title: Promiscuous RNA binding by PRC2

city: Boulder

last: Davidovich

state: Colorado

address: 596 UCB, JSCBB, 3415 Colorado Ave

institute: CU Boulder

summary: Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Long non-coding RNAs (lncRNAs) recruit PRC2 to chromatin, but the general role of RNA in maintaining repressed chromatin is unknown. ChIP-seq, combined with RNA-seq, indicating that PRC2 is also associated with active genes, but most of these are not regulated by PRC2. These results were complemented by in vitro binding assays measuring the binding constant of human PRC2 to various RNAs and find comparable affinity for human lncRNAs targeted by PRC2 and irrelevant transcripts from ciliates and bacteria. PRC2 binding is size-dependent, with lower affinity for shorter RNAs. These findings support a model in which promiscuous binding of PRC2 to RNA transcripts allows it to scan for target genes that have escaped repression, leading to maintenance of the repressed state. Such RNAs may also provide a decoy for PRC2.

overall_design: Cell culture: HEK293T/17 cells were cultured in DMEM with 10% FBS for no longer than 15 passages. ON-TARGETplus SMARTpool for human SUZ12 (Thermo Scientific, Dharmacon cat # L-006957-00-0005) was used to knockdown SUZ12 (siSUZ12) and ON-TARGETplus Non-targeting Pool (Dharmacon cat # D-001810-10-05) was used as a negative control (siCtrl). A total of 25 nM siRNA was transfected in 6-well dishes using Lipofectamine™ RNAiMAX Reagent (Life Technologies, Invitrogen) following the manufacturer’s recommendations. RNA-seq: Polyadenylated RNA was purified from 4 ug of RNA. cDNA libraries were prepared and double-stranded cDNA was fragmented using DNase I according to Illumina specifications, prior to adaptor ligation. Sequencing libraries were amplified and sequenced using an Illumina HiSeq 2000 sequencer. ChIP-seq: 30 million cells at 80% to 90% confluent culture were crosslinked in 1% v/v formaldehyde for 10 min, quenched in 150 mM glycine, washed with cold 1xPBS and harvested by scraping. Cells were lysed in Lysis Buffer (1% w/v SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1) with 1x Complete® protease inhibitors (Roche). Cells were sonicated for 10 to 15 min using a Bioruptor™ UCD-200 (Diagenode) with 30 sec pulses at maximum power. Lysates were diluted to 10 ml in IP Buffer (0.01% w/v SDS, 1.1% v/v Triton-X, 1.2 mM EDTA, 16.7 mM Tris-HCL pH 8.0, 167 mM NaCl) and 10 to 20 ng of antibodies were added and incubated overnight at 4 ⁰C with rotation. Antibodies were immunoprecipitated with 60 µl protein G Plus/protein A Agarose Suspension (Calbiochem cat # IP05) and washed sequentially with 1 ml Low Salt Wash Buffer (0.1% w/v SDS, 1% v/v Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), 1 ml High Salt Wash Buffer (0.1% w/v SDS, 1% v/v Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), 1 ml LiCl Wash Buffer (0.25 M LiCl, 1% v/v Nonidet P-40, 1% w/v deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0) and 1 ml TE buffer (Qiagen). DNA was eluted with 0.4 ml of 0.1 M NaHCO3 and 1% w/v SDS. Eluent was transferred into a new tube and NaCl was added to a final concentration of 200 mM. Crosslinks were reversed by incubation at 65 ⁰C for 2 hours. Next, proteins and RNA were digested by adding 33 µl of Digestion Reagent (0.6 M Tris-HCl pH 6.5, 152 mM EDTA, 61 ng/ml RNase A (Invitrogen cat # AM2274) and 0.61 mg/ml Proteinase K (NEB cat # P8102)) following by 1 hour incubation at 37 ⁰C. DNA was extracted by phenol:chloroform and ethanol precipitated. DNA was resuspended in Milli-Q pure water and concentration was measured using Qubit™ (Invitrogen). At least 10 ng of recovered DNA was used to synthesize sequencing libraries using the ChIP-seq Sample Preparation kit (Illumina). Between 6 and 10 pmoles were used for sequencing on the HiSeq2000 sequencer.

first: Thomas

middle: R

last: Cech


title: Ago2

status: Public on Sep 25 2013

city: Aurora

first: Matthew

last: Taliaferro

state: CO

address: 12801 E 17th Ave, RC1 South Room 10403D

department: Biology

country: USA

institute: University of Colorado Anschutz Medical Campus

type: Expression profiling by high throughput sequencing

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: mRNA-seq of Drosophila Ago2 mutants

summary: Transcription and pre-mRNA alternative splicing are highly regulated processes that play major roles in modulating eukaryotic gene expression. It is increasingly apparent that other pathways of RNA metabolism, including small RNA biogenesis, can regulate these processes. However, a direct link between alternative pre- mRNA splicing and small RNA pathways has remained elusive. Here we show that the small RNA pathway protein Argonaute-2 (Ago-2) regulates alternative pre-mRNA splicing patterns of specific transcripts in the Drosophila nucleus using genome-wide methods in conjunction with RNAi in cell culture and Ago-2 deletion or catalytic site mutations in Drosophila adults. Moreover, we show that nuclear Argonaute-2 binds to specific chromatin sites near gene promoters and negatively regulates the transcription of the Ago-2-associated target genes. These transcriptional target genes are also bound by Polycomb group (PcG) transcriptional repressor proteins and change during development, implying that Ago-2 may regulate Drosophila development. Impor- tantly, both of these activities were independent of the catalytic activity of Ago-2, suggesting new roles for Ago-2 in the nucleus. Finally, we determined the nuclear RNA-binding profile of Ago-2, found it bound to several splicing target transcripts, and identified a G-rich RNA-binding site for Ago-2 that was enriched in these transcripts. These results suggest two new nuclear roles for Ago-2: one in pre-mRNA splicing and one in transcriptional repression.

overall_design: 2 Ago2 mutants, 51B and V966M, heterozygotes and homozygotes of both each sequenced in duplicate

first: Donald

last: Rio


title: Highly efficient methane biocatalysis revealed in a methanotrophic bacterium

city: Seattle

first: David

middle: A. C.

last: Beck

state: WA

address: Benjamin Hall IRB, Rm 440, UW Box 355014

department: ChemE & eScience Institute

institute: University of Washington

summary: RNA-Seq profiling of Methylomicrobium alcaliphilum strain 20Z grown in batch on methane.  The RNA-Seq work is one part of a systems approach to characterizing metabolism of 20Z during growth on methane.  We demonstrate that methane assimilation is coupled with a highly efficient pyrophosphate-mediated glycolytic pathway, which under O2 limitation participates in a novel form of fermentation-based methanotrophy. This surprising discovery suggests a novel mode of methane utilization in oxygen-limited environments, and opens new opportunities for a modular approach towards producing a variety of excreted chemical products using methane as a feedstock.

overall_design: Four replicates of batch growth

first: Marina

middle: G

last: Kalyuzhnaya

middle: A


title: ChIP-seq of endogenous Ago2 in Drosophila S2 cells

overall_design: Input chromatin, 2 replicates of Ago2 ChIP-seq


title: Structural and functional characterization of the N-terminus of Schizosaccharomyces pombe Cwf10

city: Nashville

first: Melanie

last: Ohi

state: TN

address: 465 21st Avenue South

department: Cell and Developmental Biology

institute: Vanderbilt University Medical School

summary: The spliceosome is a dynamic macromolecular machine that catalyzes the removal of introns from pre-mRNA to make mature message. Schizosaccharomyces pombe Cwf10 (homolog Saccharomyces cerevisiae Snu114 and of Human U5-116K), an integral member of the U5 snRNP, is a GTPase that shares sequence homology with the eukaryotic translation elongation factor EF2. Cwf10 is required for pre-mRNA splicing; however, its mechanism(s) of action is still not understood. Cwf10/Snu114 family members contain a conserved N-terminal extension (NTE) that lacks homology with EF2 and has been predicted to be an intrinsically unfolded domain. Using S. pombe as a model system, we show that the NTE is not essential, but cells lacking this domain are defective in pre-mRNA splicing at all temperatures. Genetic interactions between cwf10-ΔNTE and other pre-mRNA splicing mutants are consistent with a role for the NTE in spliceosome activation. Characterization of Cwf10-NTE by various biophysical techniques shows the NTE contains both regions of structure and disorder in solution. The first twenty-three highly-conserved amino acids of the NTE are essential for its role in splicing, but are not sufficient to restore pre-mRNA splicing to wild-type levels in cwf10-∆NTE cells. When the NTE is overexpressed in the cwf10-ΔNTE background, it can complement the truncated Cwf10 protein in trans, and it also immunoprecipitates a complex similar in composition to the late-stage U5.U2/U6 spliceosome. These data show that the structurally flexible NTE is capable of making specific contacts within the spliceosome that may facilitate Cwf10’s overall role facilitating spliceosome rearrangements.

overall_design: Interrogation of the S. pombe transcriptome using poly-A enriched RNA sequencing (Illumina HiSeq 2500) in wild type and cwf10-ΔNTE cultures. A total of 4 samples were analysed: two biological repeats of wild-type strain and two biological repeats of cwf10-ΔNTE

first: S

middle: B

last: Livesay

first: Scott

middle: E

last: Collier

first: Danny

last: Bitton

first: Jürg

last: Bähler

middle: D


title: The Aurora B kinase and the polycomb protein Ring1B combine to regulate active promoters in quiescent lymphocytes [RNA-Seq]

status: Public on Sep 27 2013

city: London

first: Gopuraja

last: Dharmalingam

address: Faculty of Medicine, Imperial College, Hammersmith Hospital Campus

department: Epigenetics section

country: United Kingdom

institute: MRC London Institute of Medical Sciences

summary: Expression profiling of resting B cells to classify active and silent genes based on expression levels

overall_design: 4 biological replicates of mRNA extracted from freshly purified mouse CD43 negative resting B cells

first: Niall

last: Dillon

first: Alberto

last: Frangini


title: The Aurora B kinase and the polycomb protein Ring1B combine to regulate active promoters in quiescent lymphocytes [ChIP-seq]

summary: Genome-wide distribution of proteins and histone marks in CD43 negative mouse resting B cells

overall_design:
ChIP-seq analyses of Aurora B, Ring1B, Bcx7, USP16, H3K27me3,  and Ezh2 were carried out on wild-typeCD43 negative resting B cells. ChIP-seq datasets obtained from  Aurora B knockout and RingiB knockout cells were used as  negative controls to validate the signals for Aurora B and Ring1B  from wild-type cells. 8WG16 ChIP-seq datasets were generated from WT (Cre-ERt2 homozygous) and Ring1B KO (Cre-ERt2/Rnf2 flox homozygous). RNAP-S5ph ChIP-seq datasets were generated from WT (Cre-ERt2 homozygous) and Aurkb KO (Cre-ERt2/Aurkb flox homozygous).

ChIP-seq analyses of H3S28 phosphorylation (H3S28ph) and H2AK119 monoubiquitination (H2Aub1) in wild-type (Cre-ERt2 homozygous) and Aurora B KO (Cre-ERt2/Aurkb flox homozygous) CD43 negative resting B cells treated with 250nM tamoxifen (4-hydroxytamoxifen) for 48 hours. ChIP-seq analyses of the levels of unphosphorylated RNA Pol II (8WG16) and serine 5-phosphorylated RNA Pol II (RNAP-S5ph) were conducted in Aurkb KO (Cre-ERt2/Aurkb flox homozygous) and Ring1B KO (Cre-ERt2/Rnf2 flox homozygous) CD43 negative resting B cells treated with 250nM tamoxifen (4-hydroxytamoxifen) for 48 hours respectively). All libraries were prepared from sonicated, formaldehyde-crossilinked chromatin. For H2Aub1, ChIP was performed using micrococcal nuclease-digested, unfixed chromatin, instead.


title: RNA sequencing of Kineococcus radiotolerans transcriptome in response to ionizing radiation

status: Public on Sep 24 2013

city: Hangzhou

first: Jiangfeng

last: Guo

state: Zhejiang

address: Xiasha No.2 Road

department: College of Life Science

country: China

institute: Zhengjiang Sci-Tech University

summary: In this study, approximately 36 and 29 million raw reads of two samples, namely radiation treated strain and its untreated control, are acquired from the sequencing platform. And 143 genes are screened out with the differential expression (DE) analysis.

overall_design: Investigation of the differential expression between the radiation reduced sample and the wild-type sample in control.

first: Lufeng

last: Li


title: Transcription factor binding in human cells occurs in dense clusters formed around cohesin anchor sites [Validation Set]

city: Stockholm

first: Martin

last: Enge

address: CCK, Z4

department: Dep of Oncology-Pathology

country: Sweden

institute: Karolinska Institute

summary: During cell division, transcription factors (TFs) are removed from chromatin twice, during DNA synthesis, and during condensation of chromosomes. How TFs can efficiently find their sites following these stages has been unclear. Here, we have analyzed the binding pattern of expressed TFs in human colorectal cancer cells. We find that binding of TFs is highly clustered, and that the clusters are enriched in binding motifs for several major TF classes. Strikingly, almost all clusters are formed around cohesin, and loss of cohesin decreases both DNA accessibility and binding of TFs to clusters. We show that cohesin remains bound in S phase, holding the nascent sister chromatids together at the TF cluster sites. Furthermore, cohesin remains bound to the cluster sites when TFs are evicted in early M-phase. These results suggest that cohesin binding functions as a cellular memory that promotes re- stablishment of TF clusters after DNA replication and chromatin condensation.

overall_design: Examination of TF binding by ChIP-seq in a CRC cell-line.

first: Jian

last: Yan


title: Single cell Hi-C reveals cell-to-cell variability in chromosome structure

status: Public on Sep 26 2013

city: Rehovot

first: Yaniv

last: Lubling

address: POB 26

country: Israel

institute: Weizmann Institute of Science

summary: Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single cell Hi-C, combined with genome-wide statistical analysis and structural modeling of single copy X chromosomes, to show that individual chromosomes maintain domain organisation at the megabase scale, but show variable cell-to-cell chromosome territory structures at larger scales. Despite this structural stochasticity, localisation of active gene domains to boundaries of territories is a hallmark of chromosomal conformation, affecting most domains in most nuclei. Single cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organisation underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns.

overall_design: Mouse Th1 single-cell Hi-C maps were produced and paired-end sequenced. 10 single-cell samples and a multi-sample pool together with a population Hi-C sample are included.

first: Takashi

last: Nagano

first: Tim

middle: J

last: Stevens

first: Stefan

last: Schoenfelder

first: Eitan

last: Yaffe

first: Wendy

last: Dean

first: Ernest

last: Laue

first: Amos

last: Tanay

first: Peter

last: Fraser


title: High-throughput sequencing for microRNAs in uninjured and regenerating newt hearts

first: Jamie

middle: Ian

last: Morrison

address: Svante Arrheniusväg 20C

department: Department of Molecular Biosciences, The Wenner-Gren Institute

institute: Stockholm University

type: Non-coding RNA profiling by high throughput sequencing

summary: We have developed a standardized and reproducible heart resection/regeneration model system in the red-spotted newt. In order to ascertain the involvement of microRNAs in this amazing process, we made and sequenced cDNA libraries for microRNAs in order to provide sequence data that we could use in further quantitative and qualitative studies.

overall_design: We constructed small RNA cDNA libraries for uninjured (Uninj), 7dpi and 21dpi heart samples, which were sequenced using Illumina-Solexa technology at SciLifeLabs (Stockholm, Sweden).

middle: I

first: Nevin

middle: M

last: Witman


title: Brain transcriptomics of honey bee parasitized by Varroa destructor or Nosema ceranae

city: Avignon

first: Cynthia

last: McDonnell

address: Site Agroparc, Domaine Saint-Paul

department: UR 406 Abeilles et Environnement

country: France

institute: INRA Avignon

summary: In this study we addressed whether the transcriptome profile in the honey bee brain is similar for two major parasites of honey bee, Varroa destructor and Nosema ceranae. Honey bees parasitized by these two parasites show accelerated behavioral maturation and deficiences in orientation and learning/memory that we hoped to characterized at the transcriptomic level.

overall_design: honey bee adults infested by Varroa destructor or Nosema ceranae compared to control bees, in duplicate

first: Hugues

last: Parrinello

first: Jean-Pierre

last: Desvignes

first: Yves

last: Le Conte

first: Cédric

last: Alaux


title: p300 is a preferred client protein for Nuclear Chaperones

city: Bethesda

first: Jung

middle: S

last: Byun

state: MD

address: 37 convent drive, Bldg 37,  Rm 1042

institute: NCI/NIH


title: The polycomb protein Ezh2 regulates differentiation and plasticity of CD4 T helper type-1 and type-2 cells [ChIP-seq]

city: Chiba

first: Atsushi

last: Onodera

address: Chuo-ku Inohana 1-8-1

department: Immunology

country: Japan

institute: Chiba University

summary: Following antigen encounter by CD4 T cells, polarizing cytokines induce the expression of master regulators that control differentiation.  Inactivation of the histone methyltransferase Ezh2 was found to specifically enhance T-helper (Th)1 and Th2 cell differentiation and plasticity. Ezh2 directly bound and facilitated correct expression of Tbx21 and Gata3 in differentiating Th1 and Th2 cells, accompanied by substantial tri-methylation at lysine 27 of histone 3 (H3K27-Me3). In addition, Ezh2 deficiency resulted in spontaneous generation of discrete IFN-γ and Th2 cytokine-producing populations in non-polarizing cultures, and under these conditions IFN-γ  expression was largely dependent on enhanced expression of the transcription factor Eomesodermin. In vivo, Loss of Ezh2 caused increased pathology in a model of allergic asthma and resulted in progressive accumulation of memory phenotype Th2 cells.  This study establishes a functional link between Ezh2 and transcriptional regulation of lineage-specifying genes in terminally differentiated CD4 T cells.

overall_design: Examination of Ezh2 binding in Th1 and Th2 cells.

first: Damon

last: Tumes

first: Akane

last: Suzuki

first: Kenta

last: Shinoda

first: Yusuke

last: Endo

first: Chiaki

last: Iwamura

first: Hiroyuki

last: Hosokawa

first: Koji

last: Tokoyoda

first: Yutaka

first: Shinichiro

last: Motohashi

first: Toshinori

last: Nakayama


title: Gene expression analysis of induced pluripotent stem cells from aneuploid chromosomal syndromes

city: Beijing

first: Ruosi

last: Zhang

address: NO.1 Beichen West Road, Chaoyang District

institute: Beijing Institute of Genomics, Chinese Academy of Sciences

summary:
Background:Human aneuploidy is the leading cause of early pregnancy loss, mental retardation, and multiple congenital anomalies. Due to the high mortality associated with aneuploidy, the pathophysiological mechanisms of aneuploidy syndrome remain largely unknown. Previous studies focused mostly on whether dosage compensation occurs, and the next generation transcriptomics sequencing technology RNA-seq is expected to eventually uncover the mechanisms of gene expression regulation and the related pathological phenotypes in human aneuploidy.
Results:Using next generation transcriptomics sequencing technology RNA-seq, we profiled the transcriptomes of four human aneuploid induced pluripotent stem cell (iPSC) lines generated from monosomy X (Turner syndrome), trisomy 8 (Warkany syndrome 2), trisomy 13 (Patau syndrome), and partial trisomy 11:22 (Emanuel syndrome) as well as two umbilical cord matrix iPSC lines as euploid controls to examine how phenotypic abnormalities develop with aberrant karyotype. A total of 466 M (50-bp) reads were obtained from the six iPSC lines, and over 13,000 mRNAs were identified by gene annotation. Global analysis of gene expression profiles and functional analysis of differentially expressed (DE) genes were implemented. Over 5000 DE genes are determined between aneuploidy and euploid iPSCs respectively while 9 KEGG pathways are overlapped enriched in four aneuploidy samples.
Conclusions:Our results demonstrate that the extra or missing chromosome has extensive effects on the whole transcriptome. Functional analysis of differentially expressed genes reveals that the genes most affected in aneuploid individuals are related to central nervous system development and tumorigenesis.

overall_design: Four aneuploid iPSC (trisomy 8, trisomy 13, partial trisomy 11:22, monosomy X) and two euploid iPSC mRNA profiles were generated by deep sequencing, using SOLiD v3 platform. Gene expression values were compared.

first: Lili

last: Hao

first: Lingping

last: Wang

first: Meili

last: Chen

first: Jun

last: Yu

first: Jingfa

last: Xiao

first: Jiayan

last: Wu


title: Chromosome-wide profiling of X-inactivation status and epigenetic regulation in fetal brain

status: Public on Sep 23 2013

city: College Station

first: Kory

last: Douglas

state: TX

address: 4458 TAMU

institute: Texas A&M University

summary: Evidence from a few genes of diverse species suggests that marsupial X-chromosome inactivation (XCI) is characterized by exclusive, but leaky, inactivation of the paternally derived X chromosome. To comprehensively study the mechanism of marsupial XCI, we profiled parent-of-origin-specific-allele expression, DNA methylation, and histone modifications in opossum fetal brain and extra-embryonic membranes. The majority (152/176) of X-linked genes exhibited paternally imprinted expression with nearly 100% maternal allele expression, whereas 24 loci (14%) escaped inactivation showing varying levels of biallelic expression. In addition to regulation by the non-coding RSX transcript, strong depletion of H3K27me3 at escaper gene loci indicates that histone states also influence opossum XCI. Notably, the opossum does not show an association between X-linked gene expression and promoter DNA methylation. Our study provides the first comprehensive catalogue of parent-of-origin expression status for X-linked genes in a marsupial and sheds light on the regulation and evolution of imprinted XCI in mammals.

