title: In vivo and transcriptome-wide identification of RNA-binding protein target sites [PAR-CLIP]

first: Dominic

last: Gruen

summary: Animal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide target sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible targets and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knock-down, protein but not mRNA expression of the 439 targets was specifically and highly significantly upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites nearby the start codon of target genes. We propose that GLD-1 interacts with the translation machinery nearby the start codon, a so far unknown mode of gene regulation in eukaryotes.

overall_design: Arrested L1 worms were grown in liquid culture supplemented with 2mM 4SU or 6SG. 250,000 worms were sufficient for one iPAR-CLIP experiment. Living adult worms were transferred to NGM plates and crosslinked on ice using a Stratalinker (Stratagene) with customized 365nm UV-lamps (energy setting: 2J/cm2). Worms were lysed on ice by douncing in NP40 lysis buffer (50 mM HEPES-K pH 7.5, 150 mM KCl, 2 mM EDTA, 0.5% (v/v) NP-40, 0.5 mM DTT, protease inhibitor cocktail (Roche)). Cleared lysates were treated with RNase T1 (Fermentas) (final concentration 1U/?l) for 15 min at 22ºC. GLD-1::GFP::FLAG fusion proteins were immunoprecipitated for 1h at 4ºC using anti-FLAG antibody (Sigma, F3165) coupled to Protein G magnetic beads (Invitrogen). For one iPAR-CLIP experiment (1ml cleared lysate obtained from 250,000 worms), 300µl beads and 150µg antibody were used. Immunoprecipitates were treated with RNase T1 (100U/?l) for exactly 12 min at 22 ºC. Subsequently, PAR-CLIP was carried out as described previously (Hafner et al, 2010).  cDNA libraries  were sequenced on a Genome Analyzer II (Illumina).

first: Jungkamp

last: Anna-Carina

first: Stoeckius

last: Marlon

first: Mecenas

last: Desirea

first: Gruen

last: Dominic

first: Mastrobuoni

last: Guido

first: Kempa

last: Stefan

first: Nikolaus

last: Rajewsky


title: DNA binding factors shape the mouse methylome at distal regulatory regions.

status: Public on Dec 09 2011

city: Basel

first: Dirk

last: Schuebeler

address: Fabrikstrasse 24

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

type: Methylation profiling by high throughput sequencing

type: Genome binding/occupancy profiling by high throughput sequencing

type: Genome binding/occupancy profiling by array

type: Expression profiling by high throughput sequencing

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

overall_design: Refer to individual Series

first: Michael

middle: B

last: Stadler

first: Rabih

last: Murr

first: Lukas

last: Burger

first: Robert

last: Ivanek

first: Florian

last: Lienert

first: Anne

last: Scholer

first: Edward

middle: J

last: Oakeley

first: Dimos

last: Gaidatzis

first: Vijay

last: Tiwari

last: Schubeler


title: The Genomic Binding Sites of an Endogenous Non-Coding RNA

status: Public on Dec 07 2011

city: Boston

first: Mark

middle: L

last: Borowsky

state: MA

address: 185 Cambridge Street

department: Molecular Biology

country: USA

institute: MGH

summary: Long non-coding RNAs (lncRNAs) have important regulatory roles and can function at the level of chromatin. To determine where lncRNAs bind to chromatin, we developed CHART, a hybridization-based technique that specifically enriches endogenous RNAs along with their targets from reversibly-crosslinked chromatin extracts. CHART was used to enrich the DNA and protein targets of endogenous lncRNAs from fly and human. This analysis was extended to genome-wide mapping of roX2, a well-studied ncRNA involved in dosage-compensation in Drosophila. CHART revealed that roX2 binds at specific genomic sites that coincide with the binding sites of proteins from the MSL-complex that affects dosage compensation. These results reveal the genomic targets of roX2 and demonstrate how CHART can be used to study RNAs in a manner analogous to ChIP for proteins.

overall_design:
Examination of the binding sites of roX2 ncRNA from S2 cells using two different elution strategies compared with a sense control or input control.
Processed data file 'roX2.2.peaks.bed' (for roX2 CHART RNase eluted, combined replicates) linked below as supplementary file.

first: Matthew

middle: D

last: Simon

first: Charlotte

middle: I

last: Wang

first: Peter

last: Kharchenko

first: Jason

middle: A

last: West

first: Brad

last: Chapman

first: Artyom

last: Alekseyenko

first: Mitzi

last: Kuroda

middle: E

last: Kingston


title: modENCODE_White Lab: genome-wide ChIP data of CNC from AdultFemale on Illumina Genome Analyzer.

city: Chicago

first: Kevin

middle: P.

last: White

state: IL

address: 900 E. 57th STR. 10th FL.

department: Institute for Genomics and Systems Biology

institute: University of Chicago

summary:
This is a dataset generated by the Drosophila Regulatory Elements modENCODE Project led by Kevin P. White at the University of Chicago. It contains genome-wide binding profile of the factor CNC from AdultFemale generated by ChIP and analyzed on Illumina Genome Analyzer.

For data usage terms and conditions, please refer to http://www.genome.gov/27528022  and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design:
A validated dataset is comprised of three biological replicates for ChIP-chip experiments and two replicates for ChIP-seq and meet the modENCODE quality standards. The control sample is the chromatin Input used for ChIP. Most factors binding profiles are generated by using specific antibodies for the protein of interest. However, some factors have been tagged by GFP in a transgenic line. In that case, ChIP is generated using an anti-GFP antibody.

This submission represents the ChIP-seq component of the study.

first: Nicolas

last: Negre

first: Nicholas

last: Bild

middle: P


title: Tripartite organization of centromeric chromatin in budding yeast

city: Seattle

first: Jorja

last: Henikoff

state: WA

address: 1100 Fairview AV N, A1-162

department: Basic Sciences

institute: Fred Hutchinson Cancer Research Center

summary:
The centromere is the genetic locus that organizes the proteinaceous kinetochore and is responsible for attachment of the chromosome to the spindle at mitosis and meiosis. In most eukaryotes, the centromere consists of highly repetitive DNA sequences that are occupied by
nucleosomes containing the CenH3 histone variant, whereas in budding yeast, an ~120-bp Centromere DNA Element (CDE) that is sufficient for  centromere function is occupied by a single right-handed CenH3 (Cse4) nucleosome. However, these in vivo observations are
inconsistent with in vitro evidence for left-handed octameric CenH3 nucleosomes. To help resolve these inconsistencies, we characterized yeast centromeric chromatin at single base-pair resolution. Intact particles containing both Cse4 and H2A are precisely protected from
micrococcal nuclease over the entire CDE of all 16 yeast centromeres in both solubilized chromatin and the insoluble kinetochore. Small DNA-binding proteins protect CDEI and CDEIII and delimit the centromeric nucleosome to the ~80-bp CDEII, only enough for a single DNA wrap. As expected for a tripartite organization of centromeric chromatin, loss of Cbf1 protein, which binds to CDEI, both reduces the size of the centromere-protected region and shifts its location towards CDEIII. Surprisingly, Cse4 overproduction caused genome-wide
misincorporation of non-functional CenH3-containing nucleosomes that protect ~135 base pairs and are preferentially enriched at sites of high nucleosome turnover. Our detection of two forms of CenH3 nucleosomes in the yeast genome, a singly wrapped particle at the functional centromere and octamer-sized particles on chromosome arms, reconcile seemingly conflicting in vivo and in vitro observations.

overall_design: We used micrococcal nuclease mapping, chromatin immunoprecipitation and paired-end sequencing to determine the structure of yeast centromeres at single base-pair resolution.

middle: G

first: Kristina

last: Krassovsky

first: Steven


title: Distinct p53 Genomic Binding Patterns in Normal and Cancer-derived Human Cells

status: Public on Dec 08 2011

city: Berkeley

first: Krassimira

last: Botcheva

state: CA

address: One Cyclotron Rd MS977

department: Cell and Molecular Biology

institute: Lawrence Berkeley National Laboratory

summary: We report here genome-wide analysis of the tumor suppressor p53 binding sites in normal human cells. 743 high-confidence ChIP-seq peaks representing putative genomic binding sites were identified in normal IMR90 fibroblasts using a reference chromatin sample. More than 40 % were located within 2 kb of a transcription start site (TSS), a distribution similar to that documented for individually studied functional p53 binding sites and to date not observed by previous genome-wide studies. Nearly half of the high-confidence binding sites in the IMR90 cells reside in CpG islands, in marked contrast to sites reported in cancer-derived cells. The distinct genomic features of the IMR90 binding sites do not reflect a distinct preference for specific sequences, since the de novo developed p53 motif based on our study is similar to those reported by genome-wide studies of cancer cells. More likely the different chromatin landscape in normal compared to cancer-derived cells influences p53 binding via modulating availability of the sites. We compared the IMR90 ChIP-seq peaks to the recently published IMR90 methylome1, and demonstrated that they are enriched at hypomethylated DNA. Our study represents the first genome-wide, de novo mapping of p53 binding sites in normal human cells and reveals that p53 binding sites reside in distinct genomic landscapes in normal and cancer-derived human cells.

overall_design: Identification of genomic p53 binding sites in normal human cells by ChIP-seq.

first: Carl

last: Anderson


title: Gene expression profile of right ventricles from adult wild type and Ezh2-deficient hearts

city: San Francisco

first: Gladstone

last: Bioinformatics

address: 1650 Owens St

department: Data Science and Biotechnology

institute: Gladstone Institutes

summary: Adult-onset diseases can be associated with in utero events, but mechanisms for this remain unknown. The polycomb histone methyltransferase, Ezh2, stabilizes transcription by depositing repressive marks during development that persist into adulthood, but its function in postnatal organ homeostasis is unknown. We show that Ezh2 stabilizes cardiac gene expression and prevents cardiac pathology by repressing the homeodomain transcription factor Six1, which functions in cardiac progenitors but is stably silenced upon cardiac differentiation. Ezh2 deletion in cardiac progenitors caused postnatal myocardial pathology and destabilized cardiac gene expression with activation of Six1-dependent skeletal muscle genes. Six1 induced cardiomyocyte hypertrophy and skeletal muscle gene expression. Furthermore, genetically reducing Six1 levels rescued the pathology of Ezh2-deficient hearts. Thus, Ezh2-mediated repression of Six1 in differentiating cardiac progenitors is essential for stable postnatal heart gene expression and homeostasis. Our results suggest that epigenetic dysregulation in embryonic progenitor cells predisposes to adult disease and dysregulated stress responses.

overall_design: Four samples were analyzed. RNA was obtained from ventricles from two wild type and two Ezh2-deficient hearts.

first: Benoit

last: Bruneau

first: Paul

last: Delgado-Olguin

first: Danos

last: Christodoulou

first: Christine

last: Seidman

first: J


title: In vivo and transcriptome-wide identification of RNA-binding protein target sites [RNA-Seq]

city: Berlin

first: Dominic

last: Gruen

address: Robert-Roessle-Strasse 10

country: Germany

institute: Max-Delbrueck-Center

overall_design: PolyA mRNA was extracted from young adult wildtype (N2) worms and young adult germline less worms (glp-4(bn2) TS) to identify and quantify genes expressed in the young adult germline by sequencing. 2x100 paired end sequencing was performed according to the protocol on the Illumina HiSeq 2000.

first: Jungkamp

last: Anna-Carina

first: Stoeckius

last: Marlon

first: Mecenas

last: Desirea

first: Gruen

last: Dominic

first: Mastrobuoni

last: Guido

first: Kempa

last: Stefan

first: Nikolaus

last: Rajewsky


title: Rice endosperm: mRNA profiling and H3K27me3 occupancy

status: Public on Dec 06 2011

city: Mississippi State

first: Feng

last: Tan

state: MS

address: Dorman Hall 402

department: Biochemistry and Molecular Biology

institute: Mississippi State University


title: Genome-wide maps of chromatin state in rice endosperm

summary: We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone H3 trimethylation in rice endosperm. By obtaining about four hundred million bases of sequence from rice chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of rice endosperm. We find that the presence of H3K27me3 in either upstream or downstream of a gene is predominately associated with repression of the gene, while its absence is mainly associated with high gene expression.

overall_design: Examination of Histone H3 lysine 27 trimethylation in rice endosperm.

first: Guosheng

last: Li

first: Brandon

last: Malone

first: Zhaohua

last: Peng


title: Identification and characterization of Hoxa9 binding sites in hematopoietic cells

status: Public on Dec 05 2011

city: Ann Arbor

first: Yongsheng

last: Huang

state: MI

address: 100 Washtenaw Ave

department: Bioinformatics

institute: University of Michigan

type: Genome binding/occupancy profiling by genome tiling array


title: The transcriptional coactivator Cbp regulates self-renewal and differentiation in adult hematopoietic stem cells

status: Public on Dec 15 2011

city: Cambridge

first: Wai-In

last: Chan

address: Wellcome Trust/MRC Building, Hills Road

department: Haematology

country: United Kingdom

institute: Cambridge Institute for Medical Research


title: Tissue-specific nuclei purification from animal models for genome-wide expression and chromatin profiling.

first: Jorja

last: Henikoff

type: Expression profiling by genome tiling array

summary: Here we describe a method for purifying nuclei from specific tissues of animal models that allows simultaneous analysis of both expression and chromatin profiles. The method is based on in vivo labeling of the nuclear envelope and subsequent affinity purification. We describe the use of the method to isolate nuclei from muscle.

overall_design: We determined genes preferentially expressed in muscle cells of adult C. elegans by analyzing RNA from total and affinity-puridied muscle nuclei using microarrays. We also determined the nucleosome occupancy in these nuclei by micrococcal nuclease mapping and paired-end sequencing.

first: Florian

last: Steiner

first: Steven

first: Paul

last: Talbert

first: Sivakanthan

last: Kasinathan

first: Roger

last: Deal

first: James

middle: R

last: Priess


title: IKK-epsilon regulates the balance between the type I and type II interferon responses

status: Public on Dec 01 2011

city: Huntsville

first: Jason

last: Gertz

state: AL

address: 601 Genome Way

institute: HudsonAlpha Institute

summary: Virus infection induces the production of type I and type II interferons (IFN-I and IFN-II), cytokines that mediate the antiviral response.  IFN-I (IFN-a and -b) induces the assembly of ISGF3 (interferon-stimulated gene factor 3), a multimeric transcriptional activation complex comprised of STAT1, STAT2 and IRF9.   IFN-II (IFN-g) induces the homodimerization of STAT1 to form the GAF (gamma-activated factor) complex.   ISGF3 and GAF bind specifically to distinct regulatory DNA sequences located upstream of IFN-I and II inducible genes, respectively, and activate the expression of distinct set of antiviral genes.   The balance between the type I and type II IFN pathways plays a critical role in orchestrating the innate and adaptive immune systems.  Here, we show that the phosphorylation of STAT1 by IKKε (IkB-related kinase epsilon) inhibits STAT1 homodimerization, and thus GAF formation, but does not disrupt ISGF3 formation.  Therefore, virus and/or IFN-I activation of IKKε suppresses GAF-dependent transcription and promotes ISGF3-dependent transcription.  In the absence of IKKε, GAF-dependent transcription is enhanced at the expense of ISGF3-mediated transcription, rendering cells less resistant to infection.  We conclude that IKKε plays a critical role in regulating the balance between the IFN-I and IFN-II signaling pathways.

overall_design: ChIP-seq libraries were constructed with an antibody targeting STAT1 from bone marrow macrophages treated with interferon

first: J

first: S

last: Ng


title: Large-Scale Discovery of Enhancers from Human Heart Tissue

status: Public on Dec 04 2011

first: Matthew

middle: James

last: Blow

address: 1 Cyclotron Road

department: Genomics Division

summary: Development and function of the human heart depend on the dynamic control of tissue-specific gene expression by distant-acting transcriptional enhancers.  While large numbers of heart enhancers have been identified using the mouse as a model system, many of these regulatory sequences are poorly conserved in the human genome.  To generate an accurate genome-wide map of human heart enhancers, we used an epigenomic enhancer discovery approach and identified ~6,200 candidate enhancer sequences directly from fetal and adult human heart tissue.  Consistent with their predicted function, these elements were markedly enriched near genes implicated in heart development, function and disease.  To further validate their in vivo enhancer activity, we tested 65 of these human sequences in a transgenic mouse enhancer assay and observed that 43 (66%) drove reproducible reporter gene expression in the heart.  These results support the discovery of a genome-wide set of non-coding sequences highly enriched in human heart enhancers which is likely to facilitate down-stream studies of the role of enhancers in development and pathological conditions of the heart.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Examination of AcCBP/p300 binding in human adult heart, human fetal (16wk) heart and mouse postnatal day 2 heart

first: Dalit

last: May

first: Axel

last: Visel


title: Analysis of Caenorhabditis elegans intestinal gene expression and alternative polyadenylation using fluorescence-activated nuclei sorting (FANS) and 3' end deep sequencing (3'end-seq)

first: Zhe

last: Ji

state: ILLINOIS

address: 303 E Superior St, Lurie 6-107

institute: Northwestern University

summary: Caenorhabditis elegans is a major eukaryotic experimental system employed to unravel a broad range of cellular and biological processes. Despite the many advantages of C. elegans, biochemical approaches to study tissue-specific gene expression in postembryonic stages are challenging. Here we report a novel experimental approach that enables the efficient determination of tissue-enriched transcriptomes by rapidly releasing nuclei from major tissues of postembryonic animals followed by fluorescence-activated nuclei sorting (FANS). Furthermore, we developed and applied a deep sequencing method, named 3'end-seq, which is designed to examine gene expression and identify 3' ends of transcripts using a small quantity of input RNA. In agreement with intestinal specific gene expression, promoter elements of highly expressed genes are enriched for GATA elements and their functional properties are associated with processes that are characteristic for the intestine. In addition, we systematically mapped pre-mRNA cleavage and polyadenylation sites, or polyA sites, including >3,000 sites that have previously not been identified. The analysis of nuclear mRNA revealed widespread alternative polyA site use in intestinally expressed genes. We describe several novel approaches that will be of significance to the analysis of tissue specific gene expression using small quantity RNA samples from C. elegans and beyond.

overall_design: 3'end-seq of transcriptomes for input and sorted nuclei

first: Simon

last: Haenni

first: Mainul

last: Hoque

first: Bin

last: Tian

first: AndrÃ©

last: Furger


title: Nucleosome positioning for 4 yeast species

city: Toronto

first: Kyle

last: Tsui

address: 160 College Street

country: Canada

institute: University of Toronto

summary: We describe the genome-wide nucleosome profiles of four related yeast species. All species display the same global organization features first described in S. cerevisiae: a stereotypical nucleosome organization along genes, and the classification of promoters into these which contain or lack a pronounced Nucleosome Depleted region (NDR), with the latter displaying a more dynamic pattern of gene expression. This global similarity, however, does not reflect a static evolutionary pattern, as nucleosome positioning at specific genes diverged rapidly leaving practically no similarity between S. cerevisiae and C. glabrata orthologs (~50 Myr). We show that this rapid divergence in nucleosome positioning contrasts a conserved pattern of gene expression, consistent with the idea that divergence of nucleosome patterns has a limited effect on gene expression as many different configurations can support the same regulatory outcome.

sample_organism:
Saccharomyces bayanus
Saccharomyces cerevisiae
Nakaseomyces glabratus
Saccharomyces kudriavzevii

overall_design: Nucleosomes from 4 different yeast species were isolated and sequenced using the Illumina GAII platform.  Replicates were performed for 3 of the species

first: Sebastien

last: Dubuis

first: Marinella

last: Gebbia

first: Randy

last: Morse

first: Naama

last: Barkai

first: Itay

last: Tirosh

first: Corey

last: Nislow


title: Identification and Characterization of Hoxa9 Binding Sites in Hematopoietic Cells

first: Jay

last: Hess

address: 1301 Catherine

department: Pathology

summary: The clustered homeobox proteins play crucial roles in development, hematopoiesis and leukemia yet the targets they regulate and their mechanisms of action are poorly understood.  Here, we identified the binding sites for Hoxa9 and the Hox cofactor Meis1 on a genome-wide level and profiled their associated epigenetic modifications and transcriptional targets. Hoxa9 and the Hox cofactor Meis1 co-bind at hundreds of highly evolutionarily-conserved sites, most of which are distant from transcription start sites.  These sites show high levels of histone H3K4 monomethylation and CBP/P300 binding characteristic of enhancers. Furthermore, a subset of these sites shows enhancer activity in transient transfection assays. Many Hoxa9 and Meis1 binding sites are also bound by PU.1 and other lineage-restricted transcription factors previously implicated in establishment of myeloid enhancers.  Conditional Hoxa9 activation is associated with CBP/P300 recruitment, histone acetylation and transcriptional activation of a network of proto-oncogenes including Erg, Flt3, Lmo2, Myb and Sox4. Collectively this work suggests that Hoxa9 regulates transcription by interacting with enhancers of genes important for hematopoiesis and leukemia.

overall_design: To identify the genome-wide binding sites for Hoxa9 and the Hox cofactor Meis1

first: Yongsheng

last: Huang

first: Gordon

last: Robertson


title: Sequencing of the non-ribosomal transcriptome allows the simultaneous identification of protein-coding and non-protein-coding RNAs

city: Klosterneuburg

first: Florian

middle: M

last: Pauler

address: Am Campus 1

country: Austria

institute: IST Austria

summary: Deep Sequencing of protein-coding and non-protein-coding RNAs from mouse differentiated embryonic stem cells and 14.5dpc mouse fetal head

overall_design:
Analysis of Ribominus RNA from mouse differentiated embryonic stem cells and 14.5dpc mouse fetal head

There are no processed data files for GSM566806-GSM566811. There are no fastq raw data files for GSM566812 and GSM566813 since these samples are the combined reads from all sequence lanes.

first: Ru

first: Markus

last: Jaritz

first: Andreas

last: Sommer

first: Ido

last: Tamir

first: Hendrik

last: Marks

last: Stunnenberg

first: Denise

last: Barlow


title: A key role for Mbd3 in 5-hydroxymethylcytosine-dependent gene regulation in embryonic stem cells

first: Jui-Hung

last: Hung

address: 24 cummington st

institute: Boston University

summary: Numerous chromatin regulators are required for embryonic stem (ES) cell self-renewal and pluripotency, but few have been studied in detail. Here, we examine the roles of several chromatin regulators whose loss affects the pluripotent state of ES cells. We find that Mbd3 and Brg1 antagonistically regulate a common set of genes by regulating promoter-proximal nucleosome occupancy and recruitment of RNA polymerase II. Furthermore, both Mbd3 and Brg1 play key roles in the biology of 5-hydroxymethylcytosine (5hmC): Mbd3 colocalizes with both Tet1 and 5hmC in vivo, its localization is Tet1-dependent, and binding of Mbd3/NURD to DNA in vitro is inhibited by methylcytosine, but not hydroxymethylcytosine. Finally, both Mbd3 and Brg1 are themselves required for normal levels of 5hmC in vivo. Together, our results identify an effector for 5hmC, and reveal that control of gene expression by antagonistic chromatin regulators is a surprisingly common regulatory strategy in ES cells.

overall_design: Genomic binding profiles of Mbd3 in normal ES cells, along with ES cells depleted of additional chromatin regulators, Brg1 and Tet1 were performed by ChIP-seq. In addition, we performed mapping of RNA Polymerase II in control (EGFP), Mbd3 and Brg1 knockdown ES cells by ChIP-seq.

first: Oliver

last: Rando

first: Thomas

last: Fazzio


title: Cassava sRNA libraries from non-infected and infected tissues with Xanthomonas axonopodis. pv manihotis

city: Montpellier

first: Alvaro

middle: Luis

last: Pérez-Quintero

state: Montpellier

address: 911 Avenue Agropolis

department: RBP

country: France

institute: IRD

type: Non-coding RNA profiling by high throughput sequencing

summary: microRNAs can play a crucial role in stress response in plants, including biotic stress. Some miRNAs are known to respond to bacterial infection. This work has addressed the role of miRNAs in Manihot esculenta (cassava)-Xanthomonas axonopodis pv. manihotis (Xam) interaction. Illumina sequencing was used for analyzing small RNA libraries from cassava tissue infected and non-infected with Xam. Cassava variety MBRA685 (resistant to Xam-CIO151) Six-week-old plants were inoculated with 36h-old cultures of the aggressive Xanthomonas axonopodis pv. manihotis strain CIO151 in both leaves and stems.

overall_design: Leaves were inoculated by piercing six holes in the mesophyll and placing a 5µL drop of a liquid Xam-MgCl2 culture calibrated at OD600nm = 0.002 (1 x108cfu/ml). Two leaflets per leaf and three leaves per plant were inoculated. Stems were inoculated by puncture in the stems as described previously (24). At least three plants per collection time were inoculated. Leaves and stems were collected from inoculated plants (0 hours post inoculation -hpi, 6hpi, 24hpi, 2 days post-inoculation -dpi, 5dpi, 7dpi and 15dpi) and non-inoculated plants. RNA extractions were made using a LiCl-acid phenol:chloroform method.

first: Camilo

last: Lopez

last: Perez-Quintero


title: RNA-seq characterization of Physarum macroplasmodia

city: Magdeburg

first: Israel

last: Barrantes

state: Sachsen-Anhalt

address: Pfälzerstrasse 5

department: Lehrstuhl für Regulationsbiologie

institute: Otto-von-Guericke-Universität

summary: We report a novel approach for studying the transcriptomic changes during cell differentiation, using Physarum as a model

overall_design: 3 samples examined: starved plasmodium, starved plasmodium collected 2 hours after photoinduction, and starved plasmodium collected 6 hours after induction

first: Wolfgang

last: Marwan


title: Examination of smallRNA in wildtype and transgenic Arabidopsis grown under different temperature

city: Shanghai

first: Li

last: Qun

state: Shanghai

address: 300 Fenglin Road

department: National Key Laboratory of Plant Molecular Genetics

country: China

institute: Institute of plant physiology & ecology, SIBS, CAS

summary: A thermosensory system interacts with epigenetic machinery and disarms a RNA-silencing mechanism in Arabidopsis.

overall_design: Examination of small RNA in five samples of wildtype or transgenic Arabidopsis plants grown under different temperature by high throughput sequencing.

first: Sihui

last: Zhong

first: Junzhong

last: Liu

first: Qun

last: Li

first: Zuhua

last: He


title: S.japonicum Digital Gene Expression

city: Beijing

first: Xianyu

last: Piao

address: Dong Dan San Tiao 9

department: Institute of Pathogen Biology

institute: Chinese Academy of Medical Sciences and Peking Union Medical College

summary: Schistosoma japonicum is one of the remarkable platyhelminths that are endemic in the South Asian countries. The parasite is dioecious and can parasitize inside host blood stream for many years. Rapid reproduction by producing large number of eggs and count-react or active manipulating host anti-parasite responses are the strategies that benefit the long term parasitization of the parasite. Currently, Praziquantel is the only commercial drug that is effective against the worms but not the eggs of parasite in the host and no vaccine available to prevent human or animal schistosomiasis. Development of novel antiparasite reagents and immune-prevention measures rely on the deciphering of parasite biology. The decoding of the genomic sequence of the parasite has made it possible to dissect the biological functions of genes that govern the development of the parasite inside the hosts. In this study, the polyadenylated RNA from male and female S. japonicum was isolated and deep sequenced. By comparative analysis on the transcriptomic differences of the two sexes of the parasite, potential genes or gender-specific biological pathways can be targeted.

overall_design: Messenger RNA from male and female S. japonicum parasite was selectively purified from total RNA using oligo-(dT) conjugated magnetic beads. Complementary DNA (cDNA) was synthesized guided by oligo-(dT) as a primer.

first: Pengfei

last: Cai

first: Heng

last: Wang

first: Jianwei

first: Qi

last: Jin

first: Qijun

last: Chen


title: RNA-seq analysis of K562 cells (K562-shX) with stable integration of a tet-inducible shRNA targeting a candidate (X) transcription factor from ENCODE/HudsonAlpha-MyersLab

first: Anita

last: Bansal

institute: HudsonAlpha Institute for Biotechnology

summary:
K562-shX cells are made in an effort to validate TFBS data and ChIP-seq antibodies in Myers lab (GSE32465). K562 cells are transduced with lentiviral vector having Tet-inducible shRNA targeting a transcription factor gene. Cells with stable integration of shRNA constructs are selected using puromycin in growth media. Doxycycline is added to the growth media to induce the expression of shRNA and a red fluorescent protein marker.  A successful shRNA cell line shows at least a 70% reduction in expression of the target transcription factor as measured by qPCR. For identification, we designated these cell lines as K562-shX, where X is the transcription factor targeted by shRNA and K562 denotes the parent cell line.  For example, K562-shATF3 cells are K562 derived cells selected for stable integration of shRNA targeting the transcription factor ATF3 gene and showed at least a 70% reduction in the expression of ATF3 gene when measured by qPCR. Cells growing without doxycycline (uninduced) are used as a control to measure the change in expression of target transcription factor gene after induction of shRNA using doxycycline. For detailed growth and culturing protocols for these cells please refer to http://hudsonalpha.org/sites/default/files/pdf/shRNA%20K562%20protocol.pdf. To identify the potential downstream targets of the candidate transcription factor, analyze the mRNA expression profile of the uninduced and induced K562-shX using RNA-seq.

For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: Make K562-shX cells as described in the http://hudsonalpha.org/sites/default/files/pdf/shRNA%20K562%20protocol.pdf. Measure the mRNA expression levels in uninduced K562-shX and induced K562-shX cells in two biological replicates using RNA-seq. Identify the potential downstream targets of the candidate transcription factor.

first: Florencia

last: Pauli

first: Richard

last: Myers


title: Functional and cellular constraints that shaped the PPARg binding landscape in human and mouse macrophages

first: Sebastian

last: Pott

state: Il

address: 920 58th Street

department: Human Genetics

institute: University of Chicago

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

overall_design: Refer to individual Series


title: Transcriptional landscape of Candida parapsilosis

status: Public on Nov 30 2011

city: Dublin

first: Geraldine

last: Butler

address: Belfield

department: School of Biomolecular and Biomedical Science

country: Ireland

institute: University Colege Dublin Conway Institute

summary: We used next generation sequencing (RNA-seq) to determine the transcriptional profile of C. parapsilosis growing in several conditions including different media, temperatures and oxygen concentrations.

overall_design: We have 37 Samples representing BMW and YPD media, 1% and 21% oxygen, 30 and 37 degree celsius temperature, and CLIB214 and CDupc5 strains.

first: Alessandro

last: Guida

first: Claudia

last: Lindstaedt

first: Sarah

last: Maguire

first: Chen

last: Ding

first: Desmond

last: Higgins

first: David

last: Harris

first: Matthew

last: Berriman


title: LRH-1 and PTF1-L coregulate an exocrine pancreas-specific transcriptional network for digestive function [RNA-Seq]

city: Houston

first: Scott

middle: Andrew

last: Ochsner

state: TX

address: One Baylor Plaza

department: Molecular and Cellular Biology

institute: Baylor College of Medicine

summary: Two separate pools of three Flox or two Lrh1t-/- pancreas RNA preparations was subjected to the Illumina RNA-seq protocol.

overall_design: Examination of mRNA levels in induced Lrh1 pancreas knockout and wild-type mice.

first: Sam

last: Holmstrom

first: Tye

last: Deering

first: Galvin

last: Swift

first: Steven

last: Kliewer

first: Raymond

last: MacDonald

last: Mangelsdorf


title: MIWI catalysis is required for piRNA amplification-independent LINE1 transposon silencing [deep sequencing]

city: Gneveva

first: Ramesh

last: Pillai

address: 30, Quai Ernest-Ansermet

department: Department of Molecular Biology

institute: University of Geneva

summary: Here we show that MIWI is a small RNA-guided ribonuclease (Slicer) that requires extensive complementarity for target cleavage in vitro. Disruption of its catalytic activity in mice by a single point mutation results in male infertility and displays increased accumulation of LINE1 transposon transcripts.

overall_design: MIWI-associated piRNAs from different genotypes were sequenced. Total RNA from purified round spermatids were subjected to Ribozero purification and strand-specific RNAseq lib prepared. Global 5' RACE library was prepare from indicated genotypes.

first: Michael

last: Reuter

first: Philipp

last: Berninger

first: Shinichiro

last: Chuma

first: Hardik

last: Shah

first: Mihoko

last: Hosokawa

first: Charlotta

last: Funaya

first: Claude

last: Antony

first: Ravi

last: Sachidanandam

middle: S


title: MIWI catalysis is required for piRNA amplification-independent LINE1 transposon silencing


title: Transcriptional landscape of the pathogenic yeast Candida parapsilosis

first: Geraldine

last: Butler


title: Genome-wide Runx2 occupancy in prostate cancer cells suggests a role in regulating secretion.

status: Public on Nov 29 2011

city: Detroit

first: Houtan

last: Noushmehr

address: 2799 W. Grand Blvd

institute: Henry Ford Health System

summary: Runx2 is a metastatic transcription factor (TF) increasingly expressed during prostate cancer (PCa) progression.  Using PCa cells conditionally expressing Runx2, we previously identified Runx2-regulated genes with known roles in epithelial-mesenchymal transition, invasiveness, angiogenesis, extracellular matrix proteolysis and osteolysis.  To map Runx2-occupied regions (R2ORs) in PCa cells, we first analyzed regions predicted to bind Runx2 based on the expression data, and found that recruitment to sites upstream of the KLK2 and CSF2 genes was cyclical over time.  Genome-wide ChIP-seq analysis at a time of maximum occupancy at these sites revealed 1,603 high-confidence R2ORs, enriched with cognate motifs for RUNX, GATA and ETS TFs.  The R2ORs were distributed with little regard to annotated transcription start sites (TSSs), mainly in introns and intergenic regions. Runx2-upregulated genes, however, displayed enrichment for R2ORs within 40 kb of their TSSs.  The main annotated functions enriched in 98 Runx2-upregulated genes with nearby R2ORs were related to invasiveness and membrane trafficking/secretion.  Indeed, using SDS-PAGE, mass spectrometry and western analyses, we show that Runx2 enhances secretion of several proteins, including fatty acid synthase and metastasis-associated laminins.  Thus, combined analysis of Runx2’s transcriptome and genomic occupancy in PCa cells lead to defining its novel role in regulating protein secretion.

overall_design: Examination of Runx2 in Prostate Cancer cells

first: Gillian

middle: H

last: Little

first: Sanjeev

middle: K

last: Baniwal

first: Benjamin

last: Berman

first: Gerhard

last: Coetzee

first: Baruch

last: Frenkel


title: Gene expression profiling reveals potential breast cancer prevention mechanisms associated with the rat Mcs5a allele (DGE)

city: Madison

first: Bart

middle: M. G.

last: Smits

address: 1400 University Avenue

department: Oncology

institute: University of Wisconsin-Madison

summary: Using global gene expression profiling in T-cells from untreated Mcs5a resistant and susceptible congenic rats, we asked if signatures of potential risk-decreasing mechanisms could be identified.

overall_design:
On two independent sample sets, we applied microarray (MA) or digital gene expression (DGE) analysis comparing T-cells from Mcs5a resistant and susceptible congenic rats. This file contains the DGE data.
T-cell RNA sample pools were compared between the susceptible (susc.; n=3 pools) and the resistant (Mcs5a; n=3 pools) congenic lines

first: Lisa

last: Chung

first: Ann

last: Tran

first: Alexi

last: Schoenborn

first: Deepak

last: Sharma

first: Jill

last: Haag

last: Newton

middle: N

last: Gould

first: Bob

last: Mau


title: Functional and cellular constraints that shaped the PPARg binding landscape in human and mouse macrophages: human ChIP-Seq

city: Chicago

first: Sebastian

last: Pott

summary: Genome-wide comparisons of transcription factor binding sites in different species allow for a direct evaluation of the evolutionary constraints that shape transcription factor binding landscapes. To gain insights into the evolution of the PPARg-dependent transcriptional network, we obtained binding data for PPARg, RXR and PU.1 in human macrophages and compared the profiles to matching data from mouse macrophages (Lefterova et al. 2010 (PMID 20176806); GSE21314). We found that PPARg binding was highly divergent and only 5% of the PPARg-bound regions were occupied in both species. Despite the low conservation of PPARg binding sites, conserved PPARg target genes contribute more than 30% to the functional target genes identified in human macrophages. In addition, conserved target genes are strongly enriched for lipid metabolic functions. We detected the lineage-specification factor PU.1 at the majority of human PPARg binding sites. This confirmed the juxtaposed binding configuration found in mouse macrophages and demonstrated the preservation of tissue-specific adjacent PPARg-Pu.1 binding in the absence of individual binding site conservation. Finally, based on this PPARg and PU.1 binding between human and mouse, we suggest a mechanism by which PU.1 facilitates PPARg binding site turnover in macrophages.

overall_design:
Genome-wide location analysis for 3 transcription factors (PPARg, RXR and PU.1) in a human monocytic cell line (THP-1).

This submission represents the human binding data component of the study.

first: S

first: E

middle: T

last: Liu


title: Nascent-Seq Indicates Widespread Cotranscriptional pre-mRNA Splicing in Drosophila

city: Waltham

first: Yevgenia

middle: L

last: Khodor

address: 415 South St, MS 008

department: Biology

institute: Brandeis University

summary: To determine the prevalence of cotranscriptional splicing in Drosophila, we sequenced nascent RNA transcripts from Drosophila S2 cells as well as from Drosophila heads. 87% of introns assayed manifest more than 50% cotranscriptional splicing. The remaining 13% are cotranscriptionally spliced poorly, or slowly, with ~3% being almost completely retained in nascent pre-mRNA. Although individual introns showed slight but statistically significant differences in splicing efficiency, similar global levels of splicing were seen from both sources. Importantly, introns with low cotranscriptional splicing efficiencies are present in the same primary transcript with efficiently spliced introns, indicating that splicing is intron-specific. The analysis also indicates that cotranscriptional splicing is less efficient for first introns, longer introns and introns annotated as alternative. FinallyFinally, S2 cells expressing the slow RpII215C4 mutant manifest substantially less intron retention than wild-type S2 cells.

overall_design: Examination of Total pA and Nascent RNA from 2 different cell populations and isolated fly heads.

first: Joseph

last: Rodriguez

first: Katharine

middle: C

last: Abruzzi

first: Chih-Hang

last: Tang

first: Michael

last: Marr II

last: Rosbash


title: H3K27me3 is not required for recruitment of Polycomb repressor complex 1 to target loci in mouse embryonic stem cells

city: Oxford

first: Stephen

last: Taylor

address: Headington

institute: CBRG

summary: The established hierarchical model explaining co-occupancy of Polycomb repressor complexes 1 and 2 (PRC 1 and 2) at target loci proposes that the chromodomain of the polycomb protein, a core PRC1 subunit, recognises the H3K27me3 histone modification catalysed by PRC2. We used chromatin immunoprecipitation to analyse PRC1 occupancy at target loci in Eed-/- mouse embryonic stem cells (ESCs) that lack H3K27me3. Occupancy of the core PRC1 proteins Ring1B and Mel18 was strongly reduced, consistent with the hierarchical model. However, levels of H2A ubiquitylation (H2AK119u1), the histone modification catalysed by PRC1, were similar to wild-type cells, suggesting PRC1 recruitment is independent of H3K27me3. ChIP-sequencing analysis of Ring1B occupancy genome wide substantiated this conclusion, demonstrating significant Ring1B levels at polycomb target loci in Eed-/- ESCs. Thus PRC1 and PRC2 are recruited independently to sites that they co-occupy. We conclude that the primary function of H3K27me3 is to increase the residency of PRC1 at target loci and thereby to contribute to the stability of PRC1 mediated silencing.

overall_design:
Examination of Ring1B binding in WT, Eed ko and Input of ESCs
Examination of CBX7 in WT and Eed ko of ESCs

first: Ligia

last: Tavares

first: Neil

last: Brockdorff


title: NSD2 links dimethylation of histone H3 at lysine 36 to oncogenic programming [ChIP]

first: kaifu

last: chen

address: 1 Baylor plaza, suite 450A

summary: NSD2 (also named MMSET and WHSC1) is a histone lysine methyltransferase that is implicated in diverse diseases and commonly overexpressed in multiple myeloma due to a recurrent t(4;14) chromosomal translocation.  However, the precise catalytic activity of NSD2 is obscure, preventing progress in understanding how this enzyme influences chromatin biology and myeloma pathogenesis.  Here we show that dimethylation of histone H3 at lysine 36 (H3K36me2) is the principal chromatin-regulatory activity of NSD2.  Catalysis of H3K36me2 by NSD2 is sufficient for gene activation.  In t(4;14)-positive myeloma cells, the normal genome-wide and gene-specific distribution of H3K36me2 is obliterated, creating a chromatin landscape that selects for a transcription profile favorable for myelomagenesis.  Catalytically active NSD2 confers xenograft tumor formation and invasion capacity upon t(4;14)-negative cells and NSD2 promotes oncogenic transformation of primary cells in an H3K36me2-dependent manner.  Together our findings establish H3K36me2 as the primary product generated by NSD2, and demonstrate that genomic disorganization of this canonical chromatin mark initiates oncogenic programming.

overall_design: ChIP sequencing of H3K36me2 ChIP DNA from KMS11 and TKO2 cells using Illumina Solexa Genome Analyzer II single end sequencing protocol. The experiment contains two biological replicates of H3K36me2 ChIP DNA and input materials from KMS11 and TKO2 cells.

first: Alex

middle: J

last: Kuo

first: Peggie

last: Cheung

first: Kaifu

last: Chen

first: Barry

last: Zee

first: Josh

last: Lauring

first: Yuanxin

last: Xi

first: Ben

last: Park

first: Xiaobing

last: Shi

first: Mitomu

last: Kioi

first: Benjamin

middle: A

last: Garcia

first: Wei

last: Li

first: Or

last: Gozani


title: Identification of polyA sites in Drosophila melanogaster

status: Public on Nov 23 2011

city: Farmington

first: Brenton

last: Graveley

state: CT

address: 400 Farmington Avenue

department: Genetics and Genome Sciences

institute: University of Connecticut Health Center

summary:
The 3' ends of most Drosophila melanogaster genes are poorly annotated or are determined by only a single EST or cDNA clone.  To enhance the annotation of poly(A) site use in Drosophila, we performed deep sequencing on RNA isolated from 29 dissected tissues using an approach designed to enrich for poly(A) spanning reads.  From these experiments, we identified 1.4 million poly(A) spanning reads leading to the identification of many new poly(A) sites and the identification of many tissue-specific poly(A) sites.

For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: RNA from 29 dissected Drosophila melanogaster tissues (in duplicate) were used to prepare polyA enriched RNA-Seq libraries.  Briefly, total RNA was poly(A) selected, fragmented, and ligated to 5' and 3' RNA linkers.  These libraries were amplified using Illumina paired-end primers, and subsequently reamplified using a 3' primer complementary to the 3' adapter but containing 6 Ts at the 3' end.  The libraries were also multiplexed and up to 12 samples mixed per lane and sequenced on an Illumina GAIIx using paired-end 76 bp reads, or an illumina HiSeq 2000 using paired-end 100 bp reads.  All reads were mapped to the Drosophila melanogaster genome to identify unmapped reads.  Unmapped reads containing at least 10 A residues at the 3' end were identified, the terminal A residues trimmed, realigned to the genome to identify uniquely mapped reads.  Such reads were identified as polyA spanning reads

first: Peter

last: Smibert

first: Pedro

last: Miura

first: Jakub

middle: O

last: Westholm

first: Sol

last: Shenker

first: Gemma

last: May

first: Michael

last: Duff

first: Dayu

last: Zhang

first: Brian

middle: D

last: Eads

first: Joe

last: Carlson

last: Brown

first: Robert

last: Eisman

first: Justen

last: Andrews

first: Thomas

last: Kaufman

last: Cherbas

first: Eric

last: Lai

first: Susan

middle: E

last: Celniker


title: Gene expression in the olfactory epithelia of parasitic and reproductive adult sea lampreys

city: East Lansing

first: Scot

last: Libants

address: 13 Natural Resources Bldg.

department: Fisheries and Wildlife

institute: Michigan State University

summary: To determine gene expression differences in the olfactory epithelium of sea lamprey between sequential yet behaviorally distinct adult life history stages

overall_design: 2 samples: parasitic adults removed from fish in northern Lake Huron and Lake Michigan in February and March, and reproductive adults collected from Lake Huron and Lake Michigan tributaries in June

first: Kevin

last: Carr

first: Weiming

last: Li


title: The primary transcriptome of barley (Hordeum vulgare L.) chloroplasts

city: Cologne

first: Konrad

middle: U.

last: Förstner

state: North Rhine-Westphalia

address: Gleueler Str. 60

department: Information Services

institute: ZB MED - Information Centre for Life Sciences

summary: Gene expression in plastids of higher plants is dependent on two different transcription machineries, a plastid-encoded bacterial-type RNA polymerase (PEP) and a nuclear-encoded phage-type RNA polymerase (NEP), which recognize distinct types of promoters. The division of labor between PEP and NEP during plastid development and in mature chloroplasts is unclear due to a lack of comprehensive information on promoter usage. Here we present a thorough investigation into the distribution of PEP and NEP promoters within the plastid genome of barley (Hordeum vulgare L). Using a novel differential RNA sequencing approach, which discriminates between primary and processed transcripts, we obtained a genome-wide map of transcription start sites in plastids of mature first leaves. PEP-lacking plastids of the albostrians mutant allowed for the unambiguous identifications of NEP promoters. We observed that the chloroplast genome contains many more promoters than genes. According to our data, most genes (including genes coding for photosynthesis proteins) have both PEP and NEP promoters. We also detected numerous transcription start sites within operons indicating transcriptional uncoupling of genes in polycistronic gene clusters. Moreover, we mapped many transcription start sites in intergenic regions, as well as opposite to annotated genes demonstrating the existence of numerous non-coding RNA candidates.

overall_design: dRNA-seq analysis of total RNA from green and white plastids of the barley mutant line albostrians

first: Petya

last: Zhelyazkova

first: Cynthia

last: Sharma

middle: U

first: Karsten

last: Liere

first: Jörg

last: Vogel

last: Börner


title: Polycomb Protein Ezh1 Promotes RNA Polymerase II Elongation

city: Bethesda

first: Kambiz

last: Mousavi

state: MD

address: 50 South Drive, Room 1347

department: National Institute of Arthritis and Musculoskeletal and Skin Diseases

institute: National Institutes of Health

summary: Polycomb group (PcG) proteins initiate the formation of repressed chromatin domains and regulate developmental gene expression.  A mammalian PcG protein, Enhancer of Zeste homolog 2 (Ezh2), triggers transcriptional repression by catalyzing the addition of methyl groups onto lysine-27 of histone H3 (H3K27me2/3)1.  This action facilitates the binding of other PcG proteins to histone H3 and compaction of chromatin.  Interestingly, there exists a paralog of Ezh2, termed Ezh1, whose primary function remains unclear.  Here, we provide evidence for genome-wide association of Ezh1 with active epigenetic marks, RNA polymerase II (PolII) and mRNA production.  Ezh1 depletion reduced global PolII occupancy within gene bodies and resulted in delayed transcriptional activation during differentiation of skeletal muscle cells.  Conversely, ectopic expression of wild-type Ezh1 led to premature gene activation and rescued PolII-elongation defects in Ezh1-depleted cells.  Collectively, these findings reveal an unanticipated role of a PcG protein in promoting mRNA transcription.

overall_design: Examination of 3 different histone modifications, 3 modified forms of RNA polymerase II, Ezh1, Ezh2 and mRNA levels in a skeletal muscle cells at various developmental stages.

first: Hossein

last: Zare

first: A

last: Wang

first: Vittorio

last: Sartorelli


title: DNA double-strand breaks induced by high NaCl occur predominantly in gene deserts

status: Public on Nov 21 2011

city: Cincinnati

first: Iouri

last: Chepelev

state: OH

address: 3200 Vine St

institute: US Department of Veterans Affairs Medical Center

summary: High concentration of NaCl increases DNA breaks both in cell culture and in vivo. The breaks remain elevated as long as NaCl concentration remains high and are rapidly repaired when the concentration is lowered.  Repair of the breaks after NaCl is reduced is accompanied by formation of foci containing phosphorylated H2AX (γH2AX), which occurs around DNA double-strand breaks and contributes to their repair. By chromatin immunoprecipitation using anti-γH2AX antibody, followed by massive parallel sequencing (ChIP-Seq), we find that during repair of double–strand breaks induced by high NaCl, γH2AX is predominantly localized to regions of the genome devoid of genes (“gene deserts”), indicating that the high NaCl-induced double-strand breaks are located there. Localization to gene deserts helps explain why the DNA breaks are less harmful than are the random breaks induced by genotoxic agents such as UV radiation, ionizing radiation and oxidants.  We propose that the universal presence of NaCl around animal cells has directly influenced the evolution of the structure of their genomes.

overall_design: ChIP-Seq experiment to find locations of γH2AX in mouse genome

first: Natalia

middle: I

last: Dmitrieva

first: Kairong

last: Cui

first: Daniil

last: Kitchaev

first: Keji

last: Zhao

first: Maurice

middle: B

last: Burg


title: Histone Demethylase UTX-1 Regulates the Caenorhabditis elegans Lifespan by Targeting the Insulin/IGF-1 Signaling Pathway

status: Public on Nov 22 2011

first: Dali

last: Han

address: 5801 South Ellis Avenue

department: Department of Chemisty

summary: Epigenetic modifications are thought to be important for gene expression changes during development and aging. However, besides the Sir2 histone deacetylase in somatic tissues and H3K4 trimethylation in germlines, there is scant evidence implicating epigenetic regulations in aging. The insulin/IGF-1 signaling (IIS) pathway is a major lifespan regulatory pathway. Here we show that progressive increases in gene expression and loss of H3K27me3 on IIS components are due, at least in part, to increased activity of the H3K27 demethylase UTX-1 during aging. RNAi of the utx-1 gene extended the mean lifespan of C. elegans by ~30%, dependent on DAF-16 activity and not additive in daf-2 mutants. The loss of utx-1 increased H3K27me3 on the Igf1r/daf-2 gene and decreased IIS activity leading to a more "naive" epigenetic state. Like stem cell reprogramming, our results suggest that reestablishing epigenetic marks lost during aging might help "reset" the developmental age of animal cells.

overall_design: Examination of H3K27me3 in young and old worms without or with Utx-1 RNAi.

first: Chunuyu

last: Jin

first: Jing-Dong


title: Deep sequencing of MYC DNA-binding sites in Burkitt's lymphoma

status: Public on Nov 28 2011

city: Berlin

first: Volkhard

last: Seitz

address: Hindenburgdamm 30

department: Institute of Pathology

institute: Charité Universitätsmedizin

summary:
Background: MYC is a transcription factor encoded by the c-MYC gene (thereafter termed MYC). MYC is key transcription factor involved in many central cellular processes including ribosomal biogenesis. MYC is overexpressed in the majority of human tumours including aggressive B-cell lymphoma especially Burkitt's lymphoma. Although Burkitt's lymphoma is a highlight example for MYC overexpression due to a chromosomal translocation, no global analysis of MYC binding sites by chromatin immunoprecipitation (ChIP) followed by global next generation sequencing (ChIP-Seq) has been conducted so far in Burkitt's lymphoma.
Methodology/Principal Findings: ChIP-Seq was performed with a MYC-specific antibody giving rise to 7,054 predicted MYC binding sites after bioinformatics analysis of a total of 19 million sequence reads. In line with previous findings, binding sites accumulate in gene sets known to be involved in the ribosomal biogenesis, histone acetyltransferase and methyltransferase complexes and the cell cycle demonstrating a regulatory role of MYC in these processes. Unexpectedly, MYC binding sites also accumulate in genes typically expressed in mature B-cells. To assess the functional consequences of altered MYC binding, the ChIP-Seq data were supplemented with siRNA mediated knock-downs of MYC in BL cell lines followed by gene expression profiling. Interestingly, amongst others, genes involved in B-cell function were up-regulated in response to MYC silencing.
Conclusion/Significance: The 7,054 MYC-binding sites identified by our ChIP-Seq approach greatly extend the knowledge regarding MYC binding in Burkitt's lymphoma and sheds further light on the enormous complexity of the MYC regulatory network. Especially our observation that (i) many B-cell relevant genes are targeted by MYC and (ii) that MYC down-regulation leads to an up-regulation of B-cell genes highlights an interesting aspect of Burkitt´s lymphoma biology.

overall_design:
[ChIP-Seq] Analysis of MYC DNA binding sites by ChiP-Seq in 5 Burkitt´s lymphoma cell lines (Raji, Ramos, Blue1, BL41, CA46)

[mRNA expression profiling] siRNA-mediated knock-down of MYC was done employing the BL cell lines Raji, BL41 and Blue1 in order to detect MYC-driven gene expression changes. For this purpose, the cells were Amaxa-transfected using MYC smart pool siRNA and control siRNA (Thermo Scientific/Dharmacon, Erembodegem, Belgium), respectively.

first: Peter

last: Butzhammer

first: Burkhard

last: Hirsch

first: Jochen

last: Hecht

first: Ines

last: Guetgemann

first: Anke

last: Ehlers

first: Dido

last: Lenze

first: Elisabeth

last: Oker

last: Sommerfeld

first: Edda

last: Van der Wall

first: Christoph

last: Koenig

first: Christian

last: Zinser

first: Rainer

last: Spang

first: Michael

last: Hummel


title: Estimates of total imprint number withstand transcriptome test

city: Toronto

first: Brian

last: DeVeale

state: Ontario

address: 160 College Street, rm 1110

department: Molecular Genetics

institute: University of Toronto

summary: The number of transcripts where allelic bias is dependent parent-of-origin was predicted at 100-200 until two recent studies applied RNA-Seq to brain regions from reciprocally crossed inbred mouse strains and identified over a thousand novel imprinted loci, including hundreds present in only males or females. Reanalysis revealed that the vast majority of these novel loci are explained by technical and biological variation of the approach, and are not genuine cases of general or sex-specific parent-of-origin allelic expression. Independent replication projects that, at most, a few dozen novel imprinted transcripts are present in the dataset, in line with previous projections of 100-200 total imprinted transcripts.

overall_design: Whole brain transcriptome analysis of E17.5 F1 embryos from reciprocally crossed C57BL/6J and CastEi/J parents

first: Tomas

last: Babak


title: NSD2 links dimethylation of histone H3 at lysine 36 to oncogenic programming

first: kaifu

last: chen

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

overall_design: Refer to individual Series


title: 5-hydroxymethylcytosine: the sixth DNA base

status: Public on Nov 24 2011

city: Nijmegen

last: Stunnenberg

address: Geert Grooteplein 28

department: Department of Molecular Biology (274)

institute: Radboud University

summary: We performed a meta analysis of publicly available TET1, 5mC, 5hmC and genome wide bisulfite profiling data mostly from mouse embryonic stem cells (ESC). Genome wide chromatin immunoprecipitation combined with deep sequencing (ChIP-seq) has revealed binding of the TET1 protein at CpG-island (CGI) promoters and at bivalent promoters. We show that TET1 also coincides with DNAseI hypersensitive sites (HS). Presence of TET1 at these THREE locations suggests that it may play a dual role: an active role at CpG-islands and DNAseI hypersensitive sites and a repressive role at bivalent loci. In line with the presence of TET1, significant enrichment of 5hmC but not 5mC is detected at bivalent promoters and DNaseI HS. Surprisingly, 5hmC is not detected or present at very low levels at CGI promoters notwithstanding the presence of TET1 at these loci. Our meta analysis suggest that asymmetric methylation is present at CA- and CT-repeats in the genome of some human ESC.

overall_design: Examination of the distribution of 5-methylcytosine and 5-hydroxymethylcytosine in the genome of mouse embryonic stem cells.

