title: Comparative genome-wide DNA methylation analysis of colorectal tumor and matched normal tissues

status: Public on Oct 26 2012

city: Nijmegen

first: Arjen

last: Brinkman

address: NCMLS #274, Geert Grooteplein Zuid 30

department: Molecular Biology

institute: Radboud University, Nijmegen Center for Molecular Life Sciences

summary: In our study we applied a genome-wide DNA methylation analysis approach, MethylCap-seq, to map the differentially methylated regions in 24 tumor and matched normal colon samples. In total, 2687 frequently hypermethylated and 468 frequently hypomethylated regions were identified, which include potential biomarkers for CRC diagnosis. Hypermethylation in the tumor samples was enriched at CpG islands and gene promoters, while hypomethylation was distributed throughout the genome. Using epigenetic data from human embryonic stem cells, we show that frequent differentially methylated regions (DMRs) coincide with bivalent loci in human embryonic stem cells. DNA methylation is commonly thought to lead to cancer gene related silencing, however integration of publically available expression analysis shows that 75% of the frequently hypermethylation genes were most likely already lowly or not expressed in normal tissue. Collectively, our study provides genome-wide DNA methylation maps of colon cancer, comprehensive lists of DMRs, and gives further clues on the role of aberrant DNA methylation in CRC formation.

overall_design: To investigate DNA methylation in CRC in a genome-wide unbiased fashion, we applied MethylCap-seq. This method involves capture of methylated DNA using the MBD domain of MeCP2, and subsequent next-generation Illumina sequencing of eluted DNA. In addition, we compared MethylCap with RNA-seq and ChIP-seq profiles of H3K4me3 and H3K27me3 for the colon cancer tumor cell line HCT116 (HCT116 WT) and the cell line of HCT116 with DNMT1 and DNMT3b knockout (HCT116 DKO).

first: Femke

last: Simmer

first: Arie

middle: B

first: Yassen

last: Assenov

first: Filomena

last: Matarese

first: Anita

last: Kaan

first: Lina

last: Sabatino

first: Alberto

last: Villanueva

first: Dori

last: Huertas

first: Manel

last: Esteller

first: Thomas

last: Lengauer

first: Christoph

last: Bock

first: Vittorio

last: Colantuoni

first: Lucia

last: Altucci

first: Hendrik

last: Stunnenberg


title: Evolution and dynamics of small RNA response to a retroelement invasion in Drosphila

status: Public on Oct 31 2012

city: Cold Spring Harbor

first: Nikolay

middle: V

last: Rozhkov

email: nik.rozhkov@gmail.com, rozhkov@cshl.edu

state: NY

address: 1 Bungtown Rd.

country: USA

institute: CSHL

type: Non-coding RNA profiling by high throughput sequencing

summary: Although small RNAs efficiently control transposition activity of most transposons in the host genome, such immune system is not always applicable against new transposon's invasions. Here we explored a possibility to introduce potentially mobile copy of the Penelope retroelement previously implicated in hybrid dysgenesis syndrome in Drosophila virilis into the genomes of two distant Drosophila species. The consequences of such introduction were monitored at different phases after experimental colonization as well as in D. virilis species which is apparently in the process of ongoing Penelope invasion. We investigated the expression of Penelope and biogenesis of Penelope-derived small RNAs in D. virilis and D. melanogaster strains originally lacking active copies of this element after experimental Penelope invasion. These strains were transformed by constructs containing intact Penelope copies. We show that immediately after transformation, which imitates the first stage of retroelement invasion, Penelope undergoes transposition predominantly in somatic tissues, and may produce siRNAs that are apparently unable to completely silence its activity. However, at the later stages of colonization Penelope copies may jump into one of the piRNA-clusters, which results in production of homologous piRNAs that are maternally deposited and can silence euchromatic transcriptionally active copies of Penelope in trans and, hence, prevent further amplification of the invader in the host genome. Intact Penelope copies and different classes of Penelope-derived small RNAs were found in most geographical strains of D. virilis collected throughout the world. Importantly, all strains of this species containing full-length Penelope tested do not produce gonadal sterility in dysgenic crosses and, hence, exhibit neutral cytotype. In order to understand whether RNA interference mechanism able to target Penelope operates in related species of the virilis group we correlated the presence of full-length and potentially active Penelope with the occurrence of piRNAs homologous to this TE in the ovaries of species comprising the group. It was demonstrated, that Penelope-derived piRNAs are present in all virilis group species containing full-length but transcriptionally silent copies of this element that probably represent the remnants of its previous invasions taking place in the course of the virilis species divergent evolution.

overall_design: piRNA size profile (23-29nt) was examined in D. melanogaster strains, where Penelope-piRNAs are detected by Northern blot

first: Michael

last: Evgen’ev


title: Xenobiotics shape the physiology and gene expression of the active human gut microbiome

status: Public on Oct 30 2012

city: San Francisco

first: Peter

middle: James

last: Turnbaugh

state: CA

address: 513 Parnassus Ave

department: Microbiology and Immunology

institute: University of California San Francisco

type: Expression profiling by high throughput sequencing

summary: The human gut is colonized by trillions of microorganisms that influence human health and disease through the metabolism of xenobiotics, including therapeutic drugs and antibiotics. The diversity and metabolic potential of the human gut microbiome have been extensively characterized, but it remains unclear which microorganisms are active and which perturbations can influence this activity. Here, we use flow cytometry, 16S rRNA gene sequencing, and metatranscriptomics to demonstrate that the human gut contains distinctive subsets of active and damaged microorganisms, primarily composed of Firmicutes, which display marked temporal variation. Short-term exposure to a panel of xenobiotics resulted in significant changes in the physiology and gene expression of this active microbiome. Xenobiotic-responsive genes were found across multiple bacterial phyla, encoding novel candidate proteins for antibiotic resistance, drug metabolism, and stress response. These results demonstrate the power of moving beyond DNA-based measurements of microbial communities to better understand their physiology and metabolism.

overall_design: RNA-Seq analysis of the human gut microbiome during exposure to antibiotics and therapeutic drugs.

first: Corinne

middle: F

last: Maurice

first: Henry

middle: J

last: Haiser


title: Revisiting Global Gene Expression Analysis

status: Public on Oct 25 2012

city: Cambridge

first: Richard

middle: A

last: Young

state: MA

address: 9 Cambridge Center

institute: Whitehead Institute for Biomedical Research

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

overall_design: Refer to individual Series


title: Caenorhabditis sex-specific RNAseq

city: College Park

first: Eric

middle: S.

last: Haag

state: MD

address: 4094 Campus Drive

department: Department of Biology

institute: University of Maryland

summary: This project defines the transcriptomes of XO (male) and XX (female or mutant pseudo-female) Caenorhabditis nematodes. The data allow the overall composition and sexual regulation of the transcriptome within a single species to be determined. In addition, the five related species studied allow meta-comparisons between them. Because two of the five (C. elegans and C. briggsae) produce a self-fertile XX hermaphrodite, while the XX sex in the remaining three (C. japonica, C. remanei, and C. brenneri) are true females, the data are particularly useful for inferring effects of sexual mode on genome-wide gene expression.

sample_organism:
Caenorhabditis briggsae
Caenorhabditis elegans
Caenorhabditis remanei
Caenorhabditis brenneri
Caenorhabditis japonica

overall_design: L4 larvae and adults were pooled for each sex for five species (C. elegans, C. briggsae, C. japonica, C. brenneri, and C. remanei).  Each of these 10 species-sex combinations was replicated three times, for a total of 30 samples.

first: Cristel

middle: G

last: Thomas

first: Renhua

last: Li

first: Harold

middle: E

last: Smith

first: Gavin

middle: C

last: Woodruff

first: Brian

last: Oliver

middle: S


title: Regulation of pluripotency and self-renewal of ES cells through epigenetic-threshold modulation and mRNA pruning

city: Baltimore

first: Yixian

last: Zheng

address: 3520 San Martin Drive

department: Embryology

institute: Carnegie Institution for Science

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The core pluripotency factors (Oct4, Sox2, and Nanog), the Myc network, and the chromatin-modifying complexes such as PRC2 ensure the pluripotency and self-renewal of ES cells (ESC). How these factors coordinate with one another remains poorly understood. We report that Utf1, a target of Oct4 and Sox2, is a new bivalent chromatin component that buffers poised states of bivalent genes. By limiting PRC2 loading and Histone 3 lysine-27 trimethylation, Utf1 sets proper activation thresholds for bivalent genes. It also promotes nuclear tagging of mRNAs transcribed from insufficiently silenced bivalent genes for cytoplasmic degradation through mRNA de-capping. Whereas these opposing functions of Utf1 allow proper execution of ESC pluripotency, the mRNA pruning function also ensures rapid cell proliferation by blocking the Myc-Arf feedback regulation. Thus, Utf1 is an important regulator that couples the core pluripotency factors with Myc and PRC2 networks to promote proliferation and pluripotency execution of ESCs.

overall_design: First we mapped Utf1 binding sites in ESCs using the biotin-mediated and cross-linked ChIP-sequencing. To investigate how Utf1 might regulate gene expression, we did RNA-seq on WT and Utf1-KO ES cells. Then we did ChIP-seq of Suz12 and H3K27me3 on WT and Utf1-KO ES cells to study whether Utf1 affects PRC2 loading and H3K27me3 modofication, using H3 as control. Finally, we did RNAseq on WT and Dcp1a-KD ES cells to confirm Utf1 repress gene expression by recruiting Dcp1a complex.

first: Junling

last: Jia

first: Xiaobin

first: Gangqing

last: Hu

first: Kairong

last: Cui

first: Keji

last: Zhao


title: Revisiting Global Gene Expression Analysis (RNA-Seq)

summary: Gene expression analysis is a widely used and powerful method for investigating the transcriptional behavior of biological systems, for classifying cell states in disease and for many other purposes.  Recent studies indicate that common assumptions currently embedded in experimental and analytical practices can lead to misinterpretation of global gene expression data.  We discuss these assumptions and describe solutions that should minimize erroneous interpretation of gene expression data from multiple analysis platforms.

overall_design: Polyadenylated RNA depleted of ribosomal content was used for preparation of two independent sequencing libraries (low-Myc & high-Myc). A panel of synthetic RNA's was added to these populations, based on cell number.

first: Jakob

last: Loven

first: David

last: Orlando

first: Alla

last: Sigova


title: Short-term time course of the effects of 1α,25-dihydroxyvitamin D3 treatment on open chromatin regions in THP-1 monocytic leukemia cells

city: Kuopio

first: Sami

last: Heikkinen

address: Yliopistonranta 1E

department: Institute of Biomedicine

country: Finland

institute: University of Eastern Finland

summary: Open chromatin regions have been shown to associate with the location of transcriptiotal enhancers, i.e., the binding locations of DNA-binding transcription factors. To investigate the effects of short-term treatment by the nuclear hormone 1α,25-dihydroxyvitamin D3 (VD), a specific ligand of the transcription factor vitamin D receptor, on chromatin accessibility, FAIRE-seq was utilized on the chromatin samples from THP-1 monocytic leukemia cells that were treated with 100 nM 1α,25-dihydroxyvitamin D3 for 20, 40, 60, 80, 100 and 120 min, or with vehicle (0.1% (v/v) ethanol) for 20 and 100 min.