overall_design: Profiling of four histone modifications in embryonic day 13 opossum (Monodelphis domestica) fetal brain by Illumina ChIP-seq

first: Paul

last: Samollow

first: Andrew

last: Clark

middle: C

first: John

middle: L

last: VandeBerg


title: H2AZ extended acidic patch is necessary for formation specialized chromatin states in ESCs [ChIP-Seq]

city: Cambridge

first: Laurie

last: Boyer

state: MA

address: 77 Massachusetts Avenue

institute: Massachusetts Institute of Technology

summary: The H2A variant H2AZ is essential for embryonic development and for proper execution of developmental gene expression programs in embryonic stem cells (ESCs). Divergent regions in H2AZ are likely key for its functional specialization, but we know little about how these differences contribute to chromatin regulation.   Here, we show that the extended acidic patch, specifically the three divergent residues in the C-terminal docking domain, is necessary for lineage commitment during ESC differentiation and proper execution of gene expression programs during ESC differentiation. Surprisingly, disruption of the acidic patch domain has a distinct consequence on cellular specification compared to H2AZ depletion. This is consistent with differences in gene expression profiles of H2AZ –depleted and acidic patch (AP) mutant ESCs during early lineage commitment. Interestingly, the distinct consequence of AP mutant expression on gene regulation is coincidence with an altered destabilized chromatin state and high chromatin mobility dependent on active transcription. Collectively, our data shows that the divergent residues within the acidic patch domain are key structural determinants of H2AZ function and links chromatin structure and dynamics with gene regulation and cell fate specification.

overall_design: H2AZ extended acidic patch was mutated, or H2AZ was KD in mouse embryonic stem cells and RNA-Seq analysis was performed on the resulting cultures. Characterization of H2AZ-WT and -AP3-mutant binding specificities were performed by ChIP-Seq.

first: Vidya

last: Subramanian


title: Nanog, SoxB1 and Pou5f1/Oct4 regulate widespread zygotic gene activation during the maternal-to-zygotic transition

status: Public on Sep 22 2013

city: New Haven

first: Antonio

middle: J.

last: Giraldez

state: CT

address: 333 Cedar Street

department: Genetics

institute: Yale University

summary: Upon fertilization, maternal factors direct development in a transcriptionally silent embryo. At the maternal-to-zygotic transition (MZT), a universal step in animal development, unknown maternal factors trigger zygotic genome activation (ZGA). In zebrafish, ZGA is required for gastrulation and clearance of maternal mRNAs, which is achieved in part by the conserved microRNA miR-430. However, the precise factors that activate the zygotic program remain largely unknown. Here we show that Nanog, Pou5f1 and SoxB1 are required for genome activation in zebrafish. We identified several hundred genes directly activated by maternal factors, thus constituting the first wave of zygotic transcription in zebrafish. Ribosome profiling in the pre-MZT embryo revealed that nanog, sox19b and pou5f1 are the most highly translated transcription factor mRNAs. Combined loss of function for Nanog, SoxB1 and Pou5f1 resulted in developmental arrest prior to gastrulation, and a failure to activate >75% of zygotic genes. Furthermore, we found that Nanog binds the miR-430 locus and together with Pou5f1 and SoxB1 initiate miR-430 expression and activity. Our results demonstrate that maternal Nanog, Pou5f1 and SoxB1 are required to initiate the zygotic developmental program and in turn trigger the clearance of the maternal program by activating miR-430 expression.

overall_design: Wild type and loss-of-function total mRNA sequencing of embryonic transcriptomes pre- and post-MZT; ribosome profiling pre-MZT

first: Miler

middle: T

last: Lee

first: Ashley

last: Bonneau


title: Targeted DNA demethylation using TALE-TET1 fusion proteins

city: Charlestown

first: Morgan

middle: Lee

last: Maeder

institute: Mass General Hospital

type: Methylation profiling by high throughput sequencing

summary: Recent large-scale studies have defined genomewide, cell type-specific patterns of DNA methylation, a modification known to be important for regulating gene expression in both normal development and disease states.  However, determining the functional significance of specific methylation events remains a challenging problem due to the current lack of targeted methodologies for removing these modifications.  Here we describe an approach for efficient targeted demethylation of specific CpGs in human cells using fusions of engineered transcription activator-like effector (TALE) repeat arrays and the TET1 hydroxylase catalytic domain.  Using these TALE-TET1 fusions, we demonstrate that modification of certain critical methylated promoter CpG positions can be associated with substantial increases in endogenous human gene expression.  Our results delineate a general strategy for defining the functional significance of specific CpG methylation marks in the context of endogenous gene loci and validate new programmable DNA demethylation reagents with broad utility for research and potential therapeutic applications.

overall_design: Bisulfite sequencing of three different loci in three different cell lines (Klf4 in K562s, HBB in K562s and RHOXF2 in 293s and HeLas.  Biological triplicates of all samples and controls (off-target and GFP controls).

first: J

middle: K

last: Joung


title: Expression and regulation of lincRNAs during T cell development and differentiation

city: Morgantown

first: Gangqing

last: Hu

state: West Virginia

address: 64 Medical Center Drive

department: MicroBiology, Immunology, and Cell Biology

institute: West Virginia University

summary: Although lincRNAs are implicated in regulating gene expression in various tissues, little is known about lincRNA transcriptomes in the T cell lineages. Here we identify 1,524 lincRNAs in 42 T cell samples from early T cell progenitors to terminally differentiated T helper subsets. Our analysis revealed highly dynamic and cell-specific expression patterns of lincRNAs during T cell differentiation. Importantly, these lincRNAs are located in genomic regions enriched for protein-coding genes with immune-regulatory functions.  Many of these transcripts are bound and regulated by the key T cell transcription factors, T-bet, GATA3, STAT4 and STAT6. We demonstrate that the lincRNA LincR-Ccr2-5'AS, together with GATA3, is an essential component of a regulatory circuit in Th2-specific gene expression.

overall_design: To obtain comprehensive profiles of lincRNA expression during the development and differentiation of T cell lineages, we purified CD4-CD8 double negative (DN) cells (DN1, DN2, DN3 and DN4), double positive (DP) cells (CD4+CD8+CD3low and CD4+CD8intCD69+), single positive (SP) CD4 and CD8 cells, and thymic-derived regulatory T cells (tTreg) from thymi of C57BL/6 mice. Additionally, we obtained Th1, Th2, Th17 and iTreg cells by in vitro differentiation of naïve CD4 T cells for a various length of time in culture (4 hrs, 8 hrs, 12 hrs, 24 hrs, 48 hrs, 72 hrs, 1 week, 2weeks). Total and/or polyadenylated RNAs from these cells was analyzed using RNA-Seq. To understand the regulation of lincRNAs by T cell master regulator T-bet, we compared the transcriptiomes between T-bet deficient Th1 cells and control Th1 cells. We did similar experiments and data analysis for STAT4 (Th1), GATA3 (Th2) and STAT6 (Th2). Finally, to address the funcation of a Th2-specifically expressed lincRNA, lincR-Ccr2-5'AS, we compared the transcriptomes between lincR-Ccr2-5'AS knockdown Th2 cells and control Th2 cells.

first: Hu

last: Gangqing

first: Tang

last: Qingsong

first: Sharma

last: Suveena

first: Yu

last: Fang

first: Escobar

last: Thelma

first: Muljo

last: Stefan

first: Zhu

last: Jinfang

first: Zhao

last: Keji


title: Genome wide ChIP-Seq profiles of p300 and CBP in control and cells expressing HSP70K71E

first: Jung

last: Byun

summary: There were more enriched p300 and CBP bindings at the promoter regions in the cells expresing HSP70K71E compared to the control

overall_design: HEK293 cells were overexpressed with pCI1-EGFP (control) and with HSP70K71E-EGFP (mutant) and appied for ChIP-Seq.

first: Kevin

last: Gardner


title: Genomic profiling of Anaplastic Large Cell Lymphoma

status: Public on Sep 20 2013

city: Bellinzona

first: Francesco

last: Bertoni

state: Ticino

address: Via V. Vela 6

department: Lymphoma & Genomics Research Program

country: Switzerland

institute: IOR

type:
Genome variation profiling by SNP array
SNP genotyping by SNP array

summary: Anaplastic Large Cell Lymphoma (ALCL) is a mature T-cell lymphoma that can present as a systemic or primary cutaneous disease. Systemic ALCL represents 2-5% of adult lymphoma but up to 30% of all pediatric cases. Two subtypes of systemic ALCL are currently recognized on the basis of the presence of a translocation involving the Anaplastic Lymphoma Kinase ALK gene. Despite considerable progress, several questions remain open regarding the pathogenesis of both ALCL subtypes. To investigate the molecular pathogenesis and to assess the relationship between the ALK(+) and ALK(-)ALCL subtypes, we performed a genome-wide DNA profiling using high density, single nucleotide polymorphism (SNP) arrays (SNP-array) on a series of 63 cases and seven cell lines. The commonest lesions were losses at 17p13 and at 6q21, encompassing the TP53 and PRDM1 genes respectively. The latter gene, coding for BLIMP1, was inactivated by multiple mechanisms, more frequently, but not exclusively, in ALK(-)ALCL. In vitro and in vivo experiments showed that that PRDM1 is a tumor suppressor gene in ALCL models, likely acting as an anti-apoptotic agent. Losses of TP53 and/or PRDM1 were present in 52% of ALK(-)ALCL, and in 29% of all ALCL cases with a clinical implication.

first: Andrea

last: Rinaldi

first: Ivo

last: Kwee


title: H2AZ extended acidic patch is necessary for formation specialized chromatin states in ESCs

first: Laurie

last: Boyer

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

overall_design: Refer to individual Series


title: TFAM ChIP-seq in HeLa cells

status: Public on Sep 21 2013

city: Stanford

first: Georgi

middle: Kolev

last: Marinov

state: California

address: 279 Campus Drive West, Beckman Center, B-257A/259

institute: STANFORD UNIVERSITY

summary: ChIP-seq data characterizing the occupancy of TFAM over the mitochondrial and nuclear genomes in HeLa cells.

overall_design: Characterization of mitochondrial and nuclear genome-wide TFAM binding in HeLa cells

first: Yun

last: Wang

first: Barabra

last: Wold

first: David

last: Chan


title: Gene expression profiles mas5D, ssa2D, hsf1-overexpressing and heat-stressed wild type Schizosaccharomyces pombe cells

city: Lausanne

first: Aleksandar

last: Vjestica

state: Vaud

address: Genopode Building, University of Lausanne

department: Center for Integrative Genomics

institute: University of Lausanne

summary: The cells with the impaired Hsp40/Hsp70 chaperone complex Mas5/Ssa2 exhibit a transriptional response that is simillar to that of cells with the elevated levels of the heat-shock factor 1 (Hsf1) or heat-stressed wild type fission yeast cells

overall_design: A total of 6 experimental conditions has been analyzed including control samples, no replicates were made

first: Dan

last: Zhang

first: Jianhua

last: Liu

first: Snezhana

last: Oliferenko


title: Digital gene expression (DGE) sequencing of 10 pairs samples between kidney normal tissue and cancer tissue

status: Public on Sep 29 2013

city: shenzhen

first: JACK

last: LI

address: beishangongyequ

institute: beijing genomics insititute

summary: we combined their genome-wide profiles of tumors and normal adjacent tissues in 10 ccRCC patients. The results showed that 283 miRNAs were down-regulated and 187 up-regulated, meanwhile 7473 mRNAs were up-regulated and 3439 down-regulated in ccRCC. Expressions of 12 miRNAs and genes were validated in 10 patients we studied by RT-qPCR (validation rate from 60% to 100%). Differentially expressed gene analysis showed the collective change of miR-200 and SLC22A gene family, and pathway analysis revealed down-regulation of multiple metabolic pathways and up-regulation of focal adhesion, ECM-receptor interaction, etc. Moreover, A miRNA cluster located in chromosome Xq27.3 were significantly down-regulated in ccRCC, which were verified in 53 ccRCC patients by RT-qPCR (validation rate from 63.8% to 88.6%).56 and 586 potentially novel miRNAs and mRNA we detected according to statistical analyses. In addition, 14 miRNA candidates were randomly selected to validate using qRT-PCR and Sanger sequcencing technology, 10 of out 14 could be successfully amplified,.Plus, the sequences of all 5 randomly selected amplified products were confirmed by cloned Sanger sequencing.Our data demonstrated a combined profiling of miRNAs and mRNAs in ccRCC, which showed the decreased metabolism and the perturbation of renal cell function. Moreover, this study identified the expression alteration of a miRNA cluster on Xq27.3, which suggested its potential function in carcinogenesis of ccRCC

overall_design: Examination of 10 pairs of kidney normal cells and cancer cells .

first: Jiahao

last: Chen

first: Zhizhong

last: Li


title: Genome-wide analysis of dFOXO binding sites in Drosophila

city: Providence

first: Hua

last: Bai

state: RI

address: 34 Olive St, BMC502

department: EEB

institute: Brown University

summary: To understand how reduced insulin/IGF-1 signaling extends Drosophila lifespan through its downstream transcription factor dFOXO. We conducted ChIP analysis with a dFOXO antibody followed by Illumina high-throughput sequencing from chico heterozygous mutants, which are long-lived and normal sized, and from adult flies with ablated insulin producing cells (IPCs), which are also long-lived. dFOXO bound at promoters of 273 genes common among these genotypes, thus potentially enriching for shared factors in control of aging.

overall_design: Two replicates were sequenced from chico heterozygous mutants and IPC ablated flies.

first: Marc

last: Tatar


title: H2AZ extended acidic patch is necessary for formation specialized chromatin states in ESCs [RNA-Seq]

first: Vidya

last: Subramanian


title: Intronic Non-CG DNA Hydroxymethylation and Alternative mRNA Splicing in Honey Bees

status: Public on Sep 19 2013

city: Montréal

first: Pablo

last: Cingolani

state: Quebec

address: 701 Avenue du Docteur-Penfield

country: Canada

institute: McGill University

summary: Honey bee non-CG DNA hydroxymethylation is enriched in the introns, which supplements previous findings that honey bee CG DNA methylation is enriched in exons.

sample_organism:
Apis mellifera
Apis mellifera scutellata

overall_design: Bisulfite sequencing combined with Pvu-Seq to distinguish 5-methylcytosine from 5-hydroxymethylcytosine and RNA-Seq

first: Xiaoyi

last: Cao

first: Radhika

last: Khetani

first: Chieh-Chun

first: Melissa

last: Coon

first: Alya’a

last: Sammak

first: Aliccia

last: Bollig-Fischer

first: Susan

last: Land

last: Huang

first: Matthew

last: Hudson

first: Mark

last: Garfinkel

first: Sheng

last: Zhong

first: Gene

last: Robinson

first: Douglas

last: Ruden


title: STAT3 promotes motor neuron differentiation by collaborating with motor neuron-specific LIM complex

city: Brockport

first: Rongkun

last: Shen

state: New York

address: 350 New Campus Dr

institute: SUNY Brockport

summary: The motor neuron (MN)–hexamer complex consisting of LIM homeobox 3, Islet-1, and nuclear LIM interactor is a key determinant of motor neuron specification and differentiation. To gain insights into the transcriptional network in motor neuron development, we performed a genome-wide ChIP-sequencing analysis and found that the MN–hexamer directly regulates a wide array of motor neuron genes by binding to the HxRE (hexamer response element) shared among the target genes. Interestingly, STAT3-binding motif is highly enriched in the MN–hexamer–bound peaks in addition to the HxRE. We also found that a transcriptionally active form of STAT3 is expressed in embryonic motor neurons and that STAT3 associates with the MN–hexamer, enhancing the transcriptional activity of the MN–hexamer in an upstream signal-dependent manner. Correspondingly, STAT3 was needed for motor neuron differentiation in the developing spinal cord. Together, our studies uncover crucial gene regulatory mechanisms that couple MN–hexamer and STAT-activating extracellular signals to promote motor neuron differentiation in vertebrate spinal cord.

overall_design: To explain our experimental scheme briefly, we are interested in finding target sites for the dimer of transcription factors Isl1 and Lhx3. To mimic the biological activity of Isl1/Lhx3 dimer, we made Isl1-Lhx3 fusion and found that Isl1-Lhx3 has a potent biological activity in multiple systems (i.e. generation of ectopic motor neurons). Then we made ES cell line that induces Flag-tagged Isl1-Lhx3 expression upon Dox treatment. These *mouse* ES cells differentiate to motor neurons (iMN-ESCs) when treated with Dox following EB formation. To identify genomic binding sites of Isl1-Lhx3 (Flag-tagged), we performed ChIP with Flag antibody (pull down of Flag-Isl1-Lhx3) in ES cells treated with Dox. ChIP with Flag antibody in ES cells treated with vehicle (no Dox) was done as a negative control in parallel, and sequenced along with +Dox sample. We have done these experiments twice (two sets).

first: Seunghee

last: Lee

first: Soo-Kyung


title: Taf7l cooperates with Trf2 to regulate spermiogenesis

city: Berkeley

first: Haiying

last: Zhou

state: CA

address: 188 Li Ka Shing Center

department: MCB

institute: HHMI/UCB

summary: Taf7l (a paralogue of Taf7) and Trf2 (a TBP-related protein) are components of the core promoter complex required for gene/tissue-specific transcription of protein-coding genes by RNA polymerase II. Previous studies reported that Taf7l knockout mice exhibit structurally abnormal sperm, reduced sperm count, weakened motility and compromised fertility. Here we find that continued backcrossing of Taf7l-/Y mice from N5 to N9 produced KO males that are essentially sterile. Genome-wide expression profiling by mRNA-seq analysis of wild type (WT) and Taf7l-/Y (KO) testes revealed that Taf7l ablation impairs the expression of many post-meiotic spermatogenic specific as well as metabolic genes. Importantly, histological analysis of testes revealed that Taf7l-/Y mice develop post-meiotic arrest at the first stage of spermiogenesis, phenotypically similar to Trf2-/- mice, but distinct from Taf4b-/- mice. Indeed, we find that Taf7l and Trf2 co-regulate post-meiotic genes, but none of Taf4b-regulated germ stem cell genes in testes. Genome-wide ChIP-seq studies indicate that TAF7L binds to promoters of activated post-meiotic genes in testis. Moreover, biochemical studies show that TAF7L associates with TRF2 both in vitro and in testis suggesting that TAF7L likely cooperates directly with TRF2 at promoters of a subset of post-meiotic genes to regulate spermiogenesis. Our findings thus provide a new mechanism for cell-type specific transcriptional control involving an interaction between a ‘non-prototypic’ core promoter recognition factor (Trf2) and an orphan TAF subunit (Taf7l) in mammalian testis-specific gene transcription.

overall_design: Genome-wide mapping of TAF7L and Pol II in testis tissue, and mRNA-seq expression profiling wild type and Taf7l knockout testis.

first: Ivan

last: Grubisic

first: Tommy

last: Kaplan

first: Robert

last: Tjian


title: A genome-wide analysis of open chromatin in human epididymis epithelial cells reveals novel candidate regulatory elements for male infertility

city: Cleveland

first: Ann

last: Harris

state: OH

address: 10900 Euclid Avenue

institute: Case Western Reserve University Medical School


title: Study functions of ADAR proteins using next generation sequencing of genome and transcriptome

city: Ann Arbor

first: Isabel

middle: Xiaorong

last: Wang

state: Michigan

address: 210 washtenaw ave

department: Pediatrics&Genetics

institute: HHMI/University of Michigan

summary: Adenosine deaminases, RNA specific (ADAR) are proteins that deaminate adenosine to inosine which is then recognized in translation as guanosine.  To study the roles of ADAR proteins in RNA editing and gene regulation, we carried out DNA and RNA sequencing, RNA interference and RNA-immunoprecipitation in human B-cells.  We also characterized the ADAR protein complex by mass spectrometry.  The results uncovered over 60,000 sites where the adenosines (A) are edited to guanosine (G) and several thousand genes whose expression levels are influenced by ADAR.  We also identified more than 100 proteins in the ADAR protein complex; these include splicing factors, heterogeneous ribonucleoproteins and several members of the dynactin protein family.  Our findings show that in human B-cells, ADAR proteins are involved in two independent functions: A-to-G editing and gene expression regulation.  In addition, we showed that other types of RNA-DNA sequence differences are not mediated by ADAR proteins, and thus there are co- or post-transcriptional mechanisms yet to be determined.

overall_design: Here we studied human B-cells where ADAR proteins (ADAR1 and ADAR2) are expressed but APOBECs are not.  We identified the sequence differences between DNA and the corresponding RNA in B-cells from two individuals.  Then, we carried out RNA interference, RNA-immunoprecipitation and next generation sequencing to determine the contribution of ADAR proteins in mediating A-to-G editing and other types of RNA-DNA sequence differences.

middle: X

first: Vivian

middle: G

last: Cheung


title: Re-analysis of E-MTAB-1585

city: St Louis

first: Tiandao

last: Li

state: MO

address: 4444 Forest Park Ave

institute: Washington University

summary:
Raw data from E-MTAB-1585 was normalized by using reads per million.
https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-1585/

overall_design:
Strand specific RNA-Seq data E-MTAB-1585 was normalized and subtracted control from knockdown to generate tracks that more clearly displayed the unusual pattern of RNA expression caused by knockdown of 7SK.
The following wig files were generated from multiple samples (i.e.raw data files), as indicated in the 'readme.txt' file.
7sk_3p_KD_norm.wig: 7SK 3P Knockdown normalized
7sk_3p_KDF_norm.wig: 7SK 3P Knockdown normalized (Forward)
7sk_3p_KDR_norm.wig: 7SK 3P Knockdown normalized (Reverse)
7sk_5p_KD_norm.wig: 7SK 5P Knockdown normalized
7sk_5p_KDF_norm.wig: 7SK 5P Knockdown normalized (Forward)
7sk_5p_KDR_norm.wig: 7SK 5P Knockdown normalized (Reverse)
7sk_Control_norm.wig: 7SK Control normalized
7sk_ControlF_norm.wig: 7SK Control normalized (Forward)
7sk_ControlR_norm.wig: 7SK Control normalized (Reverse)
7sk_3p_KDF-ControlF.wig: 7SK 3P Knockdown-Control (Forward)
7sk_3p_KDR-ControlR.wig: 7SK 3P Knockdown-Control (Reverse)
7sk_5p_KDF-ControlF.wig: 7SK 5P Knockdown-Control (Forward)
7sk_5p_KDR-ControlR.wig: 7SK 5P Knockdown-Control (Reverse)

first: David

middle: H

last: Price


title: In vivo T-box Transcription Factor Profiling Reveals Joint Regulation of Embryonic Neuro-mesodermal Bipotency

city: London

first: George

middle: E.

last: Gentsch

state: Greater London

address: 1 Midland Road

department: Developmental Biology Laboratory

institute: The Francis Crick Institute

summary: Brachyury (Xbra/Xbra3) knock-down embryos of the frog Xenopus tropicalis were profiled to quantify neuro-mesodermal cell fate switches at a transcriptional level.

overall_design: Transcriptional profiling of Xbra/Xbra3 double morphants at early tadpole stage (RNA-Seq) in biological triplicates.

middle: E

first: James

last: Smith


title: Targeting oncogene expression to endothelial cells induces proliferation of the myelo-erythroid lineage by repressing the notch pathway

city: Eggesteing Leopolshafen

first: marina

last: mione

address: Hermann-von-Helmholtz-Platz 1

department: Institute of Toxicology and Genomics

country: Germany

institute: Karlsruhe Institute of Technology

summary: Human oncogenes involved in the development of hematological malignancies have been widely used to model experimental leukemia. Here, we used the fli1 promoter in zebrafish to target the expression of oncogenic HRAS to endothelial cells, including the hemogenic endothelium and observed the development of a myelo-erythroid proliferative disease. In larvae, the pathological phenotype is characterized by some disruption of the vascular system with prominent expansion of the caudal hematopoietic tissue, increase of expression of stem cell markers and myelo-erythroid specific genes and production of a large number of l-plastin leukocytes. In mosaic juveniles, increased number of hematopoietic blasts and arrest of myeloid maturation was found in kidney marrow. Peripheral blood showed delays of erythrocyte maturation and increased number of circulating myeloid progenitors.   We found that the abnormal phenotype is associated with a down regulation of the Notch pathway as shown by the decrease of expression of Notch target genes, whereas overexpressing an activated form of Notch together with the oncogene prevents the expansion of the myelo-erythroid compartment. This study identifies the downregulation of the Notch pathway following an oncogenic event in the hemogenic endothelium as an important step in the pathogenesis of myelo-erythroid diseases and describes a number of potential effectors of this transformation.

overall_design: Methods:  mRNA profiles of transgenic zebrafish overexpressing the oncogene HRAS in endothelial cells (Tg(fli1ep:GAL4FF)ubs3; Tg(UAS:eGFP-HRASV12)io006); or expressing activate Notch in endothelial cells (Tg(fli1ep:GAL4FF)ubs3; tg(UAS:NICD)kca3)  were generated by deep sequencing using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed using the CLC bio Assembly Cell software (version 3.2) and the Ensembl (release 63) predicted cDNAs for the Zv9 genome assembly. qRT–PCR validation was performed using TaqMan and SYBR Green assays.

first: Elisa

last: Alghisi

first: Martin

last: Distel

first: Michele

last: Malagola

first: Christiaan

last: Henkel

first: Marina

last: Mione


title: Synergistic Activation of Inflammatory Cytokine Genes by Priming of Regulatory DNA Elements for Increased Transcription in Response to TLR Signaling

city: New York

first: Lionel

last: Ivashkiv

state: NY

address: 535 East 70th Street

institute: Hospital for Special Surgery

summary: Synergistic activation of inflammatory cytokine genes by IFN-gamma and TLR signaling is important for innate immunity and inflammatory disease pathogenesis, but underlying mechanisms are not known. By obtaining over three billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of human primary monocytes under IFN-gamma-priming and TLR stimulation. We found that IFN-gamma induced genome-wide sustained occupancy of STAT1, IRF-1 and associated histone acetylation at TSS-proximal and distal regulatory elements and provided a synergy mechanism whereby IFN-gamma creates a primed chromatin environment to augment TLR-induced gene transcription, which suggest therapeutic approaches that selectively target priming mechanisms.

overall_design: Examination and comparison of the changes in TF binding and histone modification in human primary monocytes under different conditions.

first: Yu

last: Qiao

first: Yushan

first: Eugenia

last: Giannopoulou


title: High-efficiency RNA cloning enables quantitative measurement of miRNA expression by deep sequencing

status: Public on Sep 18 2013

city: Boulder

first: Zhaojie

last: Zhang

state: CO

address: 2300 Arapahoe Ave

institute: University of Colorado at Boulder

summary: MiRNA-mediated regulation depends on the stoichiometry between miRNAs and their mRNA targets. To decipher dynamic function of this complex layer, it is critical to characterize individual miRNA species within a specific cellular context. Small RNA cloning followed by deep sequencing is uniquely positioned as a genome-wide profiling method to quantify miRNA expression with potentially unlimited dynamic range and provide single-nucleotide resolution for precise miRNA classification and de novo discovery. However, significant biases introduced by RNA ligation steps in the current RNA cloning protocol often lead to inaccurate miRNA quantification by >1000-fold deviation. As a result, it has greatly hindered the broad application of this method. Here we report a highly efficient RNA cloning method that achieves over 90% efficiency for both 5’ and 3’ ligations with diverse small RNA substrates. When applied to a pool of either equimolar or differentially mixed synthetic miRNAs, the deviation of the cloning frequency for each miRNA is minimized to less than 2-fold of the anticipated value. By using samples obtained from multiple tissues and cells, we further demonstrate the accurate quantification of miRNA expression over a dynamic range of four orders of magnitude. Our results also reveal that most cistronic miRNAs are expressed at similar levels and, in each cell population, miRNAs repress their cognate targets in a dosage dependent manner. Collectively, our high-efficiency RNA cloning method combining with deep sequencing establishes a cost-effective approach for accurate genome-wide miRNA profiling.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: We designed an artificial system composed of synthetic miRNAs for benchmarking biases in small RNA cDNA cloning for NGS.

first: Jerome

last: Lee

first: Rui

last: Yi


title: Analysis of gene expression with the Hmga2 allelic series.

first: Philip

last: East

state: London

address: 1 Midland Road, London

department: Bioinformatics

summary: In this study, we introduced a series of expression constructs into a mouse lung cancer cell line (482N1): (1) a control short hairpin RNA (shluc) plus the empty expression vector (empty); (2) an shRNA targeting Hmga2 (shHmga2) plus empty; (3) shHmga2 plus an expression vector for the full-length Hmga2 cDNA with the shRNA site mutated (shHmga2 wt); (4) shHmga2 plus the full-length shRNA-mutated Hmga2 with let-7 sites in the 3' UTR mutated (shHmga2 m7); (5) shHmga2 plus the full-length shRNA-mutated Hmga2 with the start codon mutated (shHmga2 ATG wt); and (6) shHmga2 plus the full-length shRNA-mutated Hmga2 with the start codon and let-7 sites mutated (shHmga2 ATG m7).

overall_design: Cells were plated in triplicate, RNA was prepared using RNA-Bee as per manufacturers' instructions and RNA-seq studies were performed using standard methods. For further details on library construction and next-generation sequencing, please contact the contributor.

first: Madhu

last: Kumar

first: Elena

last: Armenteros-Monterroso

first: Probir

last: Chakravorty

first: Nik

last: Matthews

first: Monte

last: Winslow

first: Julian

last: Downward


title: Genome-wide small RNA profiling and mRNA profiling of Xenopus embryos

first: Caroline

last: Hill

address: 44 Lincoln's Inn Fields

institute: Cancer Research UK London Research Institute

summary: Here we report on genome-wide small RNA and transcriptome profiling of blastula, gastrula and neurula-stage Xenopus tropicalis embryos using deep sequencing. By combining bioinformatic analysis of the small RNA-seq dataset with experimental validation, we identify novel microRNAs and Piwi-interacting RNAs. We show that microRNA expression is highly dynamic and spatially localized in early embryos in contrast to Piwi-interacting RNAs, and we validate a novel microRNA prediction pipeline. We go on to identify a new class of small intronic non-coding RNAs, that we name sincRNAs. On a genome-wide scale we find that genes containing sincRNA clusters are transcriptionally repressed. Our data suggest a novel mechanism of sincRNA-mediated gene silencing in vertebrates.

overall_design: Examination of small RNAs and mRNA at 3 stages of Xenopus embryonic development.

first: Joanne

last: Harding

first: Stuart

last: Horswell

first: Javier

last: Armisen

first: Lyle

middle: B

last: Zimmerman

first: Eric

middle: A

last: Miska


title: Transcriptome data used for gene annotation

city: Bei Jing

first: Shilin

last: Tian

address: Xueqing Road

department: genome research

institute: Novogene Bioinformatics Technology CO.ltd

summary: To optimize the genome annotation, four tissue RNA libraries (i.e. heart, liver, lung and kidney) were constructed using the Illumina mRNA-Seq Prep Kit

overall_design: We sequenced four tissues using illumina Hiseq 2000 platform

first: Mingzhou

last: Li

first: Xuewei

first: Ruiqiang


title: Molecular Hallmarks of Experimentally Acquired Immunity to Malaria [Pilot Study]

city: Rockville

first: Ravi

last: Sanka

state: Maryland

address: 9704 Medical Center Drive

institute: JCVI

summary: Sterile immunity to Plasmodium falciparum infection can be induced experimentally in humans after few exposures. An example is the induction of immunity using whole parasites by exposure of malaria-naive volunteers to infectious mosquito bites while using chloroquine prophylaxis (CPS immunization). Chloroquine kills blood-stage parasites but leaves liver-stage parasites unaffected, thereby exposing the liver-stage and early blood-stage antigens to the immune system. Upon subsequent challenge, volunteers are completely protected from infection, but protective efficacy decreases when fewer infectious mosquito bites are used for CPS immunization. Efforts to understand the mechanisms of this immunity, and how it differs from naturally-acquired immunity, may provide critical insights that could aid malaria vaccine development.  In this pilot study, transcriptomic features are derived from blood samples collected before and after challenge with infectious mosquito bites.

overall_design: 12 samples; paired pre- and post-challenge for 5 individuals, plus two controls

first: Ewen

middle: F

last: Kirkness

first: Robert

middle: W

last: Sauerwein

first: Peter

middle: D

last: Crompton

first: Marielle

last: Haks


title: High-throughput sequencing of matched colorectal normal, tumor and metastasis tissues and proof-of principal bioinformatics modeling of therapeutic consequences of miRNA applications

city: Berlin

first: Michal

middle: Ruth

last: Schweiger

address: Ihnestrasse 63-73

department: Lehrach

institute: Max Planck Institute For Molecular Genetics

summary: MiRNAs are discussed as diagnostic and therapeutic molecules. However, effective miRNA drug treatments with miRNAs are so far hampered by the complexity of the miRNA networks. To identify potential miRNA drugs in colorectal cancer, we profiled miRNA and mRNA expression in matching normal, tumor and metastasis tissues of eight patients by Illumina sequencing. We identified miRNA-1 as top candidate differentially expressed in tumor and metastasis. Furthermore, miRNA-1 was de-regulated in 16 additional tumor entities underscoring its central role in tumor pathogenesis. Functional analyses showed an additive effect of miRNA-1 with camptothecin treatment. We used a systems-biology simulation of cellular cancer models implemented in PyBios to investigate miRNA-1 function and assessed the effects of depletion as well as overexpression in terms of miRNA-1 as a potential treatment option. In this system miRNA-1 treatment reverted the disease phenotype with different effectiveness among the patients. Scoring the gene expression changes obtained through mRNA-Seq from the same patients we show that the combination of deep sequencing and systems biological modeling can help to identify patient-specific responses to miRNA treatments. We present this data as guideline for future pre-clinical assessments of new and personalized therapeutic options.

overall_design: Examination of miRNA expression values by Illumina sequencing of matched benign, tumor and metastasis tissues of 8 colorectal cancer patients. For 4 of these patients all tissues have been resequenced to obtain mRNA expression values.

first: Christina

last: Roehr

first: Martin

last: Kerick

first: Axel

last: Fischer

first: Alexander

last: Kuehn

first: Karl

last: Kashofer

first: Bernd

last: Timmermann

first: Thomas

last: Meinel

first: Dmitriy

last: Drichel

first: Stefan

last: Boerno

first: Anja

last: Nowka

last: Tusche

first: Alice

middle: C

last: McHardy

first: Sylvia

last: Krobitsch

first: Tim

last: Becker

first: Andrea

last: Wunderlich

first: Christian

last: Barmeyer

last: Viertler

first: Kurt

last: Zatloukal

first: Christoph

last: Wierling

first: Hans

last: Lehrach

middle: R


title: Large-scale hypomethylated blocks associated with Epstein-Barr virus-induced B-cell immortalization

status: Public on Sep 17 2013

city: Baltimore

first: Kasper

middle: Daniel

last: Hansen

address: 615 N Wolfe St. E3527

department: Biostatistics

institute: Johns Hopkins

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

overall_design: Refer to individual Series

first: Sarven

last: Sarbunciyan


title: In vivo T-box Transcription Factor Profiling Reveals Joint Regulation of Embryonic Neuro-mesodermal Bipotency.

first: George

last: Gentsch

summary: We defined genome-wide regulatory inputs of the T-box transcription factors Brachyury (Xbra), Eomesodermin (Eomes) and VegT that maintain neuro-mesodermal stem cells and determine their bipotential fates in the Xenopus tropicalis frog embryo.

overall_design: Binding profiles for Xbra, Eomes and VegT in X. tropicalis embryos (ChIP-Seq)

first: James

last: Smith


title: Mapping the inter- and intra-chromosomal interactions of specific insulator binding sites with circular chromosome conformation capture (4C) assay (Gondor et al., 2008 Nature Protocol)

status: Public on Sep 15 2013

city: Singapore

first: Chin-Tong

last: Ong

address: 1 Research Link

department: Developmental Biology

country: Singapore

institute: Temasek Life Sciences Lab

type: Genome binding/occupancy profiling by genome tiling array


title: eRNAs Promote Transcription by Establishing Chromatin Accessibility at Defined Genomic Loci

city: Bethesda

first: Hossein

last: Zare

address: 5o South Dr #1347

institute: NIH

summary: Transcription factors and DNA regulatory binding motifs are fundamental components of the gene regulatory network (GRN). Here, by using genome-wide occupancy profiling of master regulators of MyoGenesis (MyoD and MyoGenin), we show their extensive occupancy in the extragenic enhancer regions coinciding with RNA synthesis (i.e. eRNA). In particular, multiple regions coding for eRNAs were observed within regulatory region of MYOD1, including previously characterized Distal Regulatory Regions (DRR) and Core Enhancer (CE). While CERNA enhanced RNA polymerase II (PolII) occupancy and transcription at MYOD1, DRRRNA acted in trans to activate the downstream MyoGenic GRN. The deployment of transcriptional machinery to appropriate loci is contingent on chromatin accessibility, a rate-limiting step preceding PolII assembly. By nuclease sensitivity assay, we show that eRNAs increase genomic access to the transcriptional complex at defined regulatory regions. In conclusion, our data suggest eRNAs establish a cell-type-specific transcriptional circuitry by directing chromatin-remodeling events.

overall_design: Examination of MyoD and MyoG binding events and production of RNA at the enhancer sites during myogenic differentiation. We performed RNAi against enhancer-derived RNA (DRRi) which resulted in reduction of chromatin accessiblity and RNA polymerase II occupancy at defined regulatory elements. In complementary experiments, overexpression of DRR-RNA (pHAN_DRR1.2) resulted in early activation of MyoG. GFPi and GFP overexpression (pHAN_GFP) were used as control in these experiments, respectively.

first: Kambiz

last: Mousavi

first: Stefania

last: Dell'Orso

first: Lars

last: Grontsved

first: Gustavo

last: Gutierrez-Cruz

first: Assia

last: Derfoul

first: Gordon

middle: L

last: Hager

first: Vittorio

last: Sartorelli


title: Dampening of expression oscillations by synchronous regulation of a microRNA and its target

city: Freiburg

first: Dominic

last: Grün

address: Stübeweg 51

institute: Max Planck Institute of Immunobiology and Epigenetics

summary: The complexity of metazoan organisms requires precise spatiotemporal regulation of gene expression during development. To identify different modes of developmental gene regulation we measured the transcriptome throughout development of the nematode Caenorhabditis elegans by mRNA sequencing with high temporal resolution. We find that approximately 2,000 transcripts undergo expression oscillations synchronized with larval transitions while thousands of genes are expressed in temporal gradients, similar to known timing regulators. By counting transcripts in individual animals, we show that the pulsatile expression of the microRNA (miRNA) lin-4 maintains the temporal gradient of its target lin-14 by dampening its expression oscillations. Our results demonstrate that this insulation is optimal when pulsatile expression of the miRNA and its target is synchronous. We propose that such a miRNA-mediated incoherent feed-forward loop is a potent filter that prevents propagation of potentially deleterious gene expression fluctuations during the development of an organism.

overall_design: We analyzed RNA-seq data of wild-type worms at two different temperatures, 20C and 25C, from samples picked every 2hrs and 1.5 hrs, resspectively, spanning all larval stages (L1,L2,L3,L4). At 20C we picked samples for L1-L3 (sample DH2: 0 hrs to 38 hrs) and for L4 (sample DH5: 38 hrs to 48 hrs) from independent populations. At 25C, all samples were picked from the same worm population (sample DH3: 0 hrs to 28.5 hrs). This time course ends at 28.5 hrs since at higher temperature nematode development is accelarated. Finally, we measured mRNA expression at 20C in a lin-4 knockout mutant worm (lin-4(e912)), again spanning all larval stages (sample DH4: 0 hrs to 48 hrs). Each sequencing sample consisted of a mixture of all time points with mRNA from different time points barcoded with Illumina barcodes and was sequenced on one or more lanes (DH2: 3 lanes; DH3: 3 lanes; DH4: 4 lanes; DH5: 1 lane) of an Illumina HiSeq2000.

first: Dong

last: Kim

first: Alexander

last: van Oudenaarden


title: A genome-wide analysis of open chromatin in human epididymis epithelial cells reveals novel candidate regulatory elements for male infertility [DNase-Seq]

first: Ann

last: Harris

summary: The epithelium lining the epididymis has a pivotal role in ensuring a luminal environment that can support normal sperm maturation. Many of the individual genes that encode proteins involved in establishing the epididymal luminal fluid are well characterized. They include ion channels, ion exchangers, transporters and solute carriers. However, the molecular mechanisms that coordinate expression of these genes and modulate their activities in response to biological stimuli are less well understood. To identify cis-regulatory elements for genes expressed in human epididymis epithelial cells we generated genome-wide maps of open chromatin by DNase-seq. This analysis identified 33,542 epididymis-selective DNase I hypersensitive sites (DHS), which were not evident in five cell types of different lineages. Identification of genes with epididymis-selective DHS at their promoters revealed gene pathways that are active in immature epididymis epithelial cells. These include processes correlating with epithelial function and also others with specific roles in the epididymis including retinol metabolism and ascorbate and aldarate metabolism. Peaks of epididymis-selective chromatin were seen in the androgen receptor gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has a critical role in regulating ion transport across the epididymis epithelium. In silico prediction of transcription factor binding sites that were over-represented in epididymis-selective DHS identified epithelial transcription factors including ELF5 and ELF3, the androgen receptor, Pax2 and Sox9, as components of epididymis transcriptional networks. Active genes, which are targets of each transcription factor, reveal important biological processes in the epididymis epithelium.

first: Jared

middle: M

last: Bischof

first: Austin

last: Gillen

first: Lingyun

last: Song

first: Nehal

last: Gosalia

first: Darin

last: London

first: Terrence

middle: S

last: Furey

first: Gregory

last: Crawford


title: Transcriptome analysis of M.tuberculosis using RNA-seq indicates WhiB6 repression of dormancy associated genes

city: Umeå

first: Christer

last: Larsson

address: Bldng. 6K

institute: Umeå universitet

summary: Tuberculosis is an infectious disease, with latent infection with Mycobacterium tuberculosis (M.tb) affecting 1/3 of humanity. It can remain quiescent for long time, making eradication very difficult. To do so, M.tb need to carefully orchestrate its gene expression to survive immune response and starvation but still be able to reactivate to enable transmission to new hosts. Here we used whole transcriptome deep sequencing to compare gene expression between wild type M.tb and a strain with its whiB6, a gene encoding a putative redox-sensing transcription factor, disrupted by a transposon. We found several genes associated with dormancy such as hspX, fdxA and narK2 upregulated in the transposon mutant, indicating that WhiB6 may be a repressor of such genes. The results suggest that WhiB6 may be a complement to the dosS/dosR system in regulating genes important for dormancy.

overall_design: Triplicate cultures of a mutant with its whiB6 gene disrupted by a transposon and wild-type M. tuberculosis CDC1551 were grown in 7H9 media. RNA was extracted and depleted from rRNA. Global gene expression was measured using AB SOLID RNA sequencing.

first: Jennifer

last: Meyers

first: Timothy

last: Mosbruger


title: MicroRNA-146 function in the innate immune response of zebrafish embryos to Salmonella typhimurium infection

status: Public on Sep 16 2013

city: Leiden

first: Annemarie

middle: H.

last: Meijer

address: Einsteinweg 55

department: Animal Sciences and Health

country: Netherlands

institute: Institute of Biology, Leiden University

first: Zakia

last: Kanwal

first: Anita

last: Ordas

first: Valesca

last: Lindenberg

first: Matyas

last: Mink

first: Herman

middle: P

last: Spaink


title: MicroRNA-146 function in the innate immune response of zebrafish embryos to Salmonella typhimurium infection [RNA-seq]

summary: We used zebrafish embryos as an in vivo system to investigate the role of the microRNA-146 family (consisting of 2 members miR-146a and miR-146b) in the innate immune response to S. typhimurium infection.  To determine the role of miR-146 microRNAs in the response to S. typhimurium infection we used Illumina RNA sequencing to compare the mRNA expression profiles of control embryos versus embryos with  knockdown of miR-146a and miR-146b. RNA sequencing analysis of miR-146 knockdown embryos showed no major effects on pro-inflammatory gene expression or on the expression of transcriptional regulators and signal transduction components of the immune response. In contrast, apoliprotein-mediated lipid transport emerged as an infection-inducible pathway under miR-146 knockdown conditions, suggesting a function of miR-146 in regulating lipid metabolism during inflammation.

overall_design: Embryos were injected at the one cell stage with a combination of two morpholinos targeting miR-146a and miR-146b, or with the standard control morpholino from GeneTools. Subsequently, at 28 hours post fertilzation (hpf) they were infected by injecting 200-250 colony forming units of S. typhimurium strain SL1027 into the caudal vein, or mock-injected with PBS. RNA was isolated at 8 hours post injection (hpi) for Illumina RNAseq analysis. Two independent experiments were performed for RNAseq analysis of biological duplicates.

middle: H


title: Large-scale hypomethylated blocks associated with Epstein-Barr virus-induced B-cell immortalization [Bisulfite-seq]

first: Kasper

last: Hansen

summary: To determine what DNA methylation and gene expression changes occur following EBV transformation. B-cells were isolated from 3 donors.  Resting, CD40 activated and EBV transfromed cells from each donor was analyzed. Each sample was assayed using Affymetrix expression arrays and whole genome bisulfite sequenicng.  Additional time points during transformation and activation were sequenced as well, but not assayed for expression.

overall_design: B-cells were isolated from 3 donors.  Resting, CD40 activated and EBV transfromed cells from each donor was analyzed.  Additional B-cells were isolated from 2 additional donors and assay at days 16 and week 3 following CD40 activation and EBV infection.  Only one donor contributed to the EBV timepoints.

first: Sarven

last: Sarbunciyan


title: Differences in DNA methylation between human neuronal and glial cells are concentrated in enhancers and non-CpG sites

status: Public on Sep 14 2013

city: Bronx

first: Stella

last: Dracheva

address: James J. Peters VA Medical Center,130 W Kingsbridge Rd

department: Dept. of Psychiatry

institute: Icahn School of Medicine at Mount Sinai

first: Alexey

last: Kozlenkov


title: Specification of innate type-2 lymphocytes by the transcriptional determinant Gfi1

status: Public on Sep 13 2013

city: Chicago

first: Aly

middle: Azeem

last: Khan

state: IL

address: 6045 S. Kenwood Ave.

institute: University of Chicago

summary: Innate type-2 lymphoid cells (ILC2s) function in immune responses against helminth parasites and are implicated in allergic inflammation and asthma. ILC2s are activated by the epithelial-derived cytokines IL-33 and IL-25 and are major sources of the type-2 cytokines IL-5 and IL-13. We show that the transcription factor Gfi1 promotes the generation of ILC2s and controls their responsiveness during Nippostrongylus brasiliensis infection as well as IL-33- or IL-25-instigated inflammation. Gfi1 directly activates Il1rl1, which encodes the IL-33 receptor. IL- 33 signaling upregulates Gfi1, thereby constituting a positive feedback loop that enables rapid and robust expansion of ILC2s in response to IL-33 signaling. Loss of Gfi1 in activated ILC2s results in an unusual effector state involving derepression of the IL-17 inflammatory program and co-expression of IL-13 with IL-17. ChIPseq reveals key Gfi1 targeted genes that are activated or repressed to maintain ILC2 identity. We propose that Gfi1 functions as a shared determinant within innate and adaptive immune cells to specify type-2 responses, while actively repressing the IL-17 effector state.

overall_design: ILC2s (~3 x 10^7 cells) were sorted from the MLN of IL-25-treated mice. Chromatin fragments bound by Gfi1 were subject to ChIP using Gfi1 antibodies and followed by high-throughput sequencing.

first: Chauncey

middle: J

last: Spooner

first: Harinder

last: Singh


title: In search of epigenetic marks in testes and sperm cells of differentially fed boars

city: Berne

first: Martin

last: Braunschweig

state: Berne

address: Bremgartenstrasse 109A

department: Vetsuisse Faculty

institute: University of Bern


title: Differential RNA-sequencing of Campylobacter jejuni NCTC 11168

city: Norfolk

first: Arnoud

last: van Vliet

address: Colney Lane

department: Gut Health and Food Safety Programme

institute: Institute of Food Research

summary: We report the use of differential RNA-sequencing for the determination of the primary transcriptome of wildtype Campylobacter jejuni NCTC 11168. This allows for the genome-wide determination of transcription start sites.

sample_organism: Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819

overall_design: Campylobacter jejuni NCTC 11168 was grown to late log phase, RNA was purified and used for differential RNA-sequencing by 454 sequencing with barcoded libraries, and used for determination of genome-wide transcription start sites

first: Ida

last: Porcelli

first: Mark

last: Reuter

first: Bruce

last: Pearson

first: Thomas

last: Wilhelm


title: MicroRNA profiling of influenza A virus infected cells

city: Evanston

first: William

last: Buggele

address: 2200 Campus Dr.

institute: Northwestern University

summary: Small RNAs were profiled during influenza A virus infection of human A549 cells to identify changes in microRNA abundance during the cellular antiviral response.

overall_design: Examination of microRNA abundance during influenza A virus infection.

first: Curt

last: Horvath


title: Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs epigenetic reprogramming

city: Liverpool

first: Aditi

last: Kanhere

address: Institute of Systems, Molecular and Integrative Biology

institute: University of Liverpool

summary: Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular mechanism for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors.

overall_design: Examination of EBNA 3 protein binding (EBNA 3A, 3B and 3C) using a pan-specific antibody and EBNA 2 binding in single ChIP-seq experiments carried out in the Mutu III Burkitt's lymphoma cell-line.

first: Michael

last: McClellan

first: C

last: Wood

first: Opeoluwa

last: Ojeniyi

first: Tim

last: Cooper

first: Aaron

last: Arvey

first: Helen

last: Webb

first: Marie

last: Harth-Hertle

first: Bettina

last: Kempkes

first: Richard

middle: G

last: Jenner

first: Michelle

last: West


title: Genome-wide distribution of the three condensin complexes in C. elegans

city: New York

first: Sevinc

last: Ercan

address: 12 Waverly Place

department: Biology

institute: New York University

summary: Condensins are multi-subunit protein complexes that regulate chromosome structure throughout cell-cycle. Metazoans contain two types of condensin complexes (I and II) with essential and distinct functions. In C. elegans a third type of condensin (IDC) functions as part of the X chromosome dosage compensation complex1,2. We mapped genome-wide binding sites of the three condensin types in C. elegans embryos. Characteristics of condensin binding are similar between condensin types.

overall_design: ChIP-seq profiles of C. elegans subunits of the three condensins in 3-6 replicates from mixed stage embryos, controls are included, and RNA-Seq profiles of C. elegans in 5 replicates from mixed staged embryos. Additionally, ChIP-seq profiles of the condensin II subunit KLE-2 in 6 replicates from L3 with controls, and RNA-Seq profiles of KLE-2 mutants in 3 replicates each from L3.

first: Anna-Lena

last: Kranz

first: Lara

last: Winterkorn

first: Chen Yu

last: Jiao

first: Sarah

last: Albritton

first: Q

middle: B

last: Chen

first: Maxwell

last: Kramer


title: modENCODE_White Lab: genome-wide ChIP-chip and ChIP-seq data [D. yakuba H3K27me3]

first: Kevin

middle: P.

last: White

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

department: Institute for Genomics and Systems Biology

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 H3K27me3 from D.yak_WPP generated by ChIP and analyzed on Illumina Genome Analyzer.

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. Factors binding profiles are generated by using specific antibodies for the protein of interest.

first: N

last: Negre

first: R

middle: K

last: Arthur

middle: A

last: Bild

first: K


title: In search of epigenetic marks in testes and sperm cells of differentially fed boars [RNA-Seq]

first: Martin

last: Braunschweig

summary: We investigated the nutritional effects on gene expression in sperm cells of F0 boars from a three generation Large White pig feeding experiment. A group of experimental (E) F0 boars were fed a standard diet supplemented with high amounts of methylating micronutrients whereas a control (C) group of F0 boars received a standard diet. These differentially fed F0 boars sired F1 boars which then sired 60 F2 pigs which were investigated in a previous study. The aim of this study was to investigate if the nutrition affects gene expression in sperm cells of differentially fed boars and thus carry information in the form of RNA molecules to the next generation. Four RNA samples from sperm cells of these differentially fed boars were analyzed by RNA-Seq methodology. We found no differential RNA expression in sperm cells of the two groups based on the adjusted P-value > 0.05. Nevertheless, we performed a pathway analysis with 105 genes that differed in gene expression on the level of nominal P-value < 0.05 between the two diet groups. We found a significant number of these differentially expressed genes were enriched for the pathway maps of bacterial infections in cystic fibrosis (CF) airways, glycolysis and gluconeogenesis p.3 and cell cycle_Initiation of mitosis. The GO processes including a significant portion of differentially expressed genes were viral transcription and viral genome expression, viral infectious cycle, cellular protein localization, cellular macromolecule localization, nuclear-transcribed mRNA catabolic process and nonsense-mediated decay. In summary, the results of the pathway analysis are also inconclusive and it is concluded that RNA expression in sperm cells is not significantly affected by extensive supplementation of methylating micronutrients. Consequently, RNA molecules could not be established as epigenetic marks in this feeding experiment.

overall_design: Gene expression in sperm cells from differentially fed F0 boars was measured. F0 boars received either a standard diet or a standard diet supplemented with methylating micronutrients. These boars were used to study transgenerational epigenetic inheritance in a three generation pig pedigree. Therefore it was of interest if the diet affects gene expression in sperm cells which could then be transmitted to next generations.

first: Rémy

last: Bruggmann

first: Vidhya

last: Jagannathan


title: Active STAT5 in CD8 T cells imprints a T-Bet-dependent Tc-1 program with repressed IL-6/ TGFb1 signaling leading to enhanced anti-tumor responses

city: MARSEILLE

first: Nathalie

last: AUPHAN-ANEZIN

address: Parc Scientifique de Luminy

institute: CIML

summary: CD8 T cells (TCs) expressing active STAT5 (STAT5CA) transcription factors were found to be superior to un-manipulated counterparts in their long-term persistence, capacity to infiltrate a tumor, thrive in its microenvironment and induce its regression. STAT5CA induced sustained expression of genes controlling tissue homing, cytolytic granule composition, Tc-1-associated effector molecules (GranzymeB+/IFNg+/TNFa+/CCL3+ but IL-2-) and potential for secondary responses. Sustained expression of both T-Bet and Eomes transcription factors was correlated with STAT5 binding to their corresponding genes by ChIPSeq analyses. Additionally, STAT5CA-expressing CD8 TCs demonstrated reduced IL-6R/TGFbRII expression and dampened IL-6 and TGFb1 signaling. Altogether, concerted STAT5/T-Bet/Eomes regulation controls homing, recall responses and resistance to Tc-17 polarization in CD8 TCs.

