title: Discovering Hematopoietic Mechanisms Through Genome-Wide Analysis of GATA Factor Chromatin Occupancy

status: Public on Nov 25 2009

city: Madison

first: Emery

middle: H.

last: Bresnick

state: WI

address: 1300 University Avenue 383 MSC

department: Pharmacology

institute: University of Wisconsin-Madison

summary:
GATA factors interact with simple DNA motifs (WGATAR) to regulate critical processes, including hematopoiesis, but very few WGATAR motifs are occupied in genomes. Given the rudimentary knowledge of mechanisms underlying this restriction, and how GATA factors establish genetic networks, we used ChIP-seq to define GATA-1 and GATA-2 occupancy genome-wide in erythroid cells. Coupled with genetic complementation analysis and transcriptional profiling, these studies revealed a rich collection of targets containing a characteristic binding motif of greater complexity than WGATAR. GATA factors occupied loci encoding multiple components of the Scl/TAL1 complex, a master regulator of hematopoiesis and leukemogenic target. Mechanistic analyses provided evidence for cross-regulatory and autoregulatory interactions among components of this complex, including GATA-2 induction of the hematopoietic corepressor ETO-2 and an ETO-2 negative autoregulatory loop. These results establish fundamental principles underlying GATA factor mechanisms in chromatin and illustrate a complex network of considerable importance for the control of hematopoiesis.

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

This SuperSeries is composed of the SubSeries listed below.

relation:
SuperSeries of: GSE18868
SuperSeries of: GSE18870

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

first: Tohru

last: Fujiwara

first: Henriette

last: O'Geen

first: Sunduz

last: Keles

first: Kimberly

last: Blahnik

first: Amelia

middle: K

last: Linnemