ChIP-seq from Neurospora crassa wild type (WT) and dim-3 (severely reduced H3K9me3 levels) strains

status: Public on Mar 22 2015

city: Colorado Springs

first: Andrew

middle: David

last: Klocko

state: Colorado

address: 278 Centennial Hall, 1420 Austin Bluffs Pkwy

department: Chemistry and Biochemistry

institute: University of Colorado Colorado Springs

summary: We report the placement of the H3K9me3 heterochromatin mark from the filamentous fungus Neurospora crassa in wild type and dim-3 strains.  The dim-3 strain has a global reduction in H3K9me3, which results from a causative mutation in importin alpha (NUP-6), a component of the nuclear transport machinery. NUP-6(dim-3) compromises the heterochromatic localization of several components of the DCDC, a histone H3K9 methyltransferase complex, from their sub-nuclear chromatin targets despite appropriate nuclear transport. To determine whether the reduced level of global H3K9me3 observed from isolated histones localizes to A:T rich DNA (genomic DNA destined to become heterochromatin), we performed H3K9me3 ChIP-seq on a dim-3 strain and compared that to a wild type strain.

overall_design: Examination of H3K9me3 in two different strains

middle: D

first: Michael

last: Rountree

first: Eric

middle: U

last: Selker


title: Optimization of miRNA-seq Data Pre-Processing

status: Public on Mar 21 2015

city: Toronto

first: Shirley

last: Tam

state: Ontario

address: 661 University Avenue, Suite 510, MaRS Building, West Tower

department: Genome Technologies

country: Canada

institute: Ontario Institute for Cancer Research

summary: Next-generation sequencing is currently the platform of choice for the discovery and quantification of miRNAs.  Despite this, there is no clear consensus on how the data should be pre-processed prior to conducting downstream analyses.  Often overlooked, data pre-processing is an essential step in data analysis: the presence of unreliable features and noise can affect the conclusions drawn from downstream analyses.  Using a spike-in dilution study, we evaluated the effects of several general-purpose aligners (BWA, Bowtie, Bowtie 2 and Novoalign), and normalization methods (counts-per-million, total count scaling, upper quartile scaling, Trimmed Mean of M, DESeq, linear regression, cyclic loess and quantile) with respect to the final miRNA count data distribution, variance, bias and accuracy of differential expression analysis.

overall_design: A spike-in dataset was created using a 12x12 cyclic Latin Square design.  Twelve miRNAs from the Arabidopsis Thaliana genome that are not present in the human genome were selected as spike-in sequences.  RNA oligonucleotides were synthesized with phosphorylated 5’ ends and added at 12 different concentrations (0, 0.1, 0.2, 0.8, 1.6, 6.4, 12.8, 51.2, 102.4, 409.6, 819.2, 3276.8 amol) to 1μg Universal Human Reference RNA, with each concentration appearing once in each row and column of the design matrix.

first: Ming-Sound

last: Tsao

first: John

last: McPherson


title: SC3-seq: a method for highly parallel and quantitative measurement of single-cell gene expression.

city: Kyoto

first: Yukihiro

last: Yabuta

state: Kyoto

address: Yoshida-Konoe-cho, Sakyo-ku

department: Anatomy and Cell Biology

institute: Kyoto University, Graduate school of medicine

summary: Single-cell mRNA sequencing (RNA-seq) methods have undergone rapid development in recent years, and transcriptome analysis of relevant cell populations at single-cell resolution has become a key research area of biomedical sciences. We here present single-cell mRNA 3-prime end sequencing (SC3-seq), a practical methodology based on PCR amplification followed by 3-prime-end enrichment for highly quantitative, parallel and cost-effective measurement of gene expression in single cells. The SC3-seq allows excellent quantitative measurement of mRNAs ranging from the 10,000-cell to 1-cell level, and accordingly, allows an accurate estimate of the transcript levels by a regression of the read counts of spike-in RNAs with defined copy numbers. The SC3-seq has clear advantages over other typical single-cell RNA-seq methodologies for the quantitative measurement of transcript levels and at a sequence depth required for the saturation of transcript detection. The SC3-seq distinguishes four distinct cell types in the peri-implantation mouse blastocysts. Furthermore, the SC3-seq reveals the heterogeneity in human-induced pluripotent stem cells (hiPSCs) cultured under on-feeder as well as feeder-free conditions, demonstrating a more homogeneous property of the feeder-free hiPSCs. We propose that SC3-seq might be used as a powerful strategy for single-cell transcriptome analysis in a broad range of investigations in biomedical sciences.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design:
Examination of SC3-seq form 5 serially diluted mESC total RNA.
Examination of 38 individual blastocyst single cells
Examination of hiPSC on SNL or feeder-free at single cell level.

first: Tomonori

last: Nakamura

first: Ikuhiro

last: Okamoto

first: Shinya

last: Aramaki

first: Shihori

last: Yokobayashi

first: Kazuki

last: Kurimoto

first: Kiyotoshi

last: Sekiguchi

first: Masato

last: Nakagawa

first: Takuya

last: Yamamoto

first: Mitinori

last: Saitou


title: RNAseq comparison of gene expression profiles in ScxGFP positive cells and ScxGFP negative cells from E13.5 forelimb samples

status: Public on Mar 19 2015

city: cincinnati

first: HAN

last: LIU

state: oh

address: 3333 burnet avenue

department: DEVELOPMENTAL BIOLOGY

institute: CINCINNATI CHILDREN'S HOSPITAL

summary: RNAs were isolated from FACS sorted ScxGFP positive cells and GFP negative cells of forelimbs at E13.5, and characterized by RNAseq

overall_design: for each sample, 3 pairs of total 6 E13.5 forelimbs were pooled together, both GFP positive cells and GFP negative cells were FACS sorted, then for RNA extraction, cDNA library preparation and proceeded for RNAseq

first: Jiang

last: Rulang

first: Liu

last: Han


title: Ptf1a promotes an inhibitory peptidergic identity in cortical pyramidal cells in vivo

city: New york

first: Julia

last: Kaltschmidt

address: 1275 York Av

institute: Sloan-Kettering Institute

summary: Ptf1a has been shown to be necessary for determining an inhibitory interneuronal fate in many regions of the nervous system. In this study, we aim to investigate the sufficiency of Ptf1a to cell-autonomously promote a specific neuronal identity by misexpressing it in developing excitatory cortical pyramidal cells and studying its impact on pyramidal cell features, such as its gene expression profile. To accomplish this, we electroporate either a Ptf1a/GFP-misexpression construct or a GFP-only control construct into E12.5 mouse embryonic cortices, harvest the cortices at E15.5, and examine Ptf1a-induced changes to the pyramidal cell transcriptome, molecular expression pattern, neurotransmitter status, and morphology. We conclude that Ptf1a is sufficient to cell-autonomously promote an inhibitory peptidergic identity and alter neuronal morphology in developing cortical pyramidal cells. The results of this study provide insight into intrinsic transcriptional controls over neuronal identity, specifically implicate Ptf1a as a potent regulator of an inhibitory peptidergic identity, and may guide future studies of neuronal reprogramming for circuit repair after disease or injury.