overall_design: THP-1 monocytic leukemia cells were treated with 100 nM 1α,25-dihydroxyvitamin D3 for 20, 40, 60, 80, 100 and 120 min or with vehicle (0.1% (v/v) ethanol) for 20 and 100 min. Chromatin sample from one biological replicate for each time point was subjected to the FAIRE-seq protocol and subsequent data analysis using an Input chromatin sample as control.

first: Petri

last: Pehkonen

first: Sabine

last: Seuter

first: Carsten

last: Carlberg


title: Analysis of soybean DNA methylomes shows CHH hypermethylation enhances gene expression in cotyledons of developing seeds

city: beijing

first: Jinsong

last: Zhang

address: No.1 West Beichen Road,Chaoyang District

country: China

institute: IGDB

type: Methylation profiling by high throughput sequencing


title: GRO-seq in Drosophila melanogaster S2 cells

city: Ithaca

first: Leighton

last: Core

address: 417 Biotechnology Building

department: Moleular Biology and Genetics

institute: Cornell University

summary: Control of RNA transcription is critical for the development and homeostasis of all organisms, and can occur at multiple steps of the transcription cycle, including RNA polymerase II (Pol II) recruitment, initiation, promoter-proximal pausing, and elongation. That Pol II accumulates on many promoters in metazoans implies that steps other than Pol II recruitment are rate-limiting and regulated 1-6.  By integrating genome-wide Pol II chromatin immunoprecipition (ChIP) and Global Run-On (GRO) genomic data sets from Drosophila cells, we examined critical features of Pol II near promoters. The accumulation of promoter-proximal polymerase is widespread, occurring on 70% of active genes; and unlike elongating Pol II within the body of genes, promoter Pol II are held paused by factors like NELF, unable to transcribe unless nuclei are treated with strong detergent.  Notably, we find that the vast majority of promoter-proximal Pol II detected by ChIP are paused, thereby identifying the biochemical nature of this rate-limiting step in transcription. Finally, we demonstrate that Drosophila promoters do not have the upstream divergent Pol II that is seen so broadly and prominently on mammalian promoters.  We postulate this is a consequence of Drosophila’s extensive use of directional core promoter sequence elements, which contrasts with mammals’ lack of directional elements and prevalence of CpG island core promoters. In support of this idea, we show that the fraction of mammalian promoters containing a TATA box core element is dramatically depleted of upstream divergent transcription.

overall_design: Comparison of multiple GRO-seq data sets

first: John

middle: T

last: Lis


title: Pol II (Rpb3) ChIP-seq in Drosophila melanogaster S2 cells

overall_design: ChIP-seq data set for Pol II (rpb3) (2 replicates).

first: Karen

last: Adelman

first: Daniel

last: Gilchrist


title: Transcriptome-wide identification of LIN28A targets in mouse embryonic stem cell (A3-1) [Illumina Seq]

city: Seoul

first: Hyeshik

last: Chang

state: South Korea

address: Building 203 Room 525, School of Biological Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu

department: School of Biological Sciences

country: South Korea

institute: Seoul National University

summary: LIN28A is a highly-conserved RNA-binding protein which is known to be involved in embryonic development, stem cell maintenance and proliferation. LIN28A is expressed in various types of cancer, and they are associated with advanced tumor malignancy. In embryonic stem cell, LIN28A specifically binds to let-7 precursors to suppress biogenesis of the let-7 microRNA family. In addition, Lin28 was reported to bind several mRNAs such as Oct4, cyclin A/B and histone H2A to activate their translation. For comprehensive understanding of the interaction between LIN28A and their target RNAs, we exploited UV-crosslinking and immunoprecipitation (CLIP) to capture their in vivo binding to target RNAs. LIN28A-binding RNAs were identified in a mouse embryonic stem cell line using multiple monoclonal and polyclonal antibodies. The result shows that LIN28A preferentially binds to let-7 precursors through GGAG binding motif, which is consistent with our previous results. We also identified that LIN28A binding is enriched in a certain subset of mRNAs. To understand the function of the novel LIN28A-mRNA binding, we carried out ribosome profiling from LIN28A-depleted mouse embryonic stem cells.

overall_design: Examination of RNA binding of LIN28A and translation in mouse embryonic stem cell.

first: Jun

last: Cho

first: V

middle: N

last: Kim


title: Polypyrimidine tract-binding protein homologues from Arabidopsis are key regulators of alternative splicing with implications in fundamental developmental processes

status: Public on Oct 22 2012

city: New York

first: Andre

last: Kahles

state: New York

address: 1275 York Avenue

department: Computational Biology Center

institute: Memorial Sloan-Kettering Cancer Center

summary: Alternative splicing (AS) generates transcript variants by the definition of different exonic and intronic regions and causes a massive expansion of transcriptome diversity. Changes in AS patterns have been found to be linked to manifold biological processes, yet fundamental aspects such as the regulation of AS and the functional implications of altered AS programs largely remain to be addressed. In this work, widespread AS regulation by Arabidopsis Polypyrimidine tract-binding protein homologues (AtPTBs) was revealed. In total 452 AS events derived from 308 distinct genes were found to be responsive to the levels of the splicing factors AtPTB1 and AtPTB2, which predominantly triggered splicing of regulated introns, inclusion of cassette exons, and usage of upstream 5' splice sites. In contrast, alternative 3' splice site events were strongly underrepresented among the AtPTB1/2 targets and no major AS regulatory function of the distantly related AtPTB3 was found. Dependent on their position within the mRNA, AtPTB-regulated events can both modify the untranslated regions and give rise to alternative protein products. Gene ontology analysis revealed a connection of AtPTB-mediated AS control with diverse biological processes, and the functional implications of selected AS events were further elucidated in the context of seed germination and flowering time control. Specifically, AtPTB misexpression changes AS of the PHYTOCHROME INTERACTING FACTOR 6 (PIF6) pre-mRNA, coinciding with altered rates of abscisic acid-dependent seed germination. Furthermore, AS patterns as well as the expression of key flowering regulators were massively changed in an AtPTB1/2 level-dependent manner. In conclusion, our work has revealed widespread AS regulatory functions of the AtPTB splicing factors with important functional implications in various fundamental processes of Arabidopsis development.

overall_design: Analysis of alternative splicing patterns in plants with increased and decreased levels of the 3 Arabidopsis Polypyrimidine-tract binding protein homologues in comparison to wild type samples, determined in duplicates

first: Christina

last: Rühl

first: Eva

last: Stauffer

first: Gabriele

last: Wagner

last: Drechsel

first: Andreas

last: Wachter

first: Gunnar

last: Rätsch


title: Transcriptional Profiling of Psoriasis Using RNA-seq Reveals Previously Unidentified Differentially Expressed genes

first: Mayte

last: Suarez-Farinas

address: 1425 Madison Ave, L2-70C, Box 1077,

institute: Mount SinaiSchool of Medicine

summary: The transcriptomic profiling of psoriasis has led to an increased understanding of disease pathogenesis. Although microarray technologies have been instrumental in this regard, it is clear that these tools detect an incomplete set of DEGs. RNA-seq can be used to supplement these prior technologies. Here, the use of RNAseq methods substantially increased the number of psoriasis-related DEGs. Furthermore, DEGs that were uniquely identified by RNA-seq, but not in other published microarray studies, further supported the role of IL-17 and tumor necrosis factor-a synergy in psoriasis. Examination of one of these factors at the protein level confirmed that RNA-seq is a powerful tool that can be used to identify molecular factors present in psoriasis lesions, and may be useful in the identification of therapeutic targets that to our knowledge have not been reported previously.  Further studies are in progress to determine the biological significance of DEGs uniquely discovered by RNA-seq.

overall_design: To define the transcriptomic profile of psoriatic skin, three pairs of lesional and nonlesional skin biopsy specimens were taken from patients with untreated  moderate-to-severe plaque psoriasis.

first: James

last: Krueger

first: Ali

last: Jabbari


title: Genome-wide nucleosome positioning during embryonic stem cell development [ChIP-Seq]

status: Public on Oct 21 2012

city: Colchester

first: Vladimir

last: Teif

address: Wivenhoe Park

department: School of Life Sciences

country: United Kingdom

institute: University of Essex

summary: We determined genome-wide nucleosome occupancy in mouse embryonic stem cells and their neural progenitor and embryonic fibroblast counterparts to assess features associated with nucleosome positioning during lineage commitment. Cell type and protein specific binding preferences of transcription factors to sites with either low (e.g. Myc, Klf4, Zfx) or high (e.g. Nanog, Oct4 and Sox2) nucleosome occupancy as well as complex patterns for CTCF were identified. Nucleosome depleted regions around transcription start and termination sites were broad and more pronounced for active genes, with distinct patterns for promoters classified according to their CpG-content or histone methylation marks. Throughout the genome nucleosome occupancy was dependent on the presence of certain histone methylation or acetylation modifications. In addition, the average nucleosome-repeat length increased during differentiation by 5-7 base pairs, with local variations for specific genomic regions. Our results reveal regulatory mechanisms of cell differentiation acting through nucleosome repositioning.

overall_design: For chromatin immunoprecipitation, for each sample, 1 x 106 cells were cross-linked with 1% PFA and cell nuclei were prepared using a swelling buffer (25 mM Hepes pH 7.8, 1 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1 mM DTT). Chromatin was sheared to mononucleosomal fragments. After IgG preclearance the sheared chromatin was incubated with 4 µg of either a H3K9ac (Abcam, ab4441), a H3K27ac (Abcam, ab4729), or a H3K9me3 (Abcam ab8898) antibody over night. After washes with sonication (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% N-lauroylsarcosine, 0.1% Na-deoxycholate), high-salt- (50 mM Hepes pH 7.9, 500 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS), lithium- (20 mM Tris-HCl pH 8.0, 1mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate) and 10 mM Tris-HCl, chromatin was eluted from the protein G magnetic beads and the crosslink was reversed over night. After RNase A and proteinase K digestion, the DNA was purified and subsequently cloned into a multiplexed Illumina library according to standard protocols. Sequenced 50 bp reads were mapped with bowtie and subsequently clustered with MACS 55 implemented in the Genomatix software suite (Genomatix, Munich, Germany) using a p-value of 10-5.

first: Karsten

last: Rippe


title: Genome-wide nucleosome positioning during embryonic stem cell development [MNase-Seq]

overall_design: We have determined genome-wide nucleosome position maps in mouse embryonic stem cells (ESCs), neural progenitor cells (NPCs) derived from these ESCs by retinoic acid induced differentiation as well as mouse embryonic fibroblasts (MEFs) from the corresponding mouse strain


title: LIN28A targets, regulation of microRNA biogenesis, and effect on transcriptome in A3-1 cells


title: Analysis of soybean DNA methylomes shows CHH hypermethylation enhances gene expression in cotyledons of developing seeds [small RNA]

first: Jinsong

last: Zhang

summary: Cytosine methylation is an important mechanism for dynamical regulation of gene expression and transposon mobility during plant developmental processes. Recently, the variation of DNA methylation has been described between wild type and DNA methylation-related mutants in Arabidopsis thaliana. However, the elaborate representation of soybean DNA methylomes remains lacking. Here, we described the epigenome maps of soybean root, stem, leaf, and cotyledon of developing seed at a single-base resolution. We confirmed the transcription start sites of genes using high-throughput sequencing and reported the DNA methylation patterns in gene and transposon regions. The correlation between gene expression and DNA methylation was revealed through transcriptome sequencing. We found CHH methylation may function in promotion of gene expression and ten cotyledon-preferred genes were identified CHH hypermethylated in cotyledon. Small RNA library sequencing showed that DNA methylation was enhanced by small RNAs not by strand-specific way, and the variation of DNA methylation between the organs was highly related with expression of small RNAs.

overall_design: small RNA profiling of roots, stems, leaves, and cotyledons of developing seeds

first: Qing-Xin

last: Song

first: Jin-Song


title: Genome-wide nucleosome positioning during embryonic stem cell development [RNA-Seq]

overall_design: The Total RNA from ESCs, NPCs and MEFs was extracted by guanidinisothiocyanat/phenol extraction with the Trifast kit (Peqlab). Total RNA preparations were treated with DNase I, phenol/chloroform extracted and precipitated before further processing. RNAs were depleted of 5S, 5.8S, 18S and 28S rRNAs using the Human/Mouse/Rat Ribo-Zero rRNA Removal Kit (Epicentre) according to the manufacturer’s protocol. After rRNA depletion, RNAs were fragmented with a kit from Ambion. Libraries for Solexa sequencing were generated according to the standard Illumina protocol that comprised first strand cDNA synthesis, second strand cDNA synthesis, end repair, addition of a single A base, and adapter ligation. Sequencing was performed on the  Illumina GAIIx (replicate 1) and Illumina HiSeq 2000 (replicate 2) platforms at the sequencing core facilities of the BioQuant in Heidelberg, Germany. RNA reads were aligned with TopHat. Further expression analysis was with the Genomatix software suite (Genomatix, Munich, Germany) and the Eldorado gene annotation. For each transcript a normalized expression value was calculated from the read distribution that accounts for the length differences using the program DEseq for the analysis of differential expression.


title: Genome-wide nucleosome positioning during embryonic stem cell development

first: Yevhen

last: Vainshtein

first: Maïwen

last: Caudron-Herger

first: Jan-Philipp

last: Mallm

first: Caroline

last: Marth

first: Thomas

last: Höfer


title: CLIP-seq of eIF4AIII reveals transcriptome-wide mapping of the human exon junction complex

city: PARIS

first: Herve

last: LE HIR

address: 46 rue d'Ulm

department: Functional Genomics

country: France

institute: IBENS

summary: The exon junction complex (EJC) is a central effector of mRNAs fate, linking nuclear processing to mRNA transport, translation and surveillance. Little is known about its transcriptome-wide targets. We used high-throughput sequencing after crosslinking and immunoprecipitation (HITS-CLIP) in human cells to identify the binding sites of the DEAD-box helicase eIF4AIII, an EJC core component. CLIP reads form peaks mainly located in spliced mRNAs. Most expressed exons harbour peaks equally distributed between the canonical EJC region ∼24 nucleotides upstream of exonic junctions and other non-canonical regions. Unexpectedly, both are preferentially associated to unstructured and purine-rich sequences containing the motif GAAGA, a potential binding site of EJC-associated factors. Therefore, EJC positions vary spatially and quantitatively between exons. This transcriptome-wide mapping of human eIF4AIII reveals unanticipated aspects of the EJC and broadens its potential impact on post-transcriptional regulation.

overall_design: To identify direct RNA binding sites of the EJC core component eIF4AIII, two biological CLIP-seq replicates were performed in HeLa cells. Additionally, mRNA-seq of the HeLa transcriptome was performed to normalize for the mRNA expression levels.

first: Jérôme

last: Saulière

first: Valentine

last: Murigneux

first: Zhen

last: Wang

first: Emélie

last: Marquenet

first: Isabelle

last: Barbosa

first: Olivier

last: Le Tonquèze

first: Yann

last: Audic

first: Luc

last: Paillard

first: Hugues

last: Roest Crollius

first: Hervé

last: Le Hir


title: Human transcriptome pattern of primary cutaneous lesions from patients with localized cutaneous leishmaniasis and mucosal leishmaniasis

status: Public on Oct 24 2012

city: Los Angeles

first: Ana

last: Maretti-Mira

address: 1450 Biggy Street, NRT 3514

department: Medicine

institute: USC

summary: We evaluated the trancriptome of primary cutaneous leisions caused by infection with Leishmania braziliensis. mRNA-seq technique was used to study the trancriptome of both host and parasite. A total of 10 samples was obtained from primary skin ulcers of two extreme clinical forms of American tegumentary leishmaniasis: (i) individuals that after antimonial treatment cured completely (localized cutaneous leishmaniasis - LCL, n=5) and (ii) individuals that developed mucosal lesions in naso and oropharynx areas long after initial healing of the cutaneous lesion (mucosal leishmaniasis - ML, n=5).  The sequencing generated an average of 13+ 5 million reads per samples. The reads were aligned to Homo sapiens (USCS - hg19) and to Leishmania braziliensis (Wellcome Trust Sanger Institute - V2_29072008) genomes. Approximately, 15,000 human genes could be detected in the samples. Low amount of L. braziliensis reads did not allow the evaluation of parasite gene expression. LCL and ML samples showed different patterns of gene expression, indicating a more robust immune response in LCL individuals. In summary, this study demonstrated that next-generation sequencing can be used for identification of potentially important biological pathways and drug targets in the host-response to L. braziliensis infection and for characterization of a gene expression signature that could be used to predict the disease outcome. Moreover, we also showed the ability of this technique in, simultaneously, sequence host and pathogen mRNA.

overall_design: Examination of 10 fragments of cutaneous lesions: 5 from localized cutaneous leishmaniasis patients and 5 from mucosal leishmaniasis patients.


title: Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation

first: Jing-Yu

last: Li

address: 615 Charles E. Young Dr. South BSRB 357

institute: University of California, Los Angeles

summary: Linker histones are essential components of chromatin but the distributions and functions of many during cellular differentiation is not well understood. Here, we show that H1.5 binds to genic and intergenic regions, forming blocks of enrichment, in differentiated human cells from all three embryonic germ layers but not in embryonic stem cells. In differentiated cells, H1.5, but not H1.3, binds preferentially to genes that encode membrane and membrane-related proteins. Strikingly, 37% of H1.5 target genes belong to gene family clusters, groups of homologous genes that are located in proximity to each other on chromosomes. H1.5 binding is associated with gene repression and is required for SIRT1 binding, H3K9me2 enrichment and chromatin compaction. Depletion of H1.5 results in loss of SIRT1 and H3K9me2, increased chromatin accessibility, deregulation of gene expression and decreased cell growth. Our data reveal for the first time a specific and novel function for linker histone subtype H1.5 in maintenance of condensed chromatin at defined gene families in differentiated human cells.

overall_design: Examine mRNA expression in control and H1.5 knockdown IMR90 cells

first: Michaela

last: Patterson

first: Hanna

middle: K

last: Mikkola

first: William

last: Lowry

first: Siavash

last: Kurdistani


title: A Role for the RNA-Binding Protein MOS2 in MicroRNA Maturation in Arabidopsis

status: Public on Oct 27 2012

city: Beijing

first: Yijun

last: Qi

address: #7 Science Park Road, Zhongguancun Life Science Park

institute: National Institute of Biological Sciences

summary: microRNAs (miRNAs) play important roles in the regulation of gene expression. In Arabidopsis, mature miRNAs are processed from primary miRNA transcripts (pri-miRNAs) by nuclear HYL1/SE/DCL1 complexes that form Dicing bodies (D-bodies). Here we report that a RNA-binding protein MOS2 bound to pri-miRNAs and was required for their efficient processing. MOS2 did not interact with HYL1, SE, and DCL1 and was not localized in D-bodies. Interestingly, in the absence of MOS2, the recruitment of pri-miRNAs by HYL1 was greatly reduced and the localization of HYL1 in D-bodies was compromised. These data suggest that MOS2 facilitates pri-miRNA processing through facilitating the recruitment of pri-miRNAs by the Dicing complexes.

overall_design: Examination of gene expression profiles in Col-0 and mos2-2 plants by deep sequencing

first: Xueying

last: Wu


title: Analysis of soybean DNA methylomes shows CHH hypermethylation enhances gene expression in cotyledons of developing seeds [mRNA]

first: Jinsong

last: Zhang

overall_design: mRNA-Seq of roots, stems, leaves, and cotyledons of developing seeds

first: Qing-Xin

last: Song

first: Jin-Song


title: Profiling of Chromatin States in a Human Cancer Cell Line

status: Public on Oct 19 2012

city: Augusta

first: Keith

last: robertson

address: 1120 15th street

institute: Georgia Health Sciences University

summary: DNA methylation mediated by the combined action of three DNA methyltransferases, DNMT1, DNMT3A, and DNMT3B, is essential for mammalian development and is also a major contributor to transformation. To elucidate how DNA methylation is targeted, we map the genome-wide localization of all DNMTs and methylation, and examine relationships between these markers and histone modifications and nucleosome structure. Our findings reveal a strong link between DNA methylation/DNMTs and transcribed loci and that these marks exhibit both overlapping and unique localization patterns. Comparisons with the epigenome of embryonic stem cells demonstrate that DNMT binding is associated with transformation-associated changes in methylation. Taken together, this study sheds important new light on determinants of DNA methylation and how it may become disrupted in cancer cells.

overall_design: Examination of different marks in undifferentiated and differentiatial NCCIT cells

first: Bilian

last: Jin

first: Justin

last: Choi

middle: D

last: Robertson


title: Linking DNA Methyltransferases (DNMTs) to Epigenetic Marks and Nucleosome Structure Genome-Wide in Human Tumor Cells

overall_design: Refer to individual Series


title: Non-coding transcription within the Igh distal VH region at PAIR elements affects the 3D structure of the Igh locus in pro-B cells

status: Public on Oct 18 2012

city: La Jolla

first: Ali

last: Torkamani

address: 3344 North Torrey Pines Court Suite 300

institute: TSRI

summary: Non-coding sense and antisense germline transcription within the immunoglobulin heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germline transcription throughout the Igh locus has been done. Therefore, we performed directional RNAseq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two PAIR elements in the distal IghV region. Importantly, long-distance loops measured by 3C are observed between these two active PAIR promoters and Eμ, the start site of Iμ germline transcription, in a lineage- and stage-specific manner, even though this antisense transcription is Eμ-independent. YY1-/- pro-B cells are greatly impaired in distal VH gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-Eμ interactions. ChIP-seq shows high level YY1 binding only at Eμ, but low levels near some antisense promoters. PAIR-Eμ interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal VH genes close to the DJH rearrangement, which is adjacent to Eμ. Therefore, we hypothesize that one key role of non-coding germline transcription is to facilitate locus compaction, allowing distal VH genes to undergo efficient rearrangement.

overall_design: ChIP Seq YY1 vs. input control

first: Ann

last: Feeney


city: San Diego

first: Lana

last: Schaffer

address: 10550 North torrey Pines Road

department: DNA Array Core

overall_design: In order to determine the amount and location of sense and antisense non-coding RNA in the Igh locus, we prepared total RNA from CD19+ RAG1-/- pro-B cells.  Samples were either pre-enriched in custom Agilent arrays, or directly sequenced.  Data from one sample for each condition is included as reflected in the publication.

first: Jiyoti

last: Verma-Gaur

first: Steven

middle: R

last: Head

first: Nicholas

last: Schork


title: The Cellular EJC Interactome Reveals Higher-Order mRNP Structure and an EJC-SR Protein Nexus

city: Worcester

first: Alper

last: Kucukural

address: Lazare Medical Research Building, 870P

department: Biochemistry and Molecular Pharmacology

institute: UMass Medical School

summary: To uncover exon junction complex (EJC) deposition sites on cellular mRNAs, RNA footprints of EJC immuo-purified from HEK293 cells were deep sequenced. The analysis of these data revealed that major “canonical” EJC occupancy site in vivo lies 24 nucleotides upstream of exon junctions (-24 position) and that the majority of exon junctions carry an EJC. Unexpectedly, we find that many sites further upstream of -24 position are also enriched in these EJC footprints. These "non-canonical" sites are binding sites of EJC-interacting proteins with a subset being occupied by SR proteins. Thus, an EJC-SR protein nexus exists within spliced mRNPs and is revealed here.

overall_design: Deep sequencing based profiling of EJC RNA footprints obtained by tandem RNA immunoprecipitation (RIPiT) of RNase I digested RNA:protein complexes.

first: Guramrit

last: Singh

first: Melissa

last: Moore


title: RUNX1 reshapes the epigenetic landscape at the onset of hematopoiesis

first: Rebecca

middle: Louise

last: Hannah

address: Addenbrookes Hospital, Hills Road

department: Haematology

institute: University of Cambridge

summary: Cell fate decisions during hematopoiesis are governed by lineage-specific transcription factors, such as RUNX1, SCL/TAL1, FLI1 and C/EBP family members. In order to gain insight about how these transcription factors regulate the activation of hematopoietic genes during embryonic development, we measured the genome-wide dynamics of transcription factor assembly on their target genes during the RUNX1-dependent transition from hemogenic endothelium to hematopoietic progenitors. Using a RUNX1-/- embryonic stem cell differentiation model expressing an inducible RUNX1 gene, we show that in the absence of RUNX1, SCL/TAL1, FLI1 and C/EBPβ prime hematopoietic genes and that this early priming is required for correct temporal expression of the myeloid master regulator PU.1 and its downstream targets. After induction, RUNX1 binds to numerous new sites, initiating a local increase of histone acetylation and rapid global alterations in the binding patterns of SCL/TAL1 and FLI1. The acquisition of hematopoietic fate controlled by RUNX1 therefore does not represent the establishment of a new regulatory layer on top of a pre-existing hemogenic endothelium program but instead entails global reorganization of lineage-specific transcription factor assemblies.

overall_design: ChIPseq data from transcription factors Runx1, Fli-1, Scl/Tal1 and C/EBPβ, histone modification H3K9Ac as well as RNA Pol II obtained from differentiating murine hematopoietic cells

first: Monika

last: Lichtinger

first: Richard

last: Ingram

first: Dorothee

last: Müller

first: Deborah

last: Clarke

first: Salam

last: Assi

first: Michael

last: Lie-A-Ling

first: Laura

last: Noailles

first: MS

last: Vijayabaskar

first: Mengchu

first: Daniel

last: Tenen

first: David

last: Westhead

first: Valerie

last: Kouskoff

first: Georges

last: Lacaud

first: Berthold

last: Göttgens

first: Constanze

last: Bonifer


title: Phf19 links methylated lysine 36 of histone H3 to regulation of Polycomb activity [ChIP-Seq]

status: Public on Oct 16 2012

city: Barcelona

first: Luciano

last: Di Croce

address: Dr. Aiguader, 88

country: Spain

institute: Centre for Genomic Regulation

summary: Polycomb group proteins are transcriptional repressors that play essential roles in regulating genes required for differentiation and embryonic development. The Polycomb repressive complex 2 (PRC2) contains the methyltransferase activity for lysine 27 on histone 3 (H3K27me3), which is a docking site for the PRC1 complex and leads to gene repression. However, the role of other histone modifications in regulating PRC2 activity is just beginning to be understood. Here we show that direct recognition of histone H3 methylated at lysine 36 (H3K36me), an mark associated with activation, by the PRC2 subunit Phf19 is required for the full enzymatic activity of the PRC2 complex. We provide structural evidence for this interaction by nuclear magnetic resonance spectroscopy (NMR). Using genome-wide chromatin binding analyses and expression analyses, we show that Phf19 binds to a subset of PRC2 targets in embryonic stem (ES) cells, and that this is required for their repression and for H3K27me3 deposition. These findings reveal that the H3K36me2/3-Phf19 interaction is essential for PRC2 complex activity and for proper regulation of gene repression in ES cells.

overall_design: We determined the genome binding/occupancy profile of Phf19, H3K36me3, H3K36me2, H3K27me3 and Suz12 by high throughput sequencing in mouse embryonic stem cells.  For Phf19 two independent biological replicas were performed and Phf19 binding sites were defined as those sites (ChIP-seq peaks) present in both replicas. H3K27me3 was evaluated in control ES cells and cells depleted of Phf19 (shRd and shPhf19 respectively).

first: Cecilia

last: Ballare

first: Lluis

last: Morey

first: Luca

last: Cozzuto

first: Martin

last: Lange


title: Ectopically expressed MSL2 females compared with normal females

city: Columbia

first: Lin

last: Sun

state: MO

address: 311 Tucker Hall

department: Biological Sciences

institute: University of Missouri

summary: We used H83M2 P element to ectopically expressed MSL2 protein in females to test if the novel formed MSL complex is the directly reason of dosage compensation

overall_design: Compare the MSL2 females with normal females and normal males; MSL2 males also included.

first: James

last: Birchler


title: Small RNA and mRNA profiles following TUT knock-down in HeLa

first: Hyeshik

last: Chang

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


title: mRNA profiles following TUT knock-down in HeLa

summary: The precise control of microRNA (miRNA) biogenesis is important for various cellular functions, and its dysregulation is often associated with human diseases.  We previously reported that Terminal   uridylyl transferase 4 (TUT4) down-regulates let-7 miRNA biogenesis by oligo-uridylating let-7 precursor (pre-let-7) in mouse embryonic stem cells and that a pluripotency marker Lin28 promotes   a processivity of TUT4.  Here we find that TUT4 positively controls let-7 biogenesis by adding a uridine residue to the 3’ end of pre-let-7 in the absence of Lin28. Such mono-uridylation enhances Dicer   processing by generating an optimal end structure of pre-let-7 for Dicer recognition and may protect pre-miRNA from trimming. Moreover, TUT7, TUT4 and TUT2 redundantly regulate pre-let-7   processing and simultaneous knock down of these TUTs leads to the decrease of mature let-7 and the accumulation of pre-let-7 in cells. This study provides a novel regulation mechanism of miRNA   biogenesis, which may function in development and tumorigenesis.

overall_design: HeLa cells were transfected with siRNA two times over a 4~5 day period.

first: Jaechul

last: Lim

first: Minju

last: Ha

first: V

last: Kim


title: RNA polymerase V targets transcriptional silencing components to promoters of protein-coding genes

status: Public on Oct 17 2012

city: Wilmington

first: Qi

last: Zheng

state: DE

address: 1701 Rockland Road

institute: Nemours Children's Health

summary: Transcriptional gene silencing controls transposons and other repetitive elements through RNA-directed DNA methylation (RdDM) and heterochromatin formation. A key component of the Arabidopsis RdDM pathway is ARGONAUTE4 (AGO4), which associates with sizs to mediate DNA methylation. Here, we show that AGO4 preferentially targets transposable elements embedded within promoters of protein-coding genes. This pattern of AGO4 binding cannot be simply explained by the sequences of AGO4-bound siRNAs; instead, AGO4 binding to specific gene promoters is also mediated by long non-coding RNAs (lncRNAs) produced by RNA polymerase V. lncRNA-mediated AGO4 binding to gene promoters directs asymmetric DNA methylation to these genomic regions and is involved in regulating the expression of targeted genes. Finally, AGO4 binding overlaps sites of DNA methylation affected by the biotic stress response. Based on these findings, we propose that the targets of AGO4-directed RdDM are regulatory units responsible for controlling gene expression under specific environmental conditions.

overall_design: ChIP-seq experiments using AGO4 antibody of WT (Col-0) and ago4 or nrpe1 mutant plants of Arabidopsis thaliana

first: M

last: Rowley

first: Gudrun

last: Bohmdorfer

first: Davinder

last: Sandhu

first: Brian

last: Gregory

first: Andrzej

last: Wierzbicki


title: comprehensive chromatin interaction in TNF alpha stimulated HUVECs

status: Public on Oct 13 2012

city: 4-6-1, Komaba, Meguroku

first: Takahide

last: Kohro

state: Tokyo

country: Japan

institute: LSBM, RCAST

summary: HUVECs were stimulated and samples were prepared after 0 and 30 min.  Chromatin interaction mediated by active RNA polymerase II was detected by ChIA-PET.

overall_design: Samples were made afrer 0 and 30 min after TNF alpha stimulation.

first: Yijun

last: Ruan

first: Youichiro

last: Wada

first: Huay-Mei

last: Pho

first: Mika

last: Kobayashi

first: Guoliang

last: Li

first: Shuichi

last: Tsutsumi

first: Xiaoan


title: H3K79me2 ChIP-seq in mouse proximal intestinal Lgr5(hi) stem cells and villus cells

city: Boston

first: Amit

last: Sinha

address: 44 Binney St

department: Pediatric Oncology

institute: Dana-Farber Cancer Institute

overall_design: Examination of H3K79me2 modifications between Lgr5(hi) stem cells and differentiated villus cells

first: Li-Lun

last: Ho

first: Scott

last: Armstrong

first: Ramesh

last: Shivdasani


title: Transcriptome analysis of Drosophila CNS midline cells reveals diverse peptidergic properties and a role for castor in neuronal differentiation

status: Public on Oct 12 2012

city: Chapel Hill

first: Stephen

last: Crews

state: NC

address: 133 N. Medical Dr

department: Biochemistry and Biophysics

institute: UNC Chapel Hill

summary: One of the key aspects of neuronal differentiation is the array of neurotransmitters and neurotransmitter receptors that each neuron possesses. One important goal of developmental neuroscience is to understand how these differentiated properties are established during development. In this paper, we use fluorescence activated cell sorting and RNA-seq to determine the transcriptome of the Drosophila CNS midline cells, which consist of a small number of well-characterized neurons and glia. These data revealed that midline cells express 9 neuropeptide precursor genes, 13 neuropeptide receptor genes, and 31 small-molecule neurotransmitter receptor genes. In situ hybridization and high-resolution confocal analyses were carried-out to determine the midline cell identity for these neuropeptides and the neuropeptide receptors. The results revealed a surprising level of diversity. Neuropeptide genes are expressed in a variety of midline cell types, including motoneurons, GABAergic interneurons, and midline glia. These data revealed previously unknown functional differences among the highly-related iVUM neurons. There also exist segmental differences in expression for the same neuronal sub-type. Similar experiments on midline-expressed neuropeptide receptor genes reveal considerable diversity in synaptic inputs. Multiple receptor types were expressed in midline interneurons and motoneurons, and, in one case, link feeding behavior to gut peristalsis and locomotion. There were also segmental differences, variations between the 3 iVUMs, and three hormone receptor genes were broadly expressed in most midline cells. The Drosophila Castor transcription factor is present at high levels in iVUM5, which is both GABAergic and expresses the short neuropeptide F precursor gene. Genetic and misexpression experiments indicated that castor specifically controls expression of the short neuropeptide F precursor gene, but does not affect iVUM cell fate or expression of Gad1. This indicates a novel function for castor in regulating neuropeptide gene expression.

overall_design: To study the development and differentiation of the CNS midline cells of Drosophila melanogaster on a genome-wide scale, these cells were labeled with GFP using the GAL/UAS system and FACS purified at 2 ermbryonic time-points; 6-8 hours and 14-16 hours after egg laying. Poly(A) mRNA was collected from these samples and cDNA libraries were generated.  Sequencing was performed on 6 independent samples: Two FACS purified CNS-midline cell samples and one non-midline sample taken from 6-8 hours After Egg Laying (AEL) embryos and from 14-16 hours AEL embryos.

first: Joseph

last: Fontana


title: ChIP-seq analysis of H3K27me3 histone modification in EZH2 mutant and wild type DLBCL cell lines

city: Collegeville

first: Gopinath

last: Ganji

state: PA

address: 1250 South Collegeville Road

institute: GSK

summary: In eukaryotes, epigenetic post-translational modification of histones is critical for regulation of chromatin structure and gene expression. EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) and is responsible for repressing target gene expression through methylation of histone H3 on lysine 27 (H3K27). Over-expression of EZH2 is implicated in tumorigenesis and correlates with poor prognosis in multiple tumor types. Recent reports have identified somatic heterozygous mutations of Y641 and A677 residues within the catalytic SET domain of EZH2 in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). The Y641 residue is the most frequently mutated residue, with 22% of GCB (Germinal centre B-cell) DLBCL and FL harboring mutations at this site. These lymphomas exhibit increased H3K27 tri-methylation (H3K27me3) due to altered substrate preferences of the mutant enzymes. However, it is unknown whether direct inhibition of EZH2 methyltransferase activity alone will be effective in treating lymphomas carrying activating EZH2 mutations.  Herein, we demonstrate that GSK126, a potent, highly-selective, SAM-competitive, small molecule inhibitor of EZH2 methyltransferase activity, decreases global H3K27me3 levels and reactivates silenced PRC2 target genes. GSK126 effectively inhibits the proliferation of EZH2 mutant DLBCL cell lines and dramatically inhibits the growth of EZH2 mutant DLBCL xenografts in mice. Together, these data demonstrate that pharmacological inhibition of EZH2 activity may provide a promising treatment for EZH2 mutant lymphoma.

overall_design: We performed a ChIP-seq experiment to understand the genomewide pattern of H3K27me3 enrichment in DLBCL cell lines that were differentially sensitive to GSK126. H3K27me3 bound chromatin  and input controls was immunoprecipitated and subjected to sequencing on the Illumina GA Iix. In total, 3 cell lines were profiled - 3 EZH2 mutant (Pfeiffer, KARPAS-422, WSU-DLCL2).

first: Michael

last: McCabe

first: Caretha

middle: L

last: Creasy


title: Genome-wide analysis of the SNAPc complex

city: Miami

first: Alessandro

last: Gardini

state: FL

address: 1501 NW 10 Avenue

department: Sylvester Comprehensive Cancer Center

institute: University of Miami


title: Analysis of miRNA, piRNA and siRNA in Caenorhabditis species

status: Public on Oct 11 2012

first: Zhen

last: Shi

address: 185 Cambridge Street

department: Molecular Biology

institute: Massachusetts General Hospital

summary: The nematode Caenorhabditis elegans contains each of the broad classes of eukaryotic small RNAs, including microRNAs (miRNAs), endogenous small-interfering RNAs (endo-siRNAs) and piwi-interacting RNAs (piRNAs). To better understand the evolution of these regulatory RNAs, we deep sequenced small RNAs from C. elegans and three closely related nematodes: C. briggsae, C. remanei and C. brenneri. The results reveal a fluid landscape of small RNA pathways with essentially no conservation of individual sequences aside from a subset of miRNAs. We identified 52 miRNA families that are conserved in each of the four species as well as numerous miRNAs that are species specific or shared between only two or three species. Despite a lack of conservation of individual piRNAs and siRNAs many of the features of each pathway, including genomic distribution, are conserved. We show that in each species, 26G siRNAs trigger stage-specific secondary siRNA formation. We also observe that piRNAs trigger siRNA formation from targets containing up to three mismatches in each species. Finally, we show that nematodes produce two distinct sex-specific classes of piRNAs, suggesting different roles for piRNAs in male and female germlines.

sample_organism:
Caenorhabditis briggsae
Caenorhabditis elegans
Caenorhabditis remanei
Caenorhabditis brenneri

overall_design: Sequencing small RNAs from four Caenorhabditis species: C. elegans, C. briggsae, C. remanei and C. brenneri

first: Gary

last: Ruvkun


title: Transcript expression levels of neonatal mouse cardiomyocytes mock-transfected, or transfected with cel-miR-67, hsa-miR-590-3p or hsa-miR-199a-3p

city: Trieste

first: Miguel

last: Mano

address: Padriciano 99

country: Italy

institute: International Centre for Genetic Engineering and Biotechnology - ICGEB

summary: To identify the relevant targets of the selected miRNAs, we assessed global transcriptome changes by deep-sequencing total neonatal mouse cardiomyocyte RNA after transfection with hsa-miR-590-3p or hsa-miR-199a-3p

overall_design: Four condition experiment; one replicate per condition; mouse neonatal cardiomyocytes transfected with cel-miR-67, hsa-miR-590-3p and hsa-miR-199a-3p; samples collected 72 hours after transfection

first: Ana

last: Eulalio

first: Matteo

last: Dal Ferro

first: Lorena

last: Zentilin

first: Gianfranco

last: Sinagra

first: Serena

last: Zacchigna

first: Mauro

last: Giacca


title: Comparison of GRO-seq with Pol II (Rpb3) ChIP-seq in Drosophila melanogaster S2 cells

city: Ithaca

first: Leighton

last: Core

address: 417 Biotechnology Building

institute: Cornell University

overall_design: Comparison of multiple GRO-seq data sets and a ChIP-seq data set for Pol II (rpb3). GRO-seq also performed +/- Sarkosyl and after depletion of NELF by RNAi.

first: John

last: Lis

first: Karen

last: Adelman


title: Smchd1 dependent and independent pathways determine developmental dynamics of CpG island methylation on the inactive X chromosome.

status: Public on Oct 15 2012

city: Oxford

first: Stephen

last: Taylor

address: Headington

institute: CBRG

summary: X chromosome inactivation involves multiple levels of chromatin modification, established progressively and in a stepwise manner during early development. The chromosomal protein Smchd1 has recently been shown to play an important role in DNA methylation of CpG islands (CGIs), a late step in the X inactivation pathway required for long-term stability of gene silencing. Here we show that inactive X chromosome (Xi) CGI methylation can occur via either Smchd1 dependent or independent pathways.  Smchd1 dependent CGI methylation, the primary pathway, is acquired gradually over an extended period, whereas Smchd1 independent CGI methylation occurs rapidly after the onset of X inactivation. The de novo methyltransferase Dnmt3b is required for methylation of both classes of CGI, whereas Dnmt3a and Dnmt3L are dispensable. Xi CGIs methylated by these distinct pathways differ in respect to their sequence characteristics and immediate chromosomal environment.   We discuss the implications of these results for understanding CGI methylation during development.

overall_design: Examination of CpG methylated DNA from wild type and smchd1 null male and female somatic cells and female ES cells differentiated for either 7 or 10 days. Duplicate samples of each type were generated.

first: Anne-Valerie

last: Gendrel

first: Anwyn

last: Apedaile

first: Heather

last: Coker

first: Ilona

last: Zvetkova

first: Y

last: Tang

first: Derek

last: Huntley

first: Giovanni

last: Montana

first: Eleni

last: Giannoulatou

first: Ausma

last: Termanis

first: Irina

last: Stancheva

first: Marion

last: Leleu

first: Neil

last: Brockdorff


title: Small RNA profiles following TUT knock-down in HeLa

city: Seoul

first: Hyeshik

last: Chang

address: Building 203 Room 525, School of Biological Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu

institute: Seoul National University

summary: The precise control of microRNA (miRNA) biogenesis is important for various cellular functions, and its dysregulation is often associated with human diseases. We previously reported that Terminal uridylyl transferase 4 (TUT4) down-regulates let-7 miRNA biogenesis by oligo-uridylating let-7 precursor (pre-let-7) in mouse embryonic stem cells and that a pluripotency marker Lin28 promotes a processivity of TUT4. Here we find that TUT4 positively controls let-7 biogenesis by adding a uridine residue to the 3’ end of pre-let-7 in the absence of Lin28. Such mono-uridylation enhances Dicer processing by generating an optimal end structure of pre-let-7 for Dicer recognition and may protect pre-miRNA from trimming. Moreover, TUT7, TUT4 and TUT2 redundantly regulate pre-let-7 processing and simultaneous knock down of these TUTs leads to the decrease of mature let-7 and the accumulation of pre-let-7 in cells. This study provides a novel regulation mechanism of miRNA biogenesis, which may function in development and tumorigenesis.

first: Inha

last: Heo

first: Minju

last: Ha

first: V

last: Kim


title: The viral and cellular microRNA targetome in lymphoblastoid cell lines

city: Durham

first: Rebecca

last: Skalsky

address: 430 CARL, Research Dr.

institute: Duke University

summary: Epstein-Barr virus (EBV) is a human herpesvirus linked to a number of B cell cancers and lymphoproliferative disorders. During latent infection, EBV expresses 25 viral pre-microRNAs (miRNAs) and induces the expression of specific host miRNAs, such as miR-155 and miR-21, which potentially play a role in viral oncogenesis. To date, a limited number of EBV miRNA targets have been identified; thus, the role of EBV miRNAs in viral pathogenesis is not well defined. Here, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) combined with deep sequencing and computational analysis to comprehensively examine the viral and cellular miRNA targetome in EBV B95-8-infected lymphoblastoid cell lines (LCLs). We identified 7,827 miRNA-interaction sites in 3,492 cellular 3'UTRs. 531 of these sites contained seed-matches to viral miRNAs. 24 PAR-CLIP-identified miRNA:3'UTR interactions were confirmed by reporter assays. Our results reveal that EBV miRNAs predominantly target cellular transcripts during latent infection, thereby manipulating the host environment. Furthermore, targets of EBV miRNAs are involved in multiple cellular processes that are directly relevant to viral infection, including innate immunity, cell survival, and cell proliferation. Finally, we present evidence that myc-regulated host miRNAs from the miR-17/92 cluster can regulate latent viral gene expression. This comprehensive survey of the miRNA targetome in EBV-infected B cells represents a key step towards defining the functions of EBV-encoded miRNAs, and potentially, identifying novel therapeutic targets for EBVassociated malignancies.

overall_design: Ago2 (Argonaute 2) PAR-CLIP and small RNA deep sequencing of Epstein-Barr virus B95.8-infected lymphoblastoid cell lines (LCLs).

first: Bryan

last: Cullen


title: Neonatal and adult cardiomyocytes

first: Miguel

last: Mano

sample_organism:
Mus musculus
Rattus norvegicus

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus
Human alphaherpesvirus 1
Human alphaherpesvirus 2
Human betaherpesvirus 5
Murid betaherpesvirus 1
human gammaherpesvirus 4
JC polyomavirus
Human immunodeficiency virus 1
Murid gammaherpesvirus 4
Human gammaherpesvirus 8
Merkel cell polyomavirus
Mus musculus cytomegalovirus 2
Betapolyomavirus hominis
Betapolyomavirus macacae

overall_design: Refer to individual Series


title: EZH2 Inhibition as a Therapeutic Strategy for Lymphoma with EZH2 Activating Mutations

first: Gopinath

last: Ganji

first: Michael

last: McCabe

first: Caretha

last: Creasy


title: Global Regulation of Nucleosome Organization And Transcription By The Yeast Ssn6-Tup1 Corepressor

status: Public on Oct 10 2012

city: Houston

first: kaifu

last: chen

address: 1 Baylor plaza, suite 450A

institute: Baylor College of Medicine

platform_organism:
Schizosaccharomyces pombe
Saccharomyces cerevisiae


title: Global Regulation of Nucleosome Organization And Transcription By The Yeast Ssn6-Tup1 Corepressor (MNase-Seq)

summary: The yeast Ssn6-Tup1 complex regulates gene expression through a variety of mechanisms, including positioning of nucleosomes over promoters of some target genes to limit accessibility to the transcription machinery. To further define the functions of Ssn6-Tup1 in gene regulation and chromatin remodeling, we performed genome-wide profiling of changes in nucleosome organization and gene expression that occur upon loss of SSN6 or TUP1, and observed extensive nucleosome alterations in both promoters and gene bodies of derepressed genes. Our improved nucleosome profiling and analysis approaches revealed low-occupancy promoter nucleosomes (P nucleosomes) at locations previously defined as nucleosome-free regions. In the absence of SSN6 or TUP1, this P nucleosome is frequently lost, whereas nucleosomes are gained at -1 and +1 positions, accompanying up-regulation of downstream genes. Our analysis of public ChIP-seq data revealed that Ssn6 and Tup1 preferentially bind TATA-containing promoters, which are also enriched in genes derepressed upon loss of SSN6 or TUP1. These results suggest that stabilization of the P nucleosome on TATA-containing promoters may be a central feature of the repressive chromatin architecture created by the Ssn6-Tup1 corepressor, and that releasing the P nucleosome contributes to gene activation.

overall_design: nucleosomes were prepared from isogenic wild type (BY4742), ssn6 KO and tup1 KO cells after varying degrees of micrococcal nuclease (MNase) digestion, followed by isolation of mononucleosomal DNA and sequencing.  Three replicates of each strain (9 samples) were subjected to Illumina sequencing.

first: Kaifu

last: Chen

first: Marenda

last: Wilson

first: Calley

last: Hirsch

first: Anjanette

last: Watson

first: Shoudan

last: Liang

first: Yue

last: Lu

first: Wei

last: Li

first: Sharon

middle: Y

last: Dent


title: Genome-Wide Profiling of Peroxisome Proliferator-Activated Receptor γ in Primary Epididymal, Inguinal, and Brown Adipocytes Reveals Depot-Selective Binding Correlated with Gene Expression

city: Odense M

first: Susanne

last: Mandrup

address: Campusvej 55

department: Biochemistry and Molecular Biology

country: Denmark

institute: University of Southern Denmark

summary: Here we have employed chromatin immunoprecipitation combined with deep sequencing to map and compare PPARγ  binding in in vitro differentiated primary mouse adipocytes isolated from epididymal, inguinal, and brown adipose tissues. While these PPARγ binding profiles are overall similar, there are clear depot-selective binding sites. Most PPARγ binding sites previously mapped in 3T3-L1 adipocytes can also be detected in primary adipocytes, but there are a large number of PPARγ binding sites that are specific to the primary cells, and these tend to be located in closed chromatin regions in 3T3-L1 adipocytes. The depot-selective binding of PPARγ is associated with highly depot-specific gene expression. This indicates that PPARγ plays a role in the induction of genes characteristic of different adipocyte lineages and that preadipocytes from different depots are differentially preprogrammed to permit PPARγ lineage-specific recruitment even when  differentiated in vitro.

overall_design: Examination of PPARγ binding in in vitro differentiatied adipocytes isolated from three different adipose depots.

first: Majken

last: Siersbæk

first: Anne

last: Loft

first: Mads

middle: M

last: Aagaard

first: Ronni

last: Nielsen

first: Søren

last: Schmidt

first: Natasa

last: Petrovic

first: Jan

last: Nedergaard


title: Transcription profile analysis of S. cerevisiae ZTW1 wild-type and mutant strains

city: Hangzhou

first: Daoqiong

last: Zheng

state: Zhejiang

address: 866 Yuhangtang Road

department: Institute of Microbiology

institute: College of Life Sciences, Zhejiang University

summary: We report the application of single-molecule-based sequencing technology for transcription profile analysis of two S. cerevisiae strains.  By combining the whole genome sequence of these strains,we sought to explore the effects of genomic structure variations on the transcription diversities.

overall_design: Comparsion of transcription profiles in S. cerevisiae strains

first: Xuechang

last: Wu


title: Global changes in the nuclear positioning of chromatin domains and genomic interactions that orchestrate B cell fate

status: Public on Oct 09 2012

city: La Jolla

first: Christopher

last: Benner

state: California

address: 9500 Gilman Dr. MC 0640

institute: University of California, San Diego (UCSD)

summary: The genome is folded into domains that are located in either transcriptionally inert or permissive compartments.  Here we used genome-wide strategies to characterize domains during B cell development. Structured Interaction Matrix Analysis revealed that CTCF occupancy was primarily associated with intra-domain interactions, whereas p300, E2A, Pax5 and PU.1 were involved with intra- and inter-domain interactions that are developmentally regulated.  We identified a spectrum of genes that switched nuclear location during early B cell development. In progenitors the transcriptionally inactive Ebf1 locus was sequestered at the nuclear lamina, thereby preserving multipotency, however upon development into the pro-B cell stage Ebf1 and other genes switched compartments to establish de novo intra- and inter-domain interactions that were associated with B lineage specific transcription signatures.

overall_design: Performed Hi-C, GRO-seq, and ChIP-seq to pinpoint the underlying molecular mechanisms that link transcriptional regulation to genomic structure and architecture in lymphocyte development

first: Yin

last: Lin

first: Robert

last: Mansson

first: Sven

last: Heinz

first: Kazuko

last: Miyazaki

first: Masaki

first: Vivek

last: Chandra

first: Claudia

last: Bossen

last: Glass

first: Cornelis

last: Murre


title: Comparative profiling of chromatin state maps and transcription factor occupancy during human fetal and adult erythropoiesis

status: Public on Oct 05 2012

city: Memphis

first: Jian

last: Xu

state: Tennessee

address: 262 Danny Thomas Place, MS 345

department: Pathology

institute: St. Jude Children's Research Hospital

summary: We report the comparative investigation of genome-wide chromatin state maps, transcription factor (TF) occupancy, and gene expression profiles from developing red cell precursors at two developmental stages. Contrasting the similarities and differences between fetal and adult erythropoiesis provides important insights into the erythroid gene expression programs and gene regulatory networks. Specifically, comparative analyses of human erythropoiesis identify developmental stage-specific enhancers as primary determinants of stage-specific gene expression programs. We find that master regulators, such as GATA1 and TAL1, cooperatively act within active enhancers but have little predictive value for stage-specific enhancer activity. Instead, a set of stage-specific co-regulators collaborates with master regulators and contributes to differential gene expression. We further identify and validate IRF2, IRF6, and MYB as effectors of adult-stage expression program. Thus, the combinatorial assembly of master regulators and transcriptional co-regulators at developmental stage-specific enhancers controls gene expression programs and temporal regulation of transcriptional networks in a mammalian genome.

overall_design: Examination of various histone modifications and transcription factor occupancy by ChIP-seq in fetal and adult proerythroblasts.

first: Zhen

last: Shao

first: Stuart

middle: H

last: Orkin


title: Comparison of the genomic structures and transcription between S. cerevisiae strains ZTW1 and ZGR3

type: Genome variation profiling by genome tiling array

summary:
To determine how does genomic structural variation changed the phenotypes of yeast. aCGH and RNA-Seq were performed to reveal the differeces in the genomic structures and transcription of ZTW1 and ZGR3.

This SuperSeries is composed of the SubSeries listed below.

overall_design: In the aCGH experiment, strain BYZ1 (S288c background) was used as the control. In the RNA-Seq experiment, the total RNA from three independent cultured cells of each yeast strain was extracted. Three cDNA libraries of one sample were mixed before sequencing.


title: Normal and Cancerous Prostate Cells

city: Darlinghurst

first: Aaron

last: Statham

state: NSW

address: 384 Victoria St

department: Cancer Department

country: Australia

institute: Garvan Institute of Medical Research

type: Genome variation profiling by high throughput sequencing

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


title: DOT1L-mediated H3K79 methylation in chromatin is dispensable for Wnt pathway-specific and other intestinal epithelial functions

first: Amit

last: Sinha


title: Genome-wide DNA methylation profiles of non-small cell lung carcinomas and control lung tissues

city: Rotterdam

first: Sjaak

last: Philipsen

address: Dr. Molewaterplein 50

department: Cell Biology

country: Netherlands

institute: ErasmusMC

summary:
Purpose: Generate genome-wide methylation profiles of non-small cell lung carcinomas (NSCLC) and their matching lung tissues for detection of hypermethylated and hypomethylated regions present in the tumors.
Methods: MethylCapture followed by next-generation sequencing (Illumina GAIIx) of 7 nsclc tumor samples and paired lung tissues in replicated, plus one cell line, 2 fully artificially methylated and 2 fully artificially unmethylated controls. Normalization of methylation reads based on CpG coupling factor–method. Relative methylation scores (rms) in 500bp non-overlapping windows. 90th percentile of rpm (reads per million) values for all 500bp genome-wide windows, with rpm <1.33 were considered. Distributions of 10bp bins rms values within each 500bp genomic region were compared using both one-sided Student’s t-test and one-sided Wilcoxon rank-sum test. Testing was done separately for hypo- and hypermethylation and p-value threshold of 10-18.
Results: MethylCap-seq data revealed strong positive correlation between replicate experiments and between paired tumor/lung samples. 14472 differentially methylated regions (DMR) with non-overlapping 500 bp windows were found.  57 DMRs were present in all NSCLC tumors. 287 were unique for squamous-cell carcinomas and 26 unique for adenocarcinomas. While hypomethylated DMRs did not correlate to any particular functional category of genes, the hypermethylated DMRs were strongly associated with genes encoding transcriptional regulators. Furthermore, subtelomeric regions and satellite repeats were hypomethylated in the NSCLC samples.
Conclusion: We provide a resource containing genome-wide DNA methylation maps of NSCLC and their paired lung tissues, and comprehensive lists of known and novel DMRs and associated genes in NSCLC. The DMRs can be in further studies to develop sensitive biological markers for NSCLC, which may enable non-invasive diagnosis and early detection of the disease, and potentially allow histological classification.

overall_design: MethylCap-seq of 7 nsclc tumor samples and paired lung tissues, plus 2 fully methylated and 2 fully unmethylated controls.

first: Rejane

last: Hughes Carvalho

first: Vanja

last: Harbele

first: Jun

last: Hou

first: Teus

last: van Gent

first: Supat

last: Thongjuea

first: Wilfred

last: van IJcken

first: Christel

last: Kockx

first: Rutger

last: Brouwer

first: Erikjan

last: Rijkers

first: Anieta

last: Sieuwerts

first: John

last: Foekens

first: Mirjam

last: van Vroonhoven

first: Joachim

last: Aerts

first: Frank

last: Grosveld

first: Boris

last: Lenhard


title: Divergent Patterns of Endogenous Small RNA Populations From Seed and Vegetative Tissues of Glycine max

city: Urbana

first: Lila

middle: O.

last: Vodkin

state: IL

address: 1201 W. Gregory Dr.

department: Crop Sciences

institute: University of Illinois

summary: An overview of small RNAs sequences existing in seed development and contrasted with vegetative tissues of the soybean

overall_design: Four small RNA sequence populations from high throughput  deep sequencing-by-synthesis and representing different tissues/organs of the soybean were characterized into small RNA classes, level of expresion, genes of origin and putative targeted genes

first: Gracia

last: Zabala

first: Edhilvia

last: Campos

first: Kranthi

last: Varala

first: Sean

last: Bloomfield

first: Sarah

middle: I

last: Jones

first: Hlaing

last: Win

first: Jigyasa

last: Tuteja

first: Bernarda

last: Calla

first: Steven

middle: J

last: Clough

first: Matthew

last: Hudson

middle: O


title: Decoding human cytomegalovirus using ribosome profiling

city: San Francisco

first: Noam

last: Ginossar

address: 1700 4th street

institute: UCSF

summary: The human cytomegalovirus (HCMV) genome was sequenced 20 years ago. However, like other complex viruses, our understanding of its protein coding potential is far from complete. Here, we use ribosome profiling and transcript analysis to experimentally define the HCMV translation products and follow their temporal expression. We identified several hundred previously unidentified open reading frames and confirmed a fraction by mass spectrometry. We found that regulated use of alternative transcript start sites plays a broad role in enabling tight temporal control of HCMV protein expression and allowing multiple distinct polypeptides to be generated from a single genomic locus. Our results reveal an unanticipated complexity to the HCMV coding capacity and illustrate the role of regulated changes in transcript start sites in generating this complexity.

overall_design:
Ribosome profiling and mRNA-seq from 3 time points (5hr, 24hr, 72hr) along HCMV infection.

The supplementary file 'GSE41605_merlin_final_orfs.bed.gz' includes the 751 ORFs of HCMV that were identified in this study.

last: Stern-Ginossar

first: Ben

last: Weisburd

first: Annette

last: Michalski

first: Vu

last: Le

first: Marco

last: Hein

first: Sheng

middle: X

last: Huang

first: Ming

last: Ma

last: Shen

first: Shu

middle: B

last: Qian

first: Hartmut

last: Hengel

first: Matthias

last: Mann

first: Nicholas

last: Ingolia

first: Jonathan

last: Weissman


title: BG3 WT Repliseq

status: Public on Oct 04 2012

city: Toronto

first: DCC

last: modENCODE

state: Ontario

address: MaRS Centre, South Tower, 101 College Street, Suite 800

country: Canada

institute: Ontario Institute for Cancer Research

summary:
modENCODE_submission_5528
This submission comes from a modENCODE project of David MacAlpine. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: The relative time of replication for all unique sequences in the Drosophila genome was determined by sequencing replication intermediates that were differentially labeled with BrdU during S-phase progression.  Briefly, cells were pulse labeled with BrdU and sorted into early, middle and late S-phase fractions.  BrdU labeled sequences from each fraction of S-phase were enriched by immunoprecipitation and sequenced on the Illumina platform.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Cell Line: ML-DmBG3-c2; Tissue: CNS-derived cell-line; Developmental Stage: third instar larval stage; Genotype: y v f mal; Sex: Unknown;  EXPERIMENTAL FACTORS: read length (base pair) ; Cell Line ML-DmBG3-c2

first: David

last: MacAlpine

first: Yoav

last: Lubelsky

first: Jason

last: Belsky

first: Joseph

last: Prinz


title: KC WT Repliseq

summary:
modENCODE_submission_5526
This submission comes from a modENCODE project of David MacAlpine. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: The relative time of replication for all unique sequences in the Drosophila genome was determined by sequencing replication intermediates that were differentially labeled with BrdU during S-phase progression.  Briefly, cells were pulse labeled with BrdU and sorted into early, middle and late S-phase fractions.  BrdU labeled sequences from each fraction of S-phase were enriched by immunoprecipitation and sequenced on the Illumina platform.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Cell Line: Kc167; Tissue: embryo-derived cell-line; Developmental Stage: late embryonic stage; Genotype: se/e; Sex: Female;  EXPERIMENTAL FACTORS: read length (base pair) ; Cell Line Kc167; read length (base pair)


title: Ago HITS-CLIP in KSHV-infected primary effusion lymphoma (PEL) cell lines BCBL-1 and BC-3

city: Gainesville

first: Rolf

last: Renne

address: 2033 Mowry Road

institute: University of Florida

summary: We performed Ago HITS-CLIP to identify targets of viral and human miRNAs in latently KSHV-infected PEL cells

overall_design: Ago HITS-CLIP was performed in two latently infected PEL cell lines, BCBL-1 and BC-3; Argonaute-immunoprecipitation of UV cross-linked Ago-miRNA-mRNA complexes, followed by RNA isolation, library construction, and high-throughput sequencing (Illumina GAxII); we performed 3 biological replicates for each cell line, two technical (sequencing) replicates of BCBL-1 biological replicate 1

first: Irina

last: Haecker

first: Lauren

middle: A

last: Gay

first: Yajie

last: Yang

first: Jianhong

last: Hu

first: Alison

last: Morse

last: McIntyre


title: Paired End CNV W1 Cl.8+ Cells

summary:
modENCODE_submission_5525
This submission comes from a modENCODE project of David MacAlpine. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: Analysis of copy number variation across modENCODE Drosophila cell line by high throughput sequencing.  We observe numerous copy number variations for the different cell lines, with each cell line having a distinct ploidy signature.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: EXPERIMENT TYPE: deep sequencing analysis. BIOLOGICAL SOURCE: Cell Line: CME-W1-Cl.8+; Tissue: dorsal mesothoracic disc; Developmental Stage: third instar larval stage; Sex: Male;  EXPERIMENTAL FACTORS: Cell Line CME-W1-Cl.8+

first: Heather


title: aSyn polyA-RNAseq in PD and unaffected cortical brain samples

city: New York

first: Herve

last: RHINN

institute: Columbia University

summary: We sought to more precisely characterize the different alpha-synuclein (aSyn) 3’UTR mRNA species in normal and PD human brain.  High-throughput, whole-transcriptome sequencing of the 3’UTR ends of polyadenylated mRNA transcripts (termed pA-RNAseq; see Methods) was performed on a cohort of 17 unaffected and 17 PD cerebral cortical tissue samples.  This revealed 5 aSyn 3’UTR isoforms, with lengths of 290, 480, 560, 1070 and 2520 nt.  Of these, the 560 nt and 2520 nt forms were predominant. The existence and relative preponderance of these species was further confirmed by Northern Blot. We next hypothesized, that aSyn 3’UTR selection might be altered in PD.  Comparison of pA-RNAseq profiles from PD and unaffected cerebral cortex samples revealed an increase in the preponderance of the long 3’UTR species (>560 nt) relative to shorter species (<560 nt).  Such a relative increase in aSynL was confirmed by Quantitative real-time RT-PCR (rt-qPCR) and appeared specific for PD, as the increase was also observed by comparison to RNA from amyotrophic lateral sclerosis patient samples.  We note that the modified aSyn 3’UTR selection associated with PD patient tissue was detected in cerebral cortex tissue, which typically harbors pathological evidence of the disease process without frank cell loss; thus, this phenotype is unlikely to be a secondary consequence of neurodegeneration.

overall_design: Comparison of 3'UTR ends of alpha-synuclein in PD and unaffected brain cortex

first: Asa

last: Abeliovich

last: Rhinn


title: Gene Density, Transcription and Insulators Contribute to the Partition of the Drosophila Genome into Physical Domains (Hi-C analysis of Drosophila melanogaster Kc167 genome)

city: Atlanta

first: Chun-hui

last: Hou

address: 1510 Clifton Rd NE

institute: Emory University

summary: The mechanisms responsible for the establishment of physical domains in metazoan chromosomes are poorly understood. Here we find that physical domains in Drosophila chromosomes are demarcated at regions of active transcription and high gene density that are enriched for transcription factors and specific combinations of insulator proteins. Physical domains contain different types of chromatin defined by the presence of specific proteins and epigenetic marks, with active chromatin preferentially located at the borders and silenced chromatin in the interior. Domain boundaries participate in long-range interactions that may contribute to the clustering of regions of active or silenced chromatin in the nucleus. Analysis of transgenes suggests that chromatin is more accessible and permissive to transcription at the borders than inside domains, independent of the presence of active or silencing histone modifications. These results suggest that the higher-order physical organization of chromatin may impose an additional level of regulation over classical epigenetic marks.

overall_design:
We carried out Hi-C experiment using HindIII on Kc167 cells and generate two libraries separately. One library was sequenced once more as technical replicates (TR1 and TR2), the other library was sequenced as biological replicate (BR).
processed files contain the following:
Column 1:  chromosome ID in which the first read in a pair locates
Column 2:  coordinate of the first read
Column 3:  chromosome ID in which the second read in a pair locates
Column 4:  coordinate of the second read

first: Chunhui

first: Li

last: Li

first: Zhaohui

last: Qin

first: Victor

middle: G

last: Corces


title: Long Span DNA Paired-End-Tag (DNA-PET) Sequencing Strategy for the Interrogation of Genomic Structural Mutations

city: Cologne

first: Axel

last: HILLMER

address: Kerpener Str. 62

department: Institute of Pathology

country: Germany

institute: University of Cologne

summary: Structural variations (SVs) contribute significantly to the variability of the human genome and extensive genomic rearrangements are a hallmark of cancer. Genomic DNA paired-end-tag (DNA-PET) sequencing is an attractive approach to identify genomic SVs. The current application of PET sequencing with short insert size DNA is insufficient for the comprehensive mapping of SVs in low complexity and repeat-rich genomic regions. We have developed a robust procedure to generate PET sequencing data using large DNA inserts of 10 - 20 kb for the identification of SVs. We compared the characteristics of the large insert libraries with short insert (1 kb) libraries with the same sequencing depths and costs. Although short insert libraries bear an advantage in identifying small deletions, they do not provide a significantly better breakpoint resolution. Large inserts are superior to short inserts in providing higher physical genome coverage and therefore achieve greater sensitivity for the identification of the different types of SVs, including copy number neutral and complex events. Further, large inserts allow the identification of SVs within repetitive sequences which cannot be spanned by short inserts.

overall_design: Structural variations of three cancer cell lines using short (1 kb) and long (10 kb and 20 kb) insert size DNA fragments

first: Yijun

last: Ruan

first: Fei

last: Yao

last: Hillmer


title: The Transcription Factor T-bet Is Induced by Multiple Pathways and Prevents an Endogenous Th2 Cell Program during Th1 Cell Responses

city: Bethesda

first: Andrew

last: Oler

address: 31 Center Drive, Room 3B62E

department: Bioinformatics and Computational Biosciences Branch (BCBB)

institute: NIAID/NIH

summary: T-bet is a critical transcription factor for T helper 1 (Th1) cell differentiation. To study the regulation and functions of T-bet, we developed a T-bet-ZsGreen reporter mouse strain, in which GFP faithfully reflects the expression of T-bet.  By using this tool, we report that signals elicited by IL-12 and IFNg are redundant in inducing T-bet in mice infected with Toxoplasma gondii and that T-bet does not contribute to its own expression when induced by IL-12 and IFNg. While both T-bet and Stat4 are critical for IFNg production, IFNg signaling is dispensable.  Strikingly, loss of T-bet results in activation of an endogenous Th2 program in cells expressing T-bet-ZsGreen. Genome-wide analyses suggest T-bet directly induces Th1-related genes but indirectly suppresses Th2-related genes. Our study revealed redundancy and synergy among several Th1-inducing pathways in regulating the expression of T-bet and IFNg, and a critical role of T-bet in suppressing an endogenous Th2 program.

overall_design: RNA-Seq experiments were performed using total RNAs isolated from both wild type and Tbx21-/- Th1 cells in duplicates. Tbet ChIP-seq was performed using wild type Th1 cells.  H3K4me1 and H3K27me3 ChIP-seq was performed using both wild type and Tbx21-/- Th1 cells.

first: Jinfang

last: Zhu

first: Dragana

last: Jankovic

first: Gang

last: Wei

first: Suveena

last: Sharma

first: Gangqing

first: Liying

last: Guo

first: Ryoji

last: Yagi

first: Hidehiro

last: Yamane

first: George

last: Punkosdy

first: Lionel

last: Feigenbaum

first: Keji

last: Zhao

first: William

middle: E

last: Paul


title: Paired End CNV Kc 167

first: DCC

last: modENCODE

summary:
modENCODE_submission_5523
This submission comes from a modENCODE project of David MacAlpine. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: Analysis of copy number variation across modENCODE Drosophila cell line by high throughput sequencing.  We observe numerous copy number variations for the different cell lines, with each cell line having a distinct ploidy signature.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: EXPERIMENT TYPE: deep sequencing analysis. BIOLOGICAL SOURCE: Cell Line: Kc167; Tissue: embryo-derived cell-line; Developmental Stage: late embryonic stage; Genotype: se/e; Sex: Female;  EXPERIMENTAL FACTORS: Cell Line Kc167

first: David

last: MacAlpine

first: Heather

first: Joseph

last: Prinz


title: Genome-wide maps of KDM5A/JARID1A/RBP2 in mouse ES cells.

city: Chicago

first: Elizaveta

middle: V

last: Benevolenskaya

address: 900 S. Ashland Ave.

department: Biochemistry and Molecular Genetics

institute: University of Illinois at Chicago

summary: We report the identification of genomic regions bound by KDM5A in mouse embryonic stem (ES) cells and define the functional categories that these regions represent. KDM5A modifies methylated lysine residues on histone tails. We developed anti-KDM5A antibodies and set to detect genomic regions enriched with these antibodies using cells with normal Kdm5a or cells from Kdm5a knockout mice as a control. We found high enrichment with the KDM5A antibodies in normal cells when compared with the genomic background. We found striking identity in the regions enriched with these antibodies in normal cells when compared with the total genomic DNA or with KDM5A ChIP assays from Kdm5a KO cells, showing that the antibodies are specific.

overall_design: Examination of target genes in ES cells

first: Abul

last: Islam

last: Kaelin Jr


title: ENCODE HudsonAlpha Methyl-seq

status: Public on Oct 03 2012

city: Stanford

first: ENCODE

last: DCC

state: CA

address: 300 Pasteur Dr

institute: ENCODE DCC

summary:
This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Florencia Pauli mailto:fpauli@hudsonalpha.org). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu).
This track shows average methylation status in CpG islands. In general, methylation of CpG sites within a promoter causes silencing of the gene associated with that promoter

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

overall_design:
CpG regions were assayed via Methyl-seq, a method developed in the Myers laboratory to measure the methylation status at CpGs throughout the genome. It combines DNA digestion by a methyl-sensitive enzyme HpaII and its methyl-insensitive isoschizomer MspI with the Illumina DNA sequencing platform. The method was first applied in a collaboration with the laboratory of Dr. Julie Baker at Stanford University to study methylation and gene expression changes that occur in human embryonic stem cells before and after differentiation to definitive endoderm. A paper describing the results as well as the method has been submitted for publication [1].
This study profiled genomic DNA and mRNA samples derived from two human embryonic stem cell lines: H9 and BG02. These cells were differentiated into definitive endoderm, embryoid bodies, embryoid body-derived cells, and AFP+ (alpha-fetoprotein positive) hepatocytes. These in vitro samples were profiled with Methyl-seq and compared them with normal tissue samples from 11-week and 24-week fetal liver and adult liver.
Methyl-seq assays more than 250,000 methyl-sensitive restriction enzyme cleavage sites, representing more than 90,000 genomic regions. These regions include 35,528 annotated CpG islands, while the remaining 55,084 non-CpG island regions are distributed across the genome in promoters, genes, and intergenic regions. Sequence tags present in MspI libraries but not in HpaII libraries are derived from methylated regions. Conversely, sequence tags that occur in HpaII libraries come from at least partially unmethylated regions.
In vitro differentiation: Definitive endoderm precursor cells were generated from H9 hES cells by treating them with activin A. Embryoid bodies (EBs) were generated by growing undifferentitated H9 and BG02 hESCs in suspension. EB-derived cells were obtained by plating clumps of the cells from the EBs. AFP+ fetal hepatocytes were derived from EBs by plating EB cells with FgF, followed by fluorescence activated cell sorting (FACS) to isolate cells expressing the green fluorescent protein (GFP) reporter gene driven from the AFP promoter.
Isolation of genomic DNA: Genomic DNA is isolated from biological replicates of each cell line by using the QIAGEN DNeasy Blood & Tissue Kit according to the instructions provided by the manufacturer. DNA concentrations and a level of quality of each preparation is determined by UV absorbance.
HpaII and MspI digestions: Cleavage of DNA by restriction endonuclease HpaII is prevented by the presence of a 5-methyl group at the internal C residue of its recognition sequence CCGG. MspI, an isoschizomer of HpaII, cleaves DNA irrespective of the presence of a methyl group at this position.
For the MspI library, 5 µg genomic DNA was digested in a 100 µl reaction with 1X NEB Buffer2 and 20 units MspI restriction enzyme and incubated for 18 hr at 37°C. For the HpaII library, 5 µg genomic DNA was digested in a 100 µl reaction with 1X NEB Buffer1 and 20 units HpaII restriction enzyme and incubated for 18 hr at 37°C.
Note that in subsequent versions of the Methyl-seq protocol, which will be described later, much lower amounts of genomic DNA were used (1 µg and potentially lower).
DNA library construction and sequencing: High-throughput sequencing libraries were generated from DNA fragments of the HpaII or MspI digested genomic DNA according to the protocol posted at the website: http://myers.hudsonalpha.org/content/protocols.html. This approach was recently modified by removing the first PCR amplification step, just prior to the gel electrophoresis size-selection step, which was found to reduce a fragment-size bias in the sequencing libraries. These libraries were sequenced with an Illumina Genome Analyzer (GA2) according to the manufacturer's recommendations.
Data analysis: For this analyis, reads that align to human genome sequence version hg19 and contain the 5'-CGG-3' HpaII-cut signature on their 5' end were used. These aligned sequence reads were mapped to CCGG sites predicted in silico on hg19. Sites with four or more Msp1 tags occurring in either the forward or reverse direction were retained for analysis. These "assayable" sites were then grouped with neighboring sites that are within 35-75 bp of each other. Thus, a "region" can be comprised of between 2 and 18 digestion sites that are each within 35-75 bp of another site. Methylated and non-methylated calls were made by using HpaII tag data from all assayable cut sites. For each site across each region, the larger of either the forward read count or reverse read count was used. Regions that have an average of 0 or 1 read per cut site are called methylated, and regions with more than one sequence read per site are called unmethylated.

first: Richard

last: Myers

first: Yuya

last: Kobayashi

first: Devin

last: Absher

first: Rebekka

last: Sprouse

first: Florencia

last: Pauli


title: Identification of maternal imprinting control regions (ICRs) in the post-implantation mouse embryo.

city: London

first: Reiner

last: Schulz

address: 8th floor Guy's Tower

department: Medical & Molecular Genetics

institute: King's College London

summary: Genomewide DNA methylation profiles, generated by MeDIP-seq, for 8.5dpc wildtype and Dnmt3l-/+ mouse embryos were compared to identify differentially methylated regions (DMRs) that depend on the activity of the de novo DNA methyltransferase cofactor Dnmt3l in the oocyte. These DMRs were further characterised by their methylation state in mature mouse sperm and in the livers of inter-subspecies newborn mice. Maternal ICRs were identified by hypomethylation in Dnmt3l-/+ embryos as well as sperm, and maternal allele-specific methylation in liver.

overall_design: MeDIP-seq for two pools of wildtype and two pools of Dnmt3l-/+ mouse 8.5dpc embryos, the sperm of three sires, and 12 pools of three different embryonic livers each. Sliding window read count comparison between wildtype and Dnmt3l-/+ embryos, and between wildtype embryos and sperm samples. Read count comparison between the parental alleles at known SNP sites in inter-subspecies liver data.

first: Bourc'his

last: Deborah

first: Carbajosa

last: Guillermo

first: Cowley

last: Michael

first: Down

last: Thomas

first: Duffie

last: Rachel

first: Holland

last: Michelle

first: Oakey

last: Rebecca

first: Proudhon

last: Charlotte

first: Rakyan

last: Vardhman

first: Schulz

last: Reiner