overall_design: TCRP1A CD8 T lymphocytes were activated by their cognate P1A Ag. After 24h, an active form of Stat5 (STAT5CA) was introduced in activated cells. Culture was continued for another 48h to induce their differentiation in effector T cells. These activated T cells were injected in congeneic hosts and recovered 70 days later from hosts' spleen and lymph nodes: TCRP1A eTC-STAT5CA.

first: Grégory

last: Verdeil

first: Salvatore

last: Spicuglia

first: Aurélien

last: Griffon

first: Julien

last: Maurizio

first: Magali

last: Grange

first: Anne-Marie

last: Schmitt-Verhulst

last: Auphan-Anezin


title: Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery

first: Julius

last: Müller

address: 61 Biopolis Drive

department: IMCB

institute: A*Star Singapore


title: Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery (RNA-Seq)

summary: Protein Arginine MethylTransferase 5 (PRMT5) is known to mediate epigenetic control on chromatin and to functionally regulate components of the splicing machinery. In this study we show that selective deletion of PRMT5 in different organs leads to cell cycle arrest and apoptosis. At the molecular level, PRMT5 depletion results in reduced methylation of Sm proteins, aberrant constitutive splicing and in the Alternative Splicing (AS) of specific mRNAs. We identify Mdm4 as one of these mRNAs, which due to its weak 5’-Donor site, acts as a sensor of splicing defects and transduces the signal to activate the p53 response, providing a mechanistic explanation of the phenotype observed in PRMT5 conditional knockout mice. Our data demonstrate a key role of PRMT5, together with p53, as guardians of the transcriptome. This will have fundamental implications in our understanding of PRMT5 activity, both in physiological conditions, as well as pathological conditions, including cancer and neurological diseases.

overall_design: Total RNA was extracted from control and Prmt5 depleted Neural Stem/Progenitors Cells (NPCs) and Mouse Embryonic Fibroblasts (MEFs). Prmt5 depleted cells were treated with 4-OHT 24 hours before splitting to induce PRMT5 knockout and final libraries were sequenced in triplicates on Illumina HiSeq 2000.

first: Marco

last: Bezzi

first: Shu

middle: X

last: Teo

first: Wei

last: Mok

first: Sanjeeb

last: Sahu

first: Leah

last: Vardy

first: Zahid

last: Bonday

first: Ernesto

last: Guccione


title: Extensive divergence of transcription factor binding in Drosophila embryos with highly conserved gene expression (part 1)

status: Public on Sep 12 2013

city: Lyon

first: Mathilde

last: PARIS

address: 32-34 avenue Tony Garnier

department: Institut de Genomique Fonctionnelle de Lyon

institute: CNRS

summary:
We sequenced mRNA from blastoderm embryos of Drosophila melanogaster, Drosophila yakuba, Drosophila pseudoobscura and Drosophila virilis. Two samples contain pooled mRNA from several species, and the remaining 24 samples contain mRNA from a single species.
Methods: Retinal mRNA profiles of Blastoderm embryos

sample_organism:
Drosophila melanogaster
Drosophila pseudoobscura
Drosophila virilis
Drosophila yakuba

overall_design: Comparison of the evolution of gene expression and regulatory TF binding in early Drosophila embryos.

last: Paris

first: Tommy

last: Kaplan

first: Xiao-Yong

last: Li

first: Jacqueline

middle: E

last: Villalta

first: Susan

last: Lott

first: Michael

last: Eisen


title: Extensive divergence of transcription factor binding in Drosophila embryos with highly conserved gene expression (part 2)

summary: To better characterize how variation in regulatory sequences drives divergence in gene expression, we undertook a systematic study of transcription factor binding and gene expression in blastoderm embryos of four species, which sample much of the diversity in the 40 million-year old genus Drosophila: D. melanogaster, D. yakuba, D. pseudoobscura and D. virilis. We compared gene expression, measured by mRNA-seq, to the genome-wide binding, measured by ChIP-seq, of four transcription factors involved in early anterior-posterior patterning. We found that mRNA levels are much better conserved than individual transcription factor binding events, and that changes in a gene’s expression were poorly explained by changes in adjacent transcription factor binding. However highly bound sites, sites in regions bound by multiple factors and sites near genes are conserved more frequently than other binding, suggesting that a considerable amount of transcription factor binding is weakly or non-functional and not subject to purifying selection

sample_organism:
Drosophila melanogaster
Drosophila pseudoobscura
Drosophila virilis


title: RNA-seq analysis of broiler liver transcriptome reveals novel responses to heat stress

city: Ames

first: Derrick

middle: Jamaal

last: Coble

state: IA

address: 2254 Kildee Hall

department: Animal Science

institute: Iowa State University

summary: Background  In broilers, heat stress can result in reduced feed consumption, digestive inefficiency, impaired metabolism, and even death. The broiler sector of the U.S. poultry industry incurs approximately $52 million in heat stress-related losses annually. The objective  of this study is to characterize the effects of chronic, cyclic heat stress on the transcriptome of a metabolically active organ, the liver. Characterizing the liver  transcriptome of heat-stressed broilers will help clarify the effects of heat stress on metabolism. This information will provide a platform for future investigations that further elucidate physiologic responses to heat stress and seek methods to ameliorate  the negative impacts of heat.  Results  Transcriptome sequencing of the livers of 8 broiler males using Illumina HiSeq 2000 technology, resulted in a total of 138 million, 100 base pair single end reads, yielding 13.8 gigabases of sequence. Forty genes were differentially expressed at a significance level of P-value < 0.05 and a fold change ≥ 2 in response to chronic, cyclic heat stress (mid-point of the last day of a 7-day cyclic heat stress of 7 hours per day), with 27 down-regulated and 13 up-regulated. Two gene networks were created from the function-based Ingenuity Pathway Analysis (IPA) of the differentially expressed genes; “Cell Signaling, Molecular Transport, Small Molecule Biochemistry” and “Endocrine System Development and Function, Small Molecule  Biochemistry Cell Signaling”. Members of the MAPK signaling pathway and differentially expressed genes that are associated with MAPK-related functions were prominent in the networks. Cellular proliferation and differentiation, inflammationand  stress-related signaling, and apoptosis-associated genes were down-regulated in response to heat stress. Genes responsible for inhibiting feed intake and sphingolipidrelated signaling were up-regulated. Genes involved with the regulation of inflammation, stress, thyroid hormone level, and body temperature were both up- and down-regulated.  Conclusions  Chronic, cyclic heat stress of broilers results in metabolic changes that can be characterized through RNA-seq analysis of the liver transcriptome. The primary affected pathways included cell signaling, molecular transport, endocrine system  development and signaling, and small molecule biochemistry.

overall_design: Examination of 2 heat treatments. Four heat stressed liver samples and 4 control liver samples analyzed.

first: Damarius

last: Fleming

last: Persia

first: Christopher

last: Ashwell

first: Max

middle: F

last: Rothschild

first: Carl

last: Schmidt

last: Lamont


title: Bacterial Argonaute samples the transcriptome to identify foreign DNA

city: Pasadena

first: Ivan

last: Olovnikov

address: 1200 E California Blvd

department: Division of Biology

institute: California Institute of Technology

summary: Eukaryotic Argonautes bind small RNAs and use them as guides to find complementary RNA targets and induce gene silencing. Though homologs of eukaryotic Argonautes are present in many bacteria and archaea their small RNA partners and functions are unknown. We found that the Argonaute of Rhodobacter sphaeroides (RsAgo) associates with 15-19 nt small RNAs that correspond to the majority of transcripts. RsAgo also binds single-stranded 22-24 nt DNA molecules that are complementary to the small RNAs and enriched in sequences derived from exogenous plasmids as well as genome-encoded foreign nucleic acids such as transposons and phage genes. Expression of RsAgo in the heterologous E. coli system leads to formation of plasmid–derived small RNA and DNA and plasmid degradation. In a R. sphaeroides mutant lacking RsAgo, expression of plasmid-encoded genes is elevated, while other genes are unaffected. Our results indicate that RNAi-related processes found in eukaryotes are also conserved in bacteria and target foreign nucleic acids.

sample_organism:
Cereibacter sphaeroides ATCC 17029
Cereibacter sphaeroides ATCC 17025
Escherichia coli BL21(DE3)

overall_design:
Small RNA and small DNA from purified RsAgo expressed in Rhodobacter sphaeroides ATCC17025 ("strain 25") and E. coli BL21(DE3)
Total small RNA (13-30nt) from R. sphaeroides ATCC17025 and ATCC17029 (wild-type; RsAgo mutant; containing either empty or RsAgo-expressing plasmid)
Total ribosomal RNA-depleted RNA from wild type or RsAgo mutant R. sphaeroides ATCC17025

first: I

first: A

last: Aravin


title: Differences in DNA Methylation between Human Neuronal and Glial Cells are Concentrated in Enhancers and non-CpG Sites [Enhanced Reduced Representation Bisulfite Sequencing data]

first: Stella

last: Dracheva

summary: We applied Illumina Human Methylation450K array to perform a genomic-scale single-site resolution DNA methylation analysis in neuronal and nonneuronal (primarily glial) nuclei separated from the orbitofrontal cortex of postmortem human brain. The findings were validated using enhanced reduced representation bisulfite sequencing. We identified thousands of sites differentially methylated (DM) between neuronal and nonneuronal cells. The DM sites were depleted within CpG island–containing promoters but enriched in predicted enhancers. Classification of the DM sites into those undermethylated in neurons (neuronal type) and those undermethylated in nonneuronal cells (glial type), combined with findings of others that methylation within control elements typically negatively correlates with gene expression, yielded large sets of predicted neuron-specific and nonneuron-specific genes. These sets of predicted genes were in excellent agreement with the available direct measurements of gene expression in human and mouse. We also found a distinct set of DNA methylation patterns that were unique for neuronal cells. In particular, neuronal-type differential methylation was overrepresented in CpG island shores, enriched within gene bodies but not in intergenic regions, and preferentially harbored binding motifs for a distinct set of transcription factors, including neuron-specific activity-dependent factors. Finally, non-CpG methylation was substantially more prevalent in neurons than in nonneuronal cells.

overall_design: Extended Reduced Representation Bisulfite Sequencing (ERRBS) was performed on genomic DNA to validate the Infinium HM450K DNA methylation data (Kozlenkov et. al., 2013, Nucleic Acids Research, accepted for publication).

first: Alexey

last: Kozlenkov