overall_design: Purified GFP-expressing cells were isolated from mouse embryonic cortices electroporated with either a Ptf1a/GFP-misexpressing construct or a GFP-only control construct. 3-6 brains were collected per sample, yielding 58-106,000 cells per sample. Ptf1a/GFP-misexpressing samples were run in triplicate, GFP-only control samples were run in duplicate.

first: Jeffrey

last: Russ

first: Mark

last: Borromeo

first: Rahul

last: Kollipara

first: Praveen

last: Bommareddy

first: Jane

last: Johnson


title: The effect of Ezh2 knockdown in high-grade glioma

first: Nienke

last: de Vries

address: Plesmanlaan 121

institute: NKI

summary: To provide further insight about the effects of prolonged Ezh2 inhibition in glioblastoma using preclinical mouse models and doxycycline-inducible shRNAs that mimic the effects of a selective EZH2 inhibitor. We demonstrate that prolonged Ezh2-depletion causes a robust switch in cell fate, including significantly enhanced proliferation and DNA damage repair and activation of part of the pluripotency network, resulting in altered tumor cell identity and tumor progression.

overall_design: SVZ derived neural stem cells (NSCs) were isolated from 7 days old p53;Ink4a/Arf;Krasv12;LucR compound conditional mice and cultured in NSC specific serum-free medium supplemented with 20ng/ml of both EGF and bFGF (R&D systems). NSCs were grown adhesion-free for the first passages to eliminate non-sphere-forming cells. Next, cells were grown adherent on poly-L-Ornithine and Laminin plates and three times infected with lentiviral CMV-Cre. These floxed, tumorigenic cells are further referred as glioma initiating cells (GICs). Next, GICs were infected with a tet-inducible, doxycycline-responsive short hairpin construct (FH1-tUTG-shEzh2). After FACS sorting for GFP, GICs were injected intracranial in NOD-SCID mice and treated with or without doxycycline in the drinking water

first: Iris

last: de Rink

first: Maarten

last: van Lohuizen


title: RNAseq of Individual 4T1 Clonal Populations

city: Cold Spring Harbor

first: Simon

middle: Robert Vincent

last: Knott

state: NY

address: 1 Bungtown Rd

department: Biology

institute: Cold Spring Harbor Laboratory

summary: The transcriptional profile 23 cell lines derived from single clones of the 4T1 cell lines were assessed with RNAseq. The two clones with a strong propensity to intravasate were found to have 12 genes in common that were overexperessed relative to the other 21 clones.

overall_design:
Clone RNAseq
1) 23 clonal lines were established using single cell FACs sorting from the 4T1 mammary cancer cell line.
2) After establishing the lines the clones were assesed (in a pooled setting) for their capacity to intravasate the vascular system.
3) Transcriptional profiling was carried out using RNAseq.
4) Two clones were found to be strong intravasators and these were compared to the other clones to identify genes that were overexpressed (as compared to at least half of the other clones in both lines).

middle: R


title: Direct reprogramming of human mesenchymal stem cells into functional renal cells using cell-free extracts

city: Ranica

first: Paraskevas

last: Iatropoulos

state: BG

address: Via G.B. Camozzi, 3

country: Italy

institute: IRCCS Istituto di Ricerche Farmacologiche Mario Negri

summary: We investigated here the reprogramming of human bone marrow mesenchymal stem cells (MSC) using cell-free extracts from renal proximal tubular epithelial cells (HK2). Reprogramming of MSC is evidenced by a dramatic change in the cell structure with the formation of microvilli, the expression of specific markers and the acquisition of functional properties characteristic of the proximal tubular epithelial cells.  To better characterize the MSC treated with HK2 extracts, we next isolated clones using a limiting dilution approach. Five out of 50 clones, showing the most epithelial-like morphology, were subjected to further analysis by qRT-PCR to examine their mesenchymal and epithelial marker profile. The difference in the expression of these markers reflects an heterogeneity in the reprogramming efficiency. Cells from clone 17 (CL17) were the most similar to the HK2 cells.  RNA sequencing of CL17, MSC and HK2 cells was performed in order to evaluate the global effect of MSC reprogramming with cell extracts. RNA sequencing confirmed a switch of the reprogrammed cells toward a proximal tubular epithelial genotype along the EGF receptor pathway.

overall_design: CL 17 cells were analyzed in comparison with MSC and HK2 cells. Analysis was performed in duplicates.

first: Evangelia

last: Papadimou

first: Marina

last: Morigi

first: Christodoulos

last: Xinaris

first: Susanna

last: Tomasoni

first: Valentina

last: Benedetti

first: Lorena

last: Longaretti

first: Cinzia

last: Rota

first: Marta

last: Todeschini

first: Paola

last: Rizzo

first: Martino

last: Introna

first: Giuseppe

last: Remuzzi

first: Michael

middle: S

last: Goligorsky

first: Ariela

last: Benigni


title: RNAseq comparison of gene expression profiles in ScxGFP positive cells from E11.5, E13.5 and E15.5 hindlimb samples

first: HAN

last: LIU

summary: RNAs were isolated from FACS sorted ScxGFP positive cells of hindlimbs at E11.5, E13.5 and E15.5, and characterized by RNAseq

overall_design: 18 hindlimbs, 12 hindlimbs and 11 hindlimbs were pooled together for E11.5, E13.5 and E15.5, respectively.GFP positive cells were FACS sorted, then for RNA extraction, cDNA library preparation and proceeded for RNAseq

first: Jiang

last: Rulang

first: Liu

last: Han


title: limb tendon RNAseq

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

overall_design: Refer to individual Series


title: Quantitative Dynamics of Chromatin Remodeling during Germ Cell Specification from Mouse Embryonic Stem Cells

first: Yukihiro

last: Yabuta

summary: Germ cell specification is accompanied by epigenetic remodeling, the scale and specificity of which are unclear. Here, we quantitatively delineate chromatin dynamics during induction of mouse embryonic stem cells (ESCs) to epiblast-like cells (EpiLCs) and from there into primordial germ cell-like cells (PGCLCs), revealing large-scale reorganization of chromatin signatures including H3K27me3 and H3K9me2 patterns. EpiLCs contain abundant bivalent gene promoters characterized by low H3K27me3, indicating a state primed for differentiation. PGCLCs initially lose H3K4me3 from many bivalent genes but subsequently regain this mark with concomitant upregulation of H3K27me3, particularly at developmental regulatory genes. PGCLCs progressively lose H3K9me2, including at lamina-associated perinuclear heterochromatin, resulting in changes in nuclear architecture. T recruits H3K27ac to activate BLIMP1 and early mesodermal programs during PGCLC specification, which is followed by BLIMP1-mediated repression of a broad range of targets, possibly through recruitment and spreading of H3K27me3. These findings provide a foundation for reconstructing regulatory networks of the germline epigenome.

overall_design: Examination of 4 different histone modifications and 1 transcription factor in 5 cell types.

first: Kazuki

last: Kurimoto

first: Katsuhiko

last: Hayashi

first: Hiroshi

last: Ohta

last: Kiyonari

first: Tadahiro

last: Mitani

first: Yoshinobu

last: Moritoki

first: Kenjiro

last: Kohri

last: Kimura

first: Takuya

last: Yamamoto

first: Yuki

last: Kato

last: Shirahige

first: Mitinori

last: Saitou


title: MEF2D drives photoreceptor development through a genome-wide competition for tissue-specific enhancers

city: Seattle

first: Timothy

middle: J

last: Cherry

state: WA

department: Developmental Biology and Regenerative Medicine

institute: Seattle Children's Research Institute


title: MEF2D drives photoreceptor development through a genome-wide competition for tissue-specific enhancers (ChIP-Seq)

summary: Organismal development requires the precise coordination of genetic programs to regulate cell fate and function. MEF2 transcription factors play essential roles in this process but how these broadly expressed factors contribute to the generation of specific cell types during development is poorly understood.  Here we show that despite being expressed in virtually all mammalian tissues, in the retina MEF2D binds to retina-specific enhancers and controls photoreceptor cell development. MEF2D achieves specificity by cooperating with a retina-specific factor CRX, which recruits MEF2D away from canonical MEF2 binding sites, and redirects it to retina-specific enhancers that lack the consensus MEF2 sequence.  Once bound to retina-specific enhancers, MEF2D and CRX co-activate the expression of photoreceptor-specific genes that are critical for retinal function. These findings demonstrate that broadly expressed TFs acquire specific functions through competitive recruitment to enhancers by tissue-specific TFs, and through cooperative activation of these enhancers to regulate tissue-specific genes.

overall_design: ChIP-seq for MEF2D in WT, Mef2d KO and Crx KO retinae and WT cultured cortical neurons.  ChIP for H3K27Ac in WT and Mef2d KO retinae

first: Milena

middle: M

last: Anzelm

last: Greenberg


title: m6A-seq nuclear RNA

first: Claudio

last: Alarcon

summary: To analyze the nuclear RNAs modified by the m6A mark

overall_design: Nuclear RNA was extracted from MDA-MB-231 cells and was immunoprecipitated with an antibody against m6A or an IgG control

first: Cludio

first: Hyeseung

last: Lee

first: Hani

last: Goodarzi

first: Sohail

last: Tavazoie


title: MEF2D drives photoreceptor development through a genome-wide competition for tissue-specific enhancers (RNA-Seq)

overall_design: Total RNA-seq data from WT, Mef2d KO and Crx KO P11 mouse retinae

first: Michael


title: G-protein coupled receptor 3 (cAMP-receptor like) regulating genes involved in blastospore production of entomopathogen fungi Beauveria bassiana

city: Hangzhou

first: Shenghua

last: Ying

state: Zhejiang

address: Yu-hang-tang Road 688

institute: Zhejiang University

summary: G-protein coupled receptor 3 (BbGPCR3) played crucial roles in mediating blastospore production, a determinant to the biocontrol potential of fungal entomopathogens. We characterized for the first time that disruption of BbGPCR3 reduced  the blastospore production in glucose-based rich medium (Sabouraud dextrose broth, SDB), but the blastospore production of disruption mutant could be recovered in trehalose-modified SDB. The global transcriptome  related to blastospore production was analyzed by using high throughput sequencing (RNA-Seq). Our transcriptional profiles revealed that numerous differentially expressed genes (DEGs), of which involved in cell process, signal transduction, and stress response, were significantly involved in blastospore production.

overall_design: 1. Total RNA obtained from BbGPCR3 disruption mutant were compared to the wild type strain in glucose-based rich medium (SDB); 2. Total RNA obtained from BbGPCR3 disruption mutant in trehalose-modified SDB were compared to that of disruption mutant in SDB.

first: Sheng-Hua

first: Ming-Guang

last: Feng


title: Arabidopsis De-etiolated 1 Represses Photomorphogenesis by Positively Regulating Phytochrome Interacting Factors in the Dark

city: Beijing

first: Xu

middle: Zhao

last: Gao

state: Beijing

address: The Summer Palace Road No. 5

department: College of Biological Sciences

institute: Peking University

summary: Arabidopsis seedlings undergo photomorphogenic development even in darkness when the function of De-etiolated 1 (DET1), a repressor of photomorphogenesis, is disrupted. Our results indicate that DET1 directly interacts with a group of transcription factors known as the phytochrome-interacting factors (PIFs). Furthermore, our results suggest that DET1 positively regulates PIF protein levels primarily by stabilizing PIF proteins in the dark. Genomic analysis also revealed that DET1 may control the expression of light-regulated genes to mediate photomorphogenesis partially through PIFs.

overall_design: Total of twelve samples, two treatments and three genotypes, and each have three replicates.

first: Jie

last: Dong

first: Dafang

last: Tang

first: Zhaoxu

first: Renbo

last: Yu

first: Kunlun

last: Li

first: Hang

last: He

first: William

last: Terzaghi

first: Xing

middle: W

last: Deng

first: Haodong

last: Chen


title: Epigenetic Memory Is Not Intrinsic To Transcription Factor-Mediated Reprogramming [Bisulfite-Seq]

status: Public on Mar 18 2015

city: Duesseldorf

first: Ulf

last: Tiemann

address: Moorenstrasse 5

department: Institute for Stem Cell Research and Regenerative Medicine

institute: Heinrich Heine University Duesseldorf

summary: Somatic cells can be reprogrammed to pluripotency using different methods. In comparison to pluripotent cells obtained through somatic nuclear transfer, induced pluripotent stem cells (iPSCs) exhibit a higher number of epigenetic errors. Furthermore, most of these abnormalities have been described to be intrinsic to the iPSC technology. Here we investigate whether the aberrant epigenetic patterns detected in iPSCs are specific to transcription factor-mediated reprogramming. We used germline stem cells (GSCs), which are the only adult cell type that can be converted into pluripotent cells (gPSCs) under specific culture conditions, and compared GSC-derived iPSCs and gPSCs at the transcriptomic, epigenetic and functional level. Our results show that both reprogramming methods generate indistinguishable states of pluripotency. GSC-derived iPSCs and gPSCs retained similar levels of donor cell-type memory and exhibited comparable numbers of reprogramming errors. Therefore, our study demonstrates that the epigenetic memory detected in iPSCs is not intrinsic to transcription factor-mediated reprogramming.

overall_design: Total DNA treated for Reduced Representation Bisulfite Sequencing (RRBSeq) from 6 different in vitro mouse cell lines: germline stem cells (GSCs), GSC-derived induced pluripotent stem cells (iPSCs, 2 independent cell lines), germline-derived pluripotent stem cells (gPSCs, 2 independent cell lines), embryonic stem cells (ESCs)

first: Guangming

last: Wu

first: Adele

last: Marthaler

first: Hans

last: Schöler

first: Natalia

last: Tapia


title: Comparative genomics reveals Chd1 as a determinant of nucleosome spacing in vivo

city: Worcester

first: Oliver

last: Rando

state: MA

address: 364 Plantation St.

institute: UMass Medical School

summary: We generated S.cerevisiae strains in which endogenous copies of candidate nucleosome spacing factors were replaced with the K.lactis copies. With this candidate approach, we found that K.lactis Chd1 directed longer nucleosome repeat length in S.cerevisiae. Generating chimeric proteins revealed that the strongest contribution to this differential spacing lies in the undercharacterised N-terminus of Chd1.

overall_design: nucleosome mapping for S.cerevisiae hho1, ioc3isw1, and chd1 deletion mutants and complemented with the K.lactis copies

first: Amanda

middle: L

last: Hughes


title: Arabidopsis shade avoidance gene expression in adult plants

city: Davis

first: Kazunari

last: Nozue

state: California

address: One Shields Avenue

department: Plant Biology

institute: University of California Davis

summary: The goal of this study is to obtain genome-wide shade-responsive genes in adult plant. We grew plants under long-day condition with high R/FR ratio (simulated "sun" condition) for two weeks. Plants were treated with shade starting at ZT 4 or left in the sun. We prepared two replicates of each sample at 1 hour and 4 hours after sun and shade treatment and five plants were pooled for each replicate. Cotyledons, hypocotyls, and roots were removed from the samples, leaving leaves and apical tissue.

overall_design: SRA Study accession (SRP028684) and BioProject accession (PRJNA214254)

first: Julin

middle: N

last: Maloof


title: Epigenetic Memory Is Not Intrinsic To Transcription Factor-Mediated Reprogramming


title: The expression pattern of microRNAs in granulosa cells of subordinate and dominant follicles during the early luteal phase of bovine estrous cycle

city: Bonn

first: Dessie

last: Salilew-Wondim

state: NRW

address: Endenicher Allee 15

department: Animal breeding and husbandry group

institute: University of Bonn

summary: This study was conducted to investigate the miRNA enrichment and degradation in the granulosa cells of dominant and subordinate follicle during the early luteal phase of the bovine estrous cycle using high throughput miRNA sequencing technology.

overall_design: We used follicle samples retrieved from ovaries obtained from Simmental heifers slaughtered at day 3 and 7 of the estrous cycle. The follicles were categorized as subordinate (SF) and dominant (DF) based on their size. The granulosa cells were collected from each follicle and subjected to total RNA isolation .The RNA samples used for miRNAs deep sequencing. A total of 12 granulosa samples (three biological replicates of granulosa cells from SF or DF at day 3 or day 7 of the estrous cycle) were used for miRNA deep sequencing.

first: D

first: A

last: Ijaz

first: S

last: Sudeep

last: Gebremedhn

first: F

last: Rings

first: M

last: Hoelker

first: E

last: Tholen

first: N

last: Christiane

first: C

last: Looft

first: K

last: Schellander

last: Tesfaye


title: Differential expression of miRNAs in granulosa cells of bovine preovulatory dominant and subordinate follicles

city: Fort Collins

first: Samuel

middle: Gebremedhn

last: Etay

state: CO

address: 1683 Campus Delivery

department: Biomedical Sciences

institute: Colorado State University

summary: The study aimed to uncover differential expression pattern of regulatory microRNAs in bovine granulosa cells  derived from  preovulatory dominant and subordinate  follicles.

overall_design: We used ovarian follicle samples  of experimental heifers slaughtered at day 19 of the estrous cycle .  Follicles were catagorized as preovulatory dominant follicles (PDF)  and  anovulatory subordinate  follicles (SF).  The granulosa cells were collected from each follicle and subjected to microRNA enriched  total RNA isolation and used for miRNAs deep sequencing. A total of 6 samples (three biological replicated  from PDF and 3  biological replicates from SF) were used for miRNA deep sequencing.


title: Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice

city: Tarrytown

first: Rene

middle: Christian

last: Adam

address: 777 Old Saw Mill River Road

institute: Regeneron Pharmaceuticals

summary: Mouse hair follicles undergo synchronized cycles. Cyclical regeneration and hair growth is fueled by hair follicle stem cells (HFSCs) and transit-amplifying cells (TACs). We used ChIP-seq to unfold genome-wide chromatin landscapes of H3K27ac and Med1 to identify super-enhancers and dissect their biological relevance in cell identity and plasticity of HFSCs in vivo and in vitro.

overall_design: Telogen quiescent hair follicle stem cells (HFSCs) and transit-amplifying cells (TACs) were FACS-purified for ChIP-sequcencing. Furthermore, cultured HFSCs were used for ChIP-sequencing.

middle: C

first: Elaine

last: Fuchs


title: Genomic-scale identification of host genes regulated by EBV during lytic cycle [RNA-Seq]

city: Brighton

first: Alison

middle: Jane

last: Sinclair

state: E. Sussex

address: University of Sussex

department: School of Life Sciences

country: United Kingdom

institute: University of Sussex

summary: Infection of resting primary B-lymphocytes by Epstein Barr virus (EBV) generates a population of cells that are effectively immortal. This represents the first step in the establishment of life-long viral latency in vivo and generates precursors that can develop into the lymphoid malignancies. However, virus spread requires a switch from latency to the lytic replication cycle, a process orchestrated by the virally encoded protein Zta, an AP1-like transcription factor that interacts with a 7 base-pair DNA sequence element.  As Zta has the potential to reprogram the patterns of gene expression in the host cell, we undertook global transcriptome analyses (RNA sequencing) in a Burkitts lymphoma derived cell line in which the Zta is expressed from an inducible promoter, mimicking the switch from latency to lytic cycle. We identified 2,263 host genes whose expression levels were altered. In parallel, we performed chromatin precipitation and next-generation DNA sequencing (ChIP-Seq) to identify genes that are direct targets of Zta. Integrating these data sets revealed 277 host genes that appear to be directly regulated by Zta. Surprisingly, the frequency and distribution of the Zta binding peaks suggests that Zta regulates host genes through long-range enhancers, with a median distance of 25.8kb from the transcriptional start site, rather than equivalents of the promoter elements through which Zta regulates viral genes.

overall_design:
Akata cells transduced with a plasmid encoding Zta, NGFR and GFP from a doxycycline regulated promoter, and control cells in which the Zta sequences were in the opposite orientation, were treated with 500 ng/ml doxycycline for 24h.  The NGFR-expressing cells were isolated with anti-NGFR antibodies coupled to paramagnetic beads.  RNA was prepared from two independent populations of control and Zta-expressing cells and the poly A-selected transcripts were analyzed by direct sequencing.
ZTA: also referred to as BZLF1.

first: Sharada

last: Ramasubramanyan

first: Kay

last: Osborn

first: Jianmin

last: Zou

first: Rajaei

last: Al-Mohammad

first: Harshil

last: Patel

first: Gordon

last: Peters

first: Martin

last: Rowe

first: Richard

middle: G

last: Jenner


title: Dynamic landscape of RNA structures in vivo reveals principles of post-transcriptional regulation [icSHAPE]

city: Stanford

first: Ryan

middle: Alexander

last: Flynn

state: CA

address: 380 Roth Way, Room 265

institute: Stanford University

summary: Visualizing the physical basis for molecular behavior inside living cells is a grand challenge in biology. RNAs are central to biological regulation, and RNA’s ability to adopt specific structures intimately controls every step of the gene expression program.1 However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles predominantly viewed only two of four nucleotides that make up RNA.2,3 Here we present a novel biochemical approach, In Vivo Click SHAPE (icSHAPE), that enables the first global view of RNA secondary structures of all four bases in living cells. icSHAPE of embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguishes different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved in vitro, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA binding or modification proteins that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and N6-methyladenosine (m6A) modification genome-wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.

overall_design: Development and implementation of a chemoaffinity method to study the in vivo secondary structure of the transcriptome

first: Robert

last: Spitale

middle: A

first: Qiangfeng

last: Zhang

first: Hannes

middle: Y

last: Kuchelmeister

first: Pete

last: Crisalli

first: Eduardo

last: Torre

first: Pedro

last: Batista

first: Eric

middle: T

last: Kool

first: Howard

last: Chang


title: BCL6 orchestrates Tfh differentiation via multiple distinct mechanisms

city: New York

first: Katerina

last: Hatzi

address: 413 E 69th Street, BB-1462

department: Hematology/Oncology

institute: WCMC

summary: Tfh cells are required for T cell help to B cells, and BCL6 is the defining transcription factor of Tfh cells. However, the functions of Bcl6 in Tfh have largely remained unclear. Here we defined the BCL6 cistrome in primary human germinal center Tfh cells to assess mechanisms of BCL6 regulation of CD4 T cells, comparing and contrasting BCL6 function in T and B cells. BCL6 primarily acts as a repressor in Tfh cells, and BCL6 binding was associated with control of Tfh cell migration, TCR signaling, and repression of alternative cell fates. Interestingly, although some BCL6 bound genes possessed BCL6 DNA binding motifs, many BCL6-bound loci were instead characterized by the presence of DNA motifs for AP1 or STAT. AP1 complexes are key positive downstream mediators of TCR signaling and external stimuli. We show that BCL6 can directly bind AP1, and AP1 and BCL6 co-occupied BCL6 binding sites with AP1 motifs, suggesting that BCL6 subverts AP1 activity. These findings reveal that BCL6 has broad and multifaceted effects on Tfh biology, and provide insight into how this master regulator mediates distinct cell-context dependent phenotypes.

overall_design: ChIP-seq experiments to identify the genome wide targets of BCL6 and the profiles of histone modifications H3K4me3, H3K4me1 and H3K27ac  in CXCR5hi Tfh cells isolated from human tonsils.

first: Ari

last: Melnick


title: The Oct4/Sox2 Complex Varies in its Ability to Displace Nucleosomes at Functional Transcription Factor Sites in Murine Embryonic Stem Cells

status: Public on Mar 16 2015

city: Evanston

first: Liqun

last: Xi

state: IL

address: 2006 Sheridan Road

department: Statistics

institute: Northwestern University

summary: The binding sequence for any transcription factor can be found millions of times within a genome, yet only a small fraction of these sequences encode functional transcription factor binding sites. To study how the competition between nucleosomes and transcription factors helps determine a functional transcription factor site from a predicted transcription factor site, we compared experimentally-generated in vitro nucleosome occupancy with in vivo nucleosome occupancy and transcription factor binding in murine embryonic stem cells. Using a solution hybridization enrichment technique, we generated a high-resolution nucleosome map from targeted regions of the genome containing predicted sites and functional sites of Oct4/Sox2 regulation.  We found that at Pax6 and Nes, which are bivalently poised in stem cells, functional Oct4 and Sox2 sites show high amounts of in vivo nucleosome displacement compared to in vitro. Oct4 and Sox2, which are active, show no significant displacement of in vivo nucleosomes at functional sites, similar to nonfunctional Oct4/Sox2 binding. This study highlights the ability of Oct4 and Sox2 transcription factors to affect nucleosome occupancy in different ways, which may reflect distinct patterns of stem cell gene regulation.

overall_design: BAC-enrichment method sequencing (BEM-seq) of micrococcal nuclease digested in vivo nucleosomes and in vitro nucleosome preferences from murine embryonic stem cells

first: Amy

last: Sebeson

first: Quanwei

first: Audrey

last: Sigmund

first: Ji-Ping

last: Wang

first: Jonathan

last: Widom

first: Xiaozhong


title: Next generation sequencing analysis reveals that the ribonucleases RNase II, RNase R and PNPase affect bacterial motility and biofilm formation in E. coli

status: Public on Mar 17 2015

city: Oeiras

first: Cecilia

middle: M.

last: Arraiano

address: Av. Republica, Apt 127

country: Portugal

institute: Instituto de Tecnologia Quimica e Biologica (ITQB) / Univ. Nova de Lisboa

summary:
Background
The RNA steady-state levels in the cell are a balance between synthesis and degradation rates. Although transcription is important, RNA processing and turnover are also key factors in the regulation of gene expression. In Escherichia coli there are three main exoribonucleases (RNase II, RNase R and PNPase) involved in RNA degradation. Although there are many studies about these exoribonucleases not much is known about their global effect in the transcriptome.