first: Henk

middle: G

first: Enrique

last: Carrillo-de Santa Pau

first: Filomena

last: Matarese


title: tRNAs marked with CCACCA are targeted for degradation

status: Public on Nov 17 2011

city: Cambridge

first: Jeremy

last: Wilusz

state: MA

address: 77 Massachusetts Ave, Bldg 76-461

department: Koch Institute for Integrative Cancer Research

institute: Massachusetts Institute of Technology

summary: The CCA-adding enzyme adds CCA to the 3' ends of transfer RNAs (tRNAs), a critical step in tRNA biogenesis that generates the amino acid attachment site. We found that the CCA-adding enzyme plays a key role in tRNA quality control by selectively marking unstable tRNAs and tRNA-like small RNAs for degradation. Instead of adding CCA to the 3' ends of these transcripts, CCA-adding enzymes from all three kingdoms of life add CCACCA. Here, we report deep sequencing analysis of the 3' ends of tRNA-Ser-CGA and tRNA-Ser-UGA from S. cerevisiae strains and show that hypomodified mature tRNAs are subjected to CCACCA (or poly(A) addition) as part of a rapid tRNA decay pathway in vivo. We conjecture that CCACCA addtion is a universal mechanism for controlling tRNA levels and preventing errors in translation.

overall_design: 121 samples analyzed in total, representing time courses of 10 different yeast strains; Biological replicates for each time point are included

first: Joseph

middle: M

last: Whipple

first: Eric

last: Phizicky

first: Phillip

last: Sharp


title: Comprehensive identification of long non-coding RNAs expressed during zebrafish embryogenesis

city: Copenhagen E

first: Eivind

last: Valen

state: Select

address: Ole Maaloes Vej 4

department: Dep. of Biology

country: Denmark

institute: University of Copenhagen

first: Andrea

last: Pauli

middle: F

last: Lin

first: Manuel

last: Garber

first: Nadine

last: Vastenhouw

first: Joshua

middle: Z

last: Levin

first: Albin

last: Sandelin

first: John

last: Rinn

first: Aviv

last: Regev

first: Alexander

last: Schier


title: NKX3-1, a Novel Transcriptional Factor of AR, Promotes Prostate Cancer Cell Survival via RAB3B GTPase-mediated protein trafficking

status: Public on Nov 16 2011

city: singapore

first: Peck Yean

last: TAN

address: 60 Biopolis Street #02-01 Genome

country: Singapore

institute: Genome Institute of Singapore (GIS)

summary: Androgen receptor (AR) orchestrates an intricate transcriptional regulatory network that governs prostate cancer initiation, development and progression. To understand this network in detail, we generated genome-wide maps of AR occupancy by ChIP-seq in LNCaP cells. We found NKX3-1, an androgen-dependent homeobox protein well-characterized for its role in prostate development and differentiation, being recruited to AR binding sites (ARBS) in response to androgen signaling. We identified 6,359 NKX3-1 binding sites, most of which overlapped with AR. In addition to its novel collaborative transcriptional role at well-known prostate cancer model genes, our binding and knockdown studies further suggested that NKX3-1 potentially regulates AR in a feed-forward manner. Integrative analysis of Oncomine molecular concepts showed that these androgen-regulated AR and NKX3-1 associated genes are significantly overexpressed in prostate carcinoma as well as advanced and recurrent prostate tumors. From our transcriptomic profiling and Gene Ontology analysis, we observed that AR and NKX3-1 co-regulate genes involved in "protein trafficking" processes, which are mandatory events in the integration of oncogenic signaling pathways leading to prostate cancer development and progression. Interestingly, we found that AR and NKX3-1 co-regulate several members of the RAB GTPase family of secretory/trafficking proteins via the involvement of FoxA1 in a ternary complex and we believe that these AR/NKX3-1/FoxA1 co-regulated RAB genes could serve as expression signatures in prostate carcinogenesis.  More specifically, through functional analyses, we showed that NKX3-1, together with AR and FoxA1, could promote prostate cancer cell survival through activation of RAB3B expression. Collectively, our study has provided important insights into the hierarchical transcriptional regulatory network established between AR and NKX3-1 and sought to elucidate the important genetic-molecular-phenotypic paradigm in androgen-dependent prostate cancer.

overall_design: Genome-wide binding analyses of AR, NKX3-1 and FoxA1 in LNCaP with or without DHT (5alpha-dihydrotestosterone) stimulation using ChIP-Seq.

first: Peck

middle: Y

last: Tan

first: Cheng

middle: W

last: Chang

first: Kern

middle: R

last: Chng

first: Edwin

last: Chueng


title: Comprehensive identification of long non-coding RNAs expressed during zebrafish embryogenesis [RNA_seq]

summary: Long non-coding RNAs (lncRNAs) comprise a diverse class of transcripts that structurally resemble mRNAs but do not encode proteins. Recent genome-wide studies in human and mouse have annotated lncRNAs expressed in cell lines and adult tissues, but a systematic analysis of lncRNAs expressed during vertebrate embryogenesis has been elusive. To identify lncRNAs with potential functions in vertebrate embryogenesis, we performed a time series of RNA-Seq experiments at eight stages during early zebrafish development. We reconstructed 56,535 high-confidence transcripts in 28,912 loci, recovering the vast majority of expressed RefSeq transcripts, while identifying thousands of novel isoforms and expressed loci. We defined a stringent set of 1,133 non-coding multi-exonic transcripts expressed during embryogenesis. These include long intergenic ncRNAs (lincRNAs), intronic overlapping lncRNAs, exonic antisense overlapping lncRNAs, and precursors for small RNAs (sRNAs). Zebrafish lncRNAs share many of the characteristics of their mammalian counterparts: relatively short length, low exon number, low expression, and conservation levels comparable to introns. Subsets of lncRNAs carry chromatin signatures characteristic of genes with developmental functions. The temporal expression profile of lncRNAs revealed two novel properties: lncRNAs are expressed in narrower time windows than protein-coding genes and are specifically enriched in early-stage embryos. In addition, several lncRNAs show tissue-specific expression and distinct subcellular localization patterns. Integrative computational analyses associated individual lncRNAs with specific pathways and functions, ranging from cell cycle regulation to morphogenesis. Our study provides the first comprehensive identification of lncRNAs in a vertebrate embryo and forms the foundation for future genetic, genomic and evolutionary studies.

overall_design: RNA-Seq for 8 zebrafish developmental stages, 2 lanes for each stage (3 for shield).

first: Michael

middle: L


title: Harnessing of the Nucleosome Remodeling Deacetylase complex controls lymphocyte development and prevents leukemogenesis

status: Public on Nov 13 2011

first: Jiangwen

last: Zhang

address: 7 Divinity Ave

department: FAS Center for Systems Biology

institute: Harvard University

summary:
Cell fate decisions depend on the interplay between chromatin regulators and transcription factors. Here we show that activity of the Mi-2u Nucleosome Remodeling and Deacetylase (NuRD) complex is controlled by the Ikaros family of lymphoid-lineage determining proteins. Ikaros, an integral component of the NuRD complex in lymphocytes, tethers this complex to active lymphoid differentiation genes, but keeps it in a functionally-poised state. Loss in Ikaros DNA binding activity causes a local increase in Mi-2u chromatin remodeling, histone deacetylation and suppression of lymphoid gene expression. The NuRD complex also redistributes to transcriptionally-poised non-Ikaros gene targets, involved in proliferation and metabolism, inducing their re-activation. Thus release of NuRD from Ikaros regulation blocks lymphocyte maturation and mediates progression to a leukemic state by engaging functionally-opposing epigenetic mechanisms and genetic networks.
We used microarrays to detail the global programme of gene expression of mouse DP thymocyte after Ikaros inactivation with dominant negative of Ik at different stage.

overall_design: 8 samples (mouse DP thymocytes from wt, and different stage after Ikaros inactivation) are analyzed Mouse Microarray Expression platforms, Affymetrix Mouse 430 2.0. Examination of different histone modifications and binding sites for Ikaros, Mi2beta in wild type DP thymocytes, and Ikaros knockout thymocytes.

first: Audrey

last: Jackson

first: Taku

last: Naito

last: Seavitt

first: Elizabeth

middle: J

last: Heller

first: Feifei

last: Liu

first: Marei

last: Dose

first: Mariko

last: Kashiwagi

first: Toshimi

last: Yoshida

first: Fotini

last: Gounari

first: Howard

last: Petrie

first: Katia

last: Georgopoulos


title: Embryonic stem cell based system for the discovery and mapping of developmental transcriptional programs

status: Public on Nov 12 2011

city: University Park

first: Shaun

last: Mahony

state: PA

address: 404 South Frear Bldg

department: Biochemistry & Molecular Biology

institute: Penn State University

summary: Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) is the method of choice to study function of transcription factors in cell fate determination. This technique faces two major obstacles when applied to developmental studies: the availability of ChIP grade antibodies and access to sufficient quantities of the cells of interest. We present a robust method for the genome-wide analysis of transcription factor binding during development in highly homogenous cells in defined developmental states.  It combines efficient embryonic stem cell (ESC) differentiation protocols with an inducible system of tagged transcription factors to enable affinity based assays such as ChIP-seq during lineage specific development.  To validate the system, we compared the activity and genomic binding of native and V5-tagged Olig2 in motor neuron progenitors and Flag-tagged and V5-tagged Hoxc9 in motor neurons.  We find that tagging transcription factors and expressing them alongside their endogenous counterparts does not alter their function or genomic association. The technology presented here can be applied to known as well as novel DNA-binding proteins. In combination with a suitable ESC differentiation paradigm it can be applied to determine how lineage-specific transcriptional networks are established and regulated.

overall_design: The aim of this study is to validate a platform for analysis of transcription factor binding that combines directed differentiation of ES cells with an inducible system of tagged transcription factors. Here, we perform ChIP-seq analysis to compare binding profiles of Olig2 as found using a native antibody and anti-V5 against the epitope tag. We also compare the ChIP-seq profiles of Hoxc9 as found using two independent epitope tags (V5 and FLAG). In all, there are 6 Illumina sequence datasets in this submission, including one replicate for each of native Olig2, iOlig2-V5 and FLAG-iHoxc9, two replicates of iHoxc9-V5, and a pseudo-ChIP control using anti-V5 in a non-induced iOlig2 cell-line.  The differentiation of ventral motor neurons is induced by treating embryonic stem cell cultures with retinoic acid and hedgehog.

first: Esteban

last: Mazzoni

first: Michelina

last: Iacovino

first: Carolyn

middle: A

last: Morrison

first: George

last: Mountoufaris

last: Closser

first: Warren

last: Whyte

first: Richard

last: Young

last: Kyba

first: David

middle: K

last: Gifford

first: Hynek

last: Wichterle


title: Initiation Pausing of 80S Ribosome on mRNA Is Governed by 5'UTR and Responsive to mTORC1 Signaling (RNA-Seq)

city: Ithaca

first: Shu-Bing

last: Qian

state: NY

address: 301 Biotech Bldg.

department: Division of Nutritional Sciences

institute: Cornell University

summary: Recent studies have revealed that the mRNA translation is punctuated by ribosomal pauses through the body of transcripts.  However, little is known about its physiological significance and regulatory aspects.  Here we present a multi-dimensional ribosome profiling approach to quantify the dynamics of initiation and elongation of 80S ribosomes across the entire transcriptome in mammalian cells.  We show that a subset of transcripts have a significant pausing of 80S ribosome around the start codon, creating a major barrier to the commitment of translation elongation.  Intriguingly, genes encoding ribosome proteins themselves exhibit an exceptionally high initiation pausing on their transcripts.  Our studies also reveal that the initiation pausing is dependent on the 5’ untranslated region (5’ UTR) of mRNAs and subject to the regulation of mammalian target of rapamycin complex 1 (mTORC1).  Thus, the initiation pausing of 80S ribosome represents a novel regulatory step in translational control mediated by nutrient signaling pathway.