Results
In order to study the effects of the exoribonucleases on the transcriptome, we sequenced the total RNA (RNA-Seq) from wild-type cells and from mutants for each of the exoribonucleases (∆rnb, ∆rnr and ∆pnp). We compared each of the mutant transcriptome with the wild-type to determine the global effects of the deletion of each exoribonucleases in exponential phase. We determined that the deletion of RNase II significantly affected 187 transcripts, while deletion of RNase R affects 202 transcripts and deletion of PNPase affected 226 transcripts. Surprisingly, many of the transcripts are actually down-regulated in the exoribonuclease mutants when compared to the wild-type control. The results obtained from the transcriptomic analysis pointed to the fact that these enzymes were changing the expression of genes related with flagellum assembly, motility and biofilm formation. The three exoribonucleases affected some stable RNAs, but PNPase was the main exoribonuclease affecting this class of RNAs. We confirmed by qPCR some fold-change values obtained from the RNA-Seq data, we also observed that all the exoribonuclease mutants were significantly less motile than the wild-type cells. Additionally, RNase II and RNase R mutants were shown to produce more biofilm than the wild-type control while the PNPase mutant did not form biofilms.

Conclusions
In this work we demonstrate how deep sequencing can be used to discover new and relevant functions of the exoribonucleases. We were able to obtain valuable information about the transcripts affected by each of the exoribonucleases and compare the roles of the three enzymes. Our results show that the three exoribonucleases affect cell motility and biofilm formation that are two very important factors for cell survival, especially for pathogenic cells.

overall_design: RNA-Seq of E. coli K-12 MG1693 wild-type(wt) and three exoribonucleases mutants was done with Illumina Hi-Seq platform.

first: Vânia

last: Pobre

first: Cecília


title: Notch ChIPseq in Angptl2 treated CD34 cells

city: Boston

first: Leonard

last: Zon

address: 1 Blackfan Circle

department: Oncology/Hematology

institute: Boston Children's Hospital

summary: ANGPTL2 is a protein in the angiopoietin-like protein family and plays a role in hematopoietic and vascular physiology.  This study examined the effects of ANGPTL2 on CD34+ human hematopoetic progenitor cells.

overall_design: 2 samples total; 1 sample from ChIPseq with Notch antibody in Angptl2-stimulated CD34+ cells and the other is the input ChIP DNA

first: Michelle

last: Lin

first: Eirini

last: Trompouki


title: Determination of decay rates across the transcriptome and identification of controlling features

city: Cleveland

first: Jeffery

middle: Michael

last: Coller

state: OH

address: 2109 Adelbert Dr.

department: Center for RNA Molecular Biology

institute: Case Western Reserve University

summary: In this work, we determine andenylated and total mRNA decay rates and correlate them with codon optimality. We conclude that optimality is a major contributor to mRNA stability in budding yeast.

overall_design: mRNA profiling was done on 10 time points using two different library prep methods and sequenced using a paired end protocol on an Illumina HiSeq2000 instrument. A biological duplicate was performed.

first: Vladimir

last: Presnyak

first: Sarah

last: Olson

first: Brenton

last: Graveley

first: Jeff


title: Pancreatic cancer exosomes induce pre-metastatic niche formation in the liver

first: David

last: Lyden

department: Departments of Pediatrics, Cell & Developmental Biology

institute: Weill Medical College of Cornell University

summary: Pancreatic cancers (PCs) are highly metastatic with poor prognosis, mainly due to delayed detection. We hypothesized that intercellular communication is critical for metastatic progression. Here, we show that PC-derived exosomes induce liver pre-metastatic niche formation in naïve mice and consequently increase liver metastatic burden. Uptake of PC-derived exosomes by Kupffer cells caused transforming growth factor β secretion and upregulation of fibronectin production by hepatic stellate cells. This fibrotic microenvironment enhanced recruitment of bone marrow-derived macrophages. We found that macrophage migration inhibitory factor (MIF) was highly expressed in PC-derived exosomes, and its blockade prevented liver pre-metastatic niche formation and metastasis. Compared to patients whose pancreatic tumors did not progress, MIF was markedly higher in exosomes from stage I PC patients who later developed liver metastasis. These findings suggest that exosomal MIF primes the liver for metastasis and may be a prognostic marker for the development of PC liver metastasis.

overall_design: Normal pancreas and Pancreatic cancer exosomes education of human von Kupffer cells in vitro

first: Bruno

last: Costa-Silva

first: Nicole

last: Aiello

first: Swarnima

last: Singh

first: Haiying

last: Zhang

first: Basant

middle: K

last: Thakur

first: Annette

last: Becker

first: Ayuko

last: Hoshino

first: Milica

last: Mark

first: Henrik

last: Molina

first: Jenny

last: Xiang

first: Tuo

first: Till-Martin

last: Theilen

first: Guillermo

last: García-Santos

first: Caitlin

last: Williams

first: Yonathan

last: Ararso

first: Yujie

last: Huang

first: Gonçalo

last: Rodrigues

first: Tang-Long

last: Shen

first: Knut-Jørgen

last: Labori

first: Inger

last: Bowitz Lothe

first: Elin

middle: H

last: Kure

first: Allyson

middle: J

last: Ocean

last: Hernandez

first: Alex

last: Doussat

first: Saya

last: Ebbesen

first: Maneesh

last: Jain

first: Kavita

last: Mallya

first: Surinder

last: Batra

first: William

middle: R

last: Jarnagin

first: Robert

middle: E

last: Schwartz

first: Irina

last: Matei

first: Héctor

last: Peinado

first: Ben

middle: Z

last: Stanger

first: Jacqueline

last: Bromberg


title: Linking the aryl hydrocarbon receptor with altered DNA methylation patterns and developmentally induced aberrant antiviral T cell responses [MeDIP-Seq]

city: Rochester

first: B Paige

last: Lawrence

address: 575 Elmwood Ave.