overall_design: Monitor the translational status of transcriptome in mammalian cells under different conditions

first: Yan

last: Han

first: Botao

last: Liu

first: Huifeng

last: Jiang

first: Jun

last: Sun

first: Zhenglong

last: Gu


title: VDR/RXR and TCF4/beta-Catenin Cistromes in Colonic Cells of Colorectal Tumor Origin: Impact on c-FOS and c-MYC Gene Expression

city: Madison

first: Mark

last: Meyer

state: WI

address: 1415 Linden Dr.

department: Nutritional Sciences

institute: University of Wisconsin-Madison

summary: Many of the transcriptional and growth regulating activities of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the intestine and colon are recapitulated in the human colorectal cancer cell LS180.  We therefore used this line together with ChIP-seq and gene expression analyses to identify the vitamin D receptor (VDR)/retinoid x receptor (RXR) and TCF7L2(TCF4)/β-catenin cistromes and the genes that they regulate.  VDR and RXR co-localized to predominantly promoter distal, VDRE-containing sites in a largely ligand-dependent manner.  These regulatory sites control the expression of both known as well as novel 1,25(OH)2D3 target genes.  TCF4 and β-catenin cistromes partially overlapped, contained TCF/LEF consensus elements, and were only modestly influenced by 1,25(OH)2D3.  However, the two heterodimer complexes co-localized at sites near a limited set of genes that included c-FOS and c-MYC; the expression of both genes was modulated by 1,25(OH)2D3.  At the c-FOS gene, both VDR/RXR and TCF4/β-catenin bound to a single distal enhancer located 24 kb upstream of the transcriptional start site.  At the c-MYC locus, however, binding was noted at a cluster of sites between -139 and -165 kb and at a site located -335 kb upstream.  Examined as isolated enhancer fragments, these regions exhibited basal and 1,25(OH)2D3-inducible activities that were interlinked to both VDR and β-catenin activation.  These data reveal additional complexity in the regulation of target genes by 1,25(OH)2D3 and support a direct action of both VDR and the TCF4/β-catenin regulatory complex at c-FOS and c-MYC.

overall_design: 13 samples are analyzed by ChIP-seq. The Input was used as the normalizing factor for all transcription factor IPs. A minimum of 2 replicates were run per antibody and condition. After analysis, these lanes were combined for greater read depth. RNA was isolated from biological replicate experiments for a total of 6 samples in this study (3x 0hr, 3x 24hr), reverse transcribed and labeled according to Nimblegen Systems, Inc. (Madison, WI) protocols.

first: Paul

middle: D

last: Goetsch

first: J

last: Pike


title: Initiation Pausing of 80S Ribosome on mRNA Is Governed by 5’UTR and Responsive to mTORC1 Signaling


title: Maternal siRNAs as regulators of parental genome imbalance and gene expression in endosperm of Arabidopsis seeds.

status: Public on Nov 11 2011

city: Austin

first: Z Jeffrey

middle: Jeffrey

last: Chen

address: 2506 Speedway NMS 3.122 Stop A500

department: Molecular Biosciences

institute: The University of Texas at Austin

summary: Seed size is important to crop domestication and natural selection and is affected by the balance of maternal and paternal genomes in endosperm. Endosperm, like placenta in mammals, provides reserves to the developing embryo. Interploidy crosses disrupt the genome balance in endosperm and alter seed size. Specifically, paternal-excess crosses (2 × 4) delay endosperm cellularization (EC) and produce larger seeds, whereas maternal-excess crosses (4 × 2) promote precocious EC and produce smaller seeds. The mechanisms for responding to the parental genome dosage imbalance and for gene expression changes in endosperm are unknown. In plants, RNA polymerase IV (PolIV or p4) encoded by NRPD1a is required for biogenesis of a major class of 24-nt small interfering RNAs (also known as p4-siRNAs), which are predominately expressed in developing endosperm. Here we show that p4-siRNA accumulation depends on the maternal genome dosage, and maternal p4-siRNAs target transposable elements (TEs) and TE-associated genes (TAGs) in seeds. The p4-siRNAs correlate negatively with expression levels of AGAMOUS-LIKE (AGL) genes in endosperm of interploidy crosses. Moreover, disruption of maternal NRPD1a expression is associated with p4-siRNA reduction and AGL up-regulation in endosperm of reciprocal crosses. This is unique genetic evidence for maternal siRNAs in response to parental genome imbalance and in control of transposons and gene expression during endosperm development.

overall_design: 8 samples: 2x X 2x seed,leaf; 4x X 4x seed,leaf; 2x X 4x seed,leaf; 4x X 2x seed,leaf.

first: Jie

last: Lu

first: Changqing

last: Zhang

middle: C

last: Baulcombe

first: Zengjian


title: Genome-wide mapping of Tbx20 (GFP-tagged) in adult mouse whole heart

city: Chicago

first: Noboru

middle: Jo

last: Sakabe

state: IL

address: 920 E 58th st

department: Human Genetics

institute: University of Chicago

summary: Tbx20 is a transcription factor important for heart development. To assess the role of Tbx20 in the adult heart, we sought to identify regulatory regions, gene targets and pathways regulated by this TF. To this end, we used chromatin immunoprecipitation (ChIP) of Tbx20 fused to GFP to locate binding sites in the genome of 8 weeks whole heart mouse tissue.

overall_design: Analysis of 1 Tbx20-GFP ChIP sample of adult mouse whole heart against whole heart input

first: Ivy

last: Aneas

first: Marcelo

last: Nobrega

first: Sylvia

last: Evans

first: Tao

last: Shen


title: High-resolution profiling and analysis of viral and host small RNAs during human cytomegalovirus infection

status: Public on Nov 10 2011

city: La Jolla

first: Gene

last: Yeo

state: CA

address: 2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab

institute: UCSD

summary: Small RNA deep sequencing analysis was conducted on primary human fibroblasts infected with human cytomegalovirus (HCMV). HCMV-encoded miRNAs accumulated to ~20% of the total smRNA population at late stages of infection, and our analysis led to improvements in viral miRNA annotations and identification of novel HCMV miRNAs. Through crosslinking and immunoprecipitation of Argonaute-bound RNAs from infected cells, followed by high-throughput sequencing (Ago CLIP-seq), we obtained direct evidence for incorporation of all HCMV miRNAs into the endogenous host silencing machinery. Additionally, significant upregulation was observed during infection for a host miRNA cluster containing miR-96, miR-182 and miR-183. We also identified novel non-miRNA forms of virus-derived smRNAs, revealing greater complexity within the smRNA population during HCMV infection.

overall_design: High-throughput profiling of smRNAs,  Ago1-, and Ago2-associated miRNAs from HCMV-infected fibroblast cells. Wild-type HCMV Towne (Genbank FJ616285.1) was used for these studies.

first: G


title: Comprehensive identification of long non-coding RNAs expressed during zebrafish embryogenesis [ChIP_Seq]

first: Eivind

last: Valen

overall_design: ChIP-Seq for H3K4me3 and H3K27me3 at zebrafish shield stage.

first: Andrea

last: Pauli

first: Michael

last: Lin

first: Manuel

last: Garber

first: Nadine

last: Vastenhouw

first: Joshua

last: Levin

first: Albin

last: Sandelin

first: John

last: Rinn

first: Aviv

last: Regev

first: Alexander

last: Schier


title: The transcriptome of Aedes aegypti females from two strains (Chetumal and Rex-D Puerto Rico) either kept on a sugar diet or blood fed

city: Irvine

first: Anthony

last: James

address: 2315 McGaugh Hall

department: Molecular Biology and Biochemistry

institute: University of California, Irvine

summary: Strain variation in Aedes aegypti transcriptome

overall_design: We report RNA-seq analyses of transcriptional changes across two Ae. aegypti strains

first: Osvaldo

last: Marinotti

first: Mariangela

last: Bonizzoni

first: William

last: Augustine Dunn


title: Histone Deacetylase 3 is an Epigenomic Brake in Macrophage Alternative Activation (ChIP-Seq)

city: RTP

first: Logan

last: Everett

state: North Carolina

address: 109 T.W. Alexander Dr

department: Office of Research and Development

institute: U.S. Environmental Protection Agency

summary: We report the genomic regions enriched in Histone Deacetylase 3 (HDAC3) in mouse bone marrow derived macrophages.  Furthermore, we also report the genomic acetylation pattern on Histone 3, Lysine 9 (H3K9) in macrophages with and without HDAC3 and/or treated with Th2 cytokine IL-4.

overall_design: HDAC3 enriched genomic regions in mouse bone marrow dervied macrophages and H3K9Ac enriched genomic regions in wild-type macrophages and macrophages treated with IL-4 and/or deficient in HDAC3.

first: Shannon

middle: E

last: Mullican

first: Christine

last: Gaddis

first: Theresa

last: Alenghat

first: Meera

last: Nair

first: Paul

last: Giacomin

first: Dan

last: Feng

first: David

last: Steger

first: Jonathan

last: Schug

last: Artis

first: Mitchell

last: Lazar


title: A genome-wide map of CTCF multivalency redefines the CTCF code

status: Public on Nov 19 2011

city: bethesda

first: Seolkyoung

last: Jung

address: 10 Center Dr

department: NIAMS

institute: NIH

summary: The “CTCF code” hypothesis posits that CTCF pleotropic functions are driven by recognition of diverse DNA sequences through combinatorial use of its 11 zinc fingers (ZFs). This model however is supported by in vitro binding studies of a limited number of sequences. To directly test CTCF multivalency in vivo we here define ZF binding requirements at ~50,000 genomic sites in primary lymphocytes. We find that CTCF reads sequence diversity through ZF clustering. ZFs4-7 anchor CTCF to ~80% of targets containing the 20bp core motif. Non-conserved flanking sequences are recognized by ZFs1-2 and ZFs8- 11 clusters, which also stabilize CTCF broadly. Alternatively, CTCF employ ZFs9-11 to associate with a second phylogenetically-conserved upstream motif at ~15% of its sites. Individually, ZFs increase overall binding affinity and chromatin residence time. Unexpectedly, we also uncover a conserved downstream DNA motif that destabilizes CTCF occupancy. CTCF thus associates with a wide array of DNA modules via combinatorial clustering of its 11 ZFs.

sample_organism:
Mus musculus
Mus spretus

platform_organism:
Mus musculus
Mus spretus

overall_design: ChIP-Seq of wt and mutant CTCF in activated  mouse B cells.  For the comparison of wt and mutant CTCF binding  profiles, CTCF fused to a biotag was overexpressed in the presence of  the endogenous wt CTCF copies together with BirA.  The  exongenous CTCF was then precipitated using streptavidin beads.  For  the comparison of wt CTCF binding in Mus musculus C57BL/6 and Mus spretus, endogenous CTCF was precipitated with an anti-CTCF antibody. All these experiments were carried out in triplicate.  In addition,  several overexpressed mutants were subjected to exonuclease treatment prior to sequencing (ChIP-Exo).

first: Hirotaka

last: Nakahashi

first: Kyong-Rim

last: Kwon

first: Wolfgang

last: Resch

first: Laura

last: Vian

first: Marei

last: Dose

first: Diana

last: Stavreva

first: Ofir

last: Hakim

first: Nathanael

last: Pruett

first: Steevenson

last: Nelson

first: Arito

last: Yamane

first: Jason

last: Qian

first: Wendy

last: Dubois

first: Scott

last: Welsh

first: Robert

last: Phair

first: Franklin

last: Pugh

first: Victor

last: Lobanenkov

first: Gordon

last: Hager

first: Rafael

last: Casellas


title: Hen1 analysis in zebrafish

status: Public on Nov 09 2011

city: Groningen

first: Eugene

last: Berezikov

address: Antonius Deusinglaan 1

department: European Research Institute for the Biology of Ageing

country: Netherlands

institute: University Medical Center Groningen

summary: small RNA libraries from wild-type and Hen1 mutant testes were made with either polyA tailing (VASAGFPHen1minus/plus) or adapter ligation (Hen1Testis and WTTestis) and sequenced on an Illumina GAII platform.

overall_design: RNA was isolated from total testis tissue of both Hen1 wildtype and Hen1 mutant animals. After size selection from gel, the small RNA libraries wre made.

first: Rene

last: Ketting


title: Nucleosome repositioning-coupled expression divergence between species

first: Kyle

last: Tsui

address: 160 College Street

summary: We describe the genome-wide nucleosome profiles for S. bayanus under standard conditions

overall_design: Nucleosomes from S. bayanus was isolated and sequenced using the Illumina GAII platform.

first: Yuanfang

last: Guan

first: Vicky

last: Yao

first: Marinella

last: Gebbia

first: Amy

last: Caudy

first: Maitreya

last: Dunham

first: Corey

last: Nislow

first: Olga

last: Troyanskaya


title: Activin/Nodal signalling controls divergent transcriptional networks in pluripotent and endoderm progenitors.

first: Matthew

last: Trotter

address: West Forvie Building, Forvie Site, Robinson Way

department: Department of Surgery

institute: University of Cambridge

summary: Activin/Nodal signalling is necessary to maintain pluripotency of human Embryonic Stem Cells (hESCs) and to induce their differentiation towards endoderm. However, the mechanisms by which Activin/Nodal signalling achieves these opposite functions remain unclear. To unravel these mechanisms, we examined the transcriptional network controlled in hESCs by Smad2 and Smad3 which represent the direct effectors of Activin/Nodal signalling. These analyses reveal that Smad2/3 participate in the control of the core transcriptional network characterising pluripotency which includes Oct-4, Nanog, FoxD3, Dppa4, Tert, Myc and UTF-1. In addition, similar experiments performed on endoderm cells confirm that a broad part of the transcriptional network directing differentiation is downstream of Smad2/3. Therefore, Activin/Nodal signalling appears to control divergent transcriptional networks in hESCs and in endoderm. Importantly, we observed an overlap between the transcriptional network downstream of Nanog and Smad2/3 in hESCs while functional studies showed that both factors cooperate to control the expression of pluripotency genes. Therefore, the effect of Activin/Nodal signalling on pluripotency and differentiation could be dictated by tissue specific Smad2/3 partners such as Nanog, explaining the mechanisms by which signalling pathways can orchestrate divergent cell fate decisions.

overall_design: Identification of Smad2/3 binding sites in pluripotent hESCs. 5 ChIP-Seq samples including 1 input control sample and 4 ChIP samples (two conditions x two replicates).

first: Ludovic

last: Vallier

first: Stephanie

last: Brown


title: The Exoribonuclease Nibbler Controls 3' End Processing of MicroRNAs in Drosophila

city: Boston

first: Jui-Hung

last: Hung

address: 24 cummington st

institute: Boston University

summary: MicroRNAs (miRNAs) are endogenous noncoding small RNAs with important roles in many biological pathways; their generation and activity are under precise regulation. Emerging evidence suggests that miRNA pathways are precisely modulated with controls at the level of transcription, processing, and stability, with miRNA deregulation linked with diseases and neurodegenerative disorders. In the Drosophila miRNA biogenesis pathway, long primary miRNA transcripts undergo sequential cleavage to release the embedded miRNAs. Mature miRNAs are then loaded into Argonaute1 (Ago1) within the RNA-induced silencing complex (RISC). Intriguingly, we found that Drosophila miR-34 displays multiple isoforms that differ at the 3′ end, suggesting a novel biogenesis mechanism involving 3′ end processing. To define the cellular factors responsible, we performed an RNA interference (RNAi) screen and identified a putative 3′→5′ exoribonuclease CG9247/nibbler essential for the generation of the smaller isoforms of miR-34. Nibbler (Nbr) interacts with Ago1 and processes miR-34 within RISC. Deep sequencing analysis revealed a larger set of multi-isoform miRNAs that are controlled by nibbler. These findings suggest that Nbr-mediated 3′ end processing represents a critical step in miRNA maturation that impacts miRNA diversity.

overall_design: Total RNA was isolated using mirVana (Ambion) and size-fractionated by PAGE into 18-30nt. These were independently processed and sequenced using the Illumina GAII platform. In total, four libraries were analyzed.

first: Bo

last: Han

first: Zhiping

last: Weng

first: Phillip

last: Zamore

first: Stefan

last: Ameres


title: Role of FliL in Swarmer Cell Differentiation

status: Public on Nov 15 2011

city: Knoxville

first: Kathleen

last: Cusick

state: TN

address: 1414 West Cumberland Ave

institute: Univ of Tenn

summary: Proteus mirabilis is an opportunistic pathogen that causes urinary tract infections in immunocompromised patients. Flagellar motility coupled with the ability to differentiate from short swimmer cells to highly elongated swarmer cells constitute key parts of the infection process. The mechanism by which the cells transduce the signal to differentiate remains unknown, but is believed to involve inhibition of flagellar rotation. Recent work has implicated FliL as a critical component of the signal transduction process, possibly through interactions with the motor proteins. The objective in the current study was to define the molecular mechanism underlying the cellâs ability to perceive a surface and begin the differentiation process, with a specific emphasis as to the role of FliL. Thus, the deep-sequencing technology of RNA-Seq was combined with genetic analysis to further examine the role of FliL in swarmer cell differentiation. RNA-Seq analysis of wild-type swarmer cells confirmed prior reports of genes involved in the differentiation process while concomittantly identifying novel genes potentially involved in swarming (including PMI1628, PMI0714-0716, and PMI0735, PMI0737, PMI0739, and PMI0740-0734). In comparison, RNA-Seq analysis of a fliL mutant showed that nearly all genes associated with the flagellar cascade and chemotaxis are repressed. A phenotypic, genetic, and transcriptomic characterization was then performed on spontaneous revertants of the fliL mutant, in which motility was restored yet still resulted in cells that differentiated under non-inducing (i.e. broth) conditions. Sequencing revealed perturbations in the C-terminus of FliL in both the mutant and revertant. Collectively, the data suggest that FliL senses the torque applied to the motor proteins- such as is encountered by a solid surface - and relays this signal to the master flagellar regulator FlhD4C2, triggering the expression of genes involved in the flagellar cascade.

overall_design: Transcriptomic comparison between wild-type Proteus mirabilis swarmer cells and a fliL mutant via RNA-Seq.

first: Yi-Ying

last: Lee

first: Brian

last: Youchak

first: Robert

last: Belas


title: ChIP-seq to identify the positions of three histone modifications in the rice genome

first: Yufeng

last: Wu

address: 1575 Linden Drive

institute: UW-Madison

summary: Gene expression is controlled by the complex interaction of transcription factors binding to promoters and other regulatory DNA elements.  One common characteristic of the genomic regions associated with regulatory proteins is a pronounced sensitivity to DNase I digestion.  We generated genome-wide high resolution maps of DNase I hypersensitive (DH) sites from both seedling and callus tissues of rice.  Approximately 25% of the DH sites from both tissues were found in the putative promoters, indicating that the vast majority of gene regulatory elements in rice are not located at promoter regions.  We found 58% more DH sites in callus than in seedling.  For DH sites detected in both seedling and callus, 31% displayed significantly different levels of DNase I sensitivity within the two tissues.  Genes that were differentially expressed in seedling and callus were frequently associated with DH sites in both tissues.  The DNA sequences contained within the DH sites were hypomethylated, consistent with what is known about active gene regulatory elements.  Interestingly, tissue-specific DH sites located in the promoters showed an elevated level of DNA methylation.  A distinct elevation of H3K27me3 was associated with intergenic DH sites.  These results suggest that epigenetic modifications play a role in the dynamic changes of the numbers and DNase I sensitivity of DH sites during development.

overall_design: To do associated analysis with DNase I hypersensitive sites in rice, we performed ChIP-seq to identify the positions of three histone modifications (H3K4me2, H3K36me3 and H4K12ac) in the rice genome (leaf tissue only - not callus). The ChIP DNA from seedling of each experiment was sequenced on one lane of Illumina Genome Analyzer.

first: Wenli

last: Zhang

first: James

last: Schnable

first: Zixian

last: Zeng

first: Michael

last: Freeling

first: Gregory

last: Crawford

first: Jiming

last: Jiang


title: Transcriptome of rice seedling and callus based on RNA-seq method

summary: To support our research of epigenomics in rice genome, we conducted massively parallel pyrosequencing of mRNAs (RNA-seq) using rice seedling and callus tissues. We obtained a total of 40.1 M reads from seedling and 29.6 M reads from callus.The RNA-seq data derived from the replicates showed high correlations (Spearman’s rank correlation coefficient, SC, 0.96 for seedling, 0.88 for callus). Our RNAseq data derived from the seedling also showed a high correlation with the recently published rice RNA-seq data that was also from two-week old seeding tissue (Lu et al., 2010, PMID: 20627892) (SC=0.87).

overall_design: Two biological replicates of each tissue was applied in RNA-seq. Each biological replicate was sequenced twice in separate lane.


title: Genome-wide High Resolution Mapping of Active Regulatory DNA Elements in the Rice Genome

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

overall_design: Refer to individual Series


summary:
Gene expression is controlled by the complex interaction of transcription factors binding to promoters and other regulatory DNA elements.  One common characteristic of the genomic regions associated with regulatory proteins is a pronounced sensitivity to DNase I digestion.  We generated genome-wide high resolution maps of DNase I hypersensitive (DH) sites from both seedling and callus tissues of rice.  Approximately 25% of the DH sites from both tissues were found in the putative promoters, indicating that the vast majority of gene regulatory elements in rice are not located at promoter regions.  We found 58% more DH sites in callus than in seedling.  For DH sites detected in both seedling and callus, 31% displayed significantly different levels of DNase I sensitivity within the two tissues.  Genes that were differentially expressed in seedling and callus were frequently associated with DH sites in both tissues.  The DNA sequences contained within the DH sites were hypomethylated, consistent with what is known about active gene regulatory elements.  Interestingly, tissue-specific DH sites located in the promoters showed an elevated level of DNA methylation.  A distinct elevation of H3K27me3 was associated with intergenic DH sites.  These results suggest that epigenetic modifications play a role in the dynamic changes of the numbers and DNase I sensitivity of DH sites during development.

For RNA-seq used in this project, please check GEO data GSE33265.

overall_design: Generation of genome-wide high resolution maps of DNase I hypersensitive sites in two tissues of rice. For seedling, we constructed 3 libraries (biological replicates) and sequenced a lane of Illumina Genome Analyzer for each library. For callus, we constructed 2 libraries (biological replicates). We sequenced two lanes for one library and one lane for another library.