department: Environmental Medicine

institute: University of Rochester Medical Center

summary: Successfully fighting infection requires a properly tuned immune system. Recent epidemiological studies link exposure to pollutants that bind the aryl hydrocarbon receptor (AHR) during development with poorer immune responses later in life. Yet, how developmental triggering of AHR durably alters immune cell function remains unknown. Using a mouse model, we show that developmental activation of AHR leads to long-lasting reduction in the response of CD8+ T cells during influenza virus infection, cells critical for resolving primary infection. Combining genome-wide approaches, we demonstrate that developmental activation alters DNA methylation and gene expression patterns in isolated CD8+ T cells prior to and during infection. Altered transcriptional profiles in CD8+ T cells from developmentally exposed mice reflect changes in pathways involved in proliferation and immunoregulation, with an overall pattern that bears hallmarks of T cell exhaustion. Developmental exposure also changed DNA methylation across the genome, but differences were most pronounced following infection, where we observed inverse correlation between promoter methylation and gene expression. This points to altered regulation of DNA methylation as one mechanism by which AHR causes durable changes in T cell function. Discovering that distinct gene sets and pathways were differentially changed in developmentally exposed mice prior to and after infection further reveals that the process of CD8+ T cell activation is rendered fundamentally different by early life AHR signaling. These findings reveal a novel role for AHR in the developing immune system: regulating DNA methylation and gene expression as immune cells respond to viral infection later in life.

overall_design: In this study, two biological replicates were collected for each of four treatment groups: developmental exposure to vehicle control (Veh) and naïve, developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and naïve, developmental exposure to Veh and infected, developmental exposure to TCDD and infected. For each sample, both RNA-seq and methylated DNA immunoprecipitation (MeDIP)-seq were performed.

first: Bethany

last: Winans

first: Anusha

last: Nagari

first: Minho

last: Chae

first: Chia-I

last: Ko

first: Alvaro

last: Puga

first: W

middle: L

last: Kraus

first: B

middle: P


title: SMO variants explain the majority of drug resistance in basal cell carcinoma [Target Sequencing]

first: Jiang

last: Li

address: 269 Campus Drive, Stanford University

department: Dermatology

summary: Advanced basal cell carcinomas (BCCs) frequently acquire resistance to Smoothened (SMO) inhibitors through unknown mechanisms, providing a unique opportunity to study human tumor evolution. Here, we identify SMO mutations in 50% (22/44) of resistant BCCs compared with 5.6% (2/36) of untreated BCCs (p<0.0001), and show that these mutations maintain Hedgehog signaling in the presence of SMO inhibitors. Alterations include four ligand binding pocket (LBP) mutations that define sites of inhibitor binding and four variants that confer constitutive activity and inhibitor resistance, thus defining pivotal residues of SMO that ensure receptor autoinhibition. Finally, we show that both classes of SMO variants respond to the aPKC-ι/λ inhibitor PSI and GLI2 antagonist ATO that operate downstream of SMO

overall_design:
Identification of Smo mutations in 30 resistant basal cell cancers and 36 untreated basal cell cancers
The exonic regions of PTCH1 and SMO were amplified using the Access Array platform (Fluidigm)

first: Scott

middle: X

last: Atwood

last: Sarin

first: Geurim

last: Kim

first: Melika

last: Rezaee

first: Mina

middle: S

last: Ally

first: Jinah

first: Catherine

last: Yao

first: Ramon

last: Whitson

first: Anne

last: Chang

first: Anthony

last: Oro

first: Jean

last: Tang


title: Astroglial Connexin 43 sets the blood brain barrier immune quiescence

city: Paris

first: Laurent

last: Jourdren

address: 46 rue d'Ulm

department: Biologie

institute: École Normale Supérieure

summary: In the brain, a unique homeostatic equilibrium strictly controls immune reactions. In healthy conditions, the immune system poorly communicates with the central nervous system (CNS), immune cell migration across the blood brain barrier (BBB) is kept at a very low level and the unique CNS microenvironment strictly restricts immune reactions. Here, we identified the astroglial gap junction protein Connexin 43 has a new actor of this homeostasis. We showed that genetic inactivation of astroglial Cx43 in the mouse induces expression of a specific set of chemokines and endothelial adhesion molecules leading to neutrophils, macrophages, B and T lymphocytes and mature plasmocytes infiltration at the BBB, and to the development of antigen presentation-related mechanisms, in sterile conditions. Thus, astroglial Cx43 by decreasing the expression of leukocytes adhesion and migration mediators, promotes the immune quiescence of the BBB

overall_design: Comparison of 3 months-old Cx43 deleted mice against two distinct WT genetic background

first: Anne-Cécile

last: Boulay

first: Aurélien

last: Mazeraud

first: Salvatore

last: Cisternino

first: Bruno

last: Saubaméa

first: Phillipe

last: Mailly

last: Jourdern

first: Corinne

last: Blugeon

first: Stéphane

last: Le Crom

first: Pascal

last: Ezan

first: Patrick

last: Avé

first: Paulo

last: Vieira

first: Fabrice

last: Chrétien

first: Martine

last: Cohen-Salmon


title: SMO variants explain the majority of drug resistance in basal cell carcinoma [exome-seq]

first: Jiang

last: Li

summary: Advanced basal cell carcinomas (BCCs) frequently acquire resistance to Smoothened (SMO) inhibitors through unknown mechanisms, providing a unique opportunity to study human tumor evolution. Here, we identify SMO mutations in 50% (22/44) of resistant BCCs compared with 5.6% (2/36) of untreated BCCs (p<0.0001), and show that these mutations maintain Hedgehog signaling in the presence of SMO inhibitors. Alterations include four ligand binding pocket (LBP) mutations that define sites of inhibitor binding and four variants that confer constitutive activity and inhibitor resistance, thus defining pivotal residues of SMO that ensure receptor autoinhibition. Finally, we show that both classes of SMO variants respond to the aPKC-ι/λ inhibitor PSI and GLI2 antagonist ATO that operate downstream of SMO.

overall_design: Identification of somatic mutations in 14 resistant BCCs along with matched adjacent normal skin using exome sequencing.

first: Scott

last: Atwood

first: Kavita

last: Sarin

first: Geurim

last: Kim

first: Melika

last: Rezaee

first: Mina

last: Ally

first: Jinah

first: Catherine

last: Yao

first: Ramon

last: Whitson

last: Chang


title: SMO variants explain the majority of drug resistance in basal cell carcinoma

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

overall_design: Refer to individual Series


title: The PTBP1-associated long noncoding RNA Pnky regulates embryonic and postnatal neural stem cells

status: Public on Mar 15 2015

city: SAN FRANCISCO

first: John

last: Liu

address: 35 Medical Center Way

institute: UCSF

summary: While thousands of long noncoding RNAs (lncRNAs) have been identified, few lncRNAs that control neural stem cell (NSC) behavior are known.  Here, we identify Pinky (Pnky) as a novel, neural-specific lncRNA that regulates neurogenesis from NSCs in the embryonic and postnatal brain.  In postnatal NSCs, Pnky knockdown potentiates neuronal lineage commitment and expands the transit-amplifying cell population, increasing neuron production several-fold.  Pnky is evolutionarily conserved and expressed in NSCs of the developing human brain.  In the embryonic mouse cortex, Pnky knockdown increases neuronal differentiation and depletes the NSC population.  Pnky interacts with splicing regulator PTBP1, and PTBP1 knockdown also enhances neurogenesis.  In NSCs, Pnky and PTBP1 regulate the expression and alternative splicing of a core set of transcripts that relates to the cellular phenotype.  These data thus unveil Pnky as a conserved lncRNA that interacts with a key RNA processing factor and regulates a critical stage of neurogenesis from embryonic and postnatal NSC populations.

overall_design: Differential expression and differential splicing in Pnky or Ptbp1 shRNA knockdown vs control, in duplicates

first: Alexander

middle: D

last: Ramos

first: Rebecca

last: Andersen

first: Siyuan

first: Daniel

last: Lim


title: Dynamic landscape of RNA structures in vivo reveals principles of post-transcriptional regulation

first: Ryan

middle: Alexander

last: Flynn


title: Genomic-scale identification of host genes regulated by EBV during the lytic cycle

first: Alison

middle: Jane

last: Sinclair


title: Liver Med23 ablation improves glucose and lipid metabolism and prevent diet-induced obesity

city: Tianjin

first: Yajing

last: Chu

address: Nanjing Road 288

department: State Key Laboratory of Experimental Hematology

institute: Institute of Hematology, Chinese Academy of Medical Sciences

summary: Mediator complex function as an integrative hub for transcriptional regulation. Here we show that Mediator subunit MED23 regulate glucose and lipid metabolism via FOXO1 in liver. Here, we have generated a liver-specific Med23-knockout (LMKO) mouse and found that Med23-deletion in liver improved glucose and lipid metabolism, as well as insulin responsiveness, and prevented diet-induced obesity. Mechanistically, MED23 participated in gluconeogenesis and cholesterol synthesis by interacting with FOXO1. Disruption of this interaction by hepatic Med23-deletion impaired the Mediator and RNAP II recruitment and partially reduced the expression of the FOXO1 target genes. Remarkably, acute hepatic Med23 knockdown in db/db mice significantly improved insulin sensitivity. Overall, our data revealed Mediator MED23 as a critical regulator of glucose and lipid metabolism, suggesting novel therapeutic strategies against metabolic diseases.

overall_design: Comparison of mRNA expression profile of control and LMKO mice fasted 24 h (fasted) or fasted 24 h then re-fed 24 h (re-fed) (n = 2 per group)

first: Yichi

last: Xu

first: Xiao

first: Gang

last: Wang


title: Diabetes Enhances the Proliferation of Adult Pancreatic Multipotent Progenitor Cells and Biases Their Differentiation to More Beta-Cell Production

status: Public on Mar 13 2015

city: Montreal

first: Hamed

last: Najafabadi

state: QC

address: 740 Dr. Penfield Avenue, Room 7202

department: Human Genetics

institute: McGill University

summary: Endogenous pancreatic multipotent progenitors (PMPs) are ideal candidates for regenerative approaches to compensate for b-cell loss since their b-cell–producing capacities as well as strategic location would eliminate unnecessary invasive manipulations. However, little is known about the status and potentials of PMPs under diabetic conditions. Here we show that b-cell metabolic stress and hyperglycemia enhance the proliferation capacities of adult PMP cells and bias their production of progeny toward b-cells in mouse and human. These effects are dynamic and correlate with functional b-cell regeneration when conditions allow.

overall_design: Insulin-positive Glut2-low cell population of adult pancreatic tissue is enriched for PMP cells. Streptozocin (STZ) can enter beta-cells via Glut2 , induce cell death and consequently diabetes. Insulin-positive cells from two groups (STZ-injected experiment and vehicle-injected control, n=3/group) of MIP-GFP transgenic male mice were sorted to Glut2-low (Glut2L) and Glut2-high (Glut2H) by FACS. Total RNA from these samples were extracted for transcriptome analysis.

first: Rozita

last: Razavi


title: A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depend on histone recycling in transcribed chromatin [ChIP-exo]

city: Stockholm

first: Karl

last: Ekwall

address: Alfred Nobels Alle 7

country: Sweden

institute: Karolinska Inst

summary: Nucleosome composition actively contribute to the chromatin structure and accessibility. To preserve chromatin state during replication, transcription and DNA repair, cells have evolved mechanisms to evict or recycle histones, generating a landscape of differentially aged nucleosomes. To map the stability of nucleosomes, we have adapted the recombination induced tag exchange (RITE) method to Schizosaccharomyces pombe histone H3. The RITE method allows us to study replication-independent protein turnover both through the occurrence of, in our case, new histone H3 and the disappearance or preservation of old histone H3. We contrast the RITE system to nucleosome turnover measured by chromatin incorporation of an epitope-tagged H3 under an inducible promoter. We confirm previous findings that stable nucleosomes are found at heterochromatin, but also at coding regions of actively transcribed genes. Genome-wide comparisons with several chromatin marks showed that high turnover nucleosomes correlate with H2A.Z, acetylated H4 and H3K4me2. The histones with high turnover are primarily found at the nucleosomes on the 5´and/or 3´ edges of the transcribed unit. In addition, in this study we have determined genome-wide maps of all three methylation marks at H4K20. All methylation of H4K20 appeared in low turnover nucleosomes and particularly in euchromatic regions. H4K20me1 marks stable nucleosomes at loci proximal to nucleosome depleted regions (NDR) and H4K20me2/3 were found further inside of transcribed units, especially at coding regions of long genes expressed at low levels. Further, this transcription-dependent accumulation of histone methylations was dependent on the histone chaperone complex FACT (Facilitates Chromatin Transcription), as predicited from its role in recycling nucleosomes during transcription.

overall_design: In total 4 samples: ChIP-exo of old H3 (antibody HA) and new H3 (antibody T7) at 0h and 2h after estradiol treatment

first: J

last: Svensson

first: Pauline

last: Audergon

first: Victoria

last: Menendez-Benito

first: Manu

last: Shukla

first: Indranil

last: Sinha

first: Ulrika

last: Norman-Axelsson

first: Elisabet

last: Jemt

first: Robin

middle: C

last: Allshire


title: SMO variants explain the majority of drug resistance in basal cell carcinoma [RNA-Seq]

first: Jiang

last: Li

overall_design: Genome-wide gene expression profiling of 8 normal skin biopsies, 9 resistant basal cell cancers and 4 sensitive basal cell cancers.

first: Scott

last: Atwood

first: Kavita

middle: Y

last: Sarin

first: Geurim

last: Kim

first: Melika

last: Rezaee

first: Mina

last: Ally

first: Jinah

first: Catherine

last: Yao

first: Ramon

last: Whitson

first: Anne

last: Chang

first: Anthony

last: Oro

first: Jean

last: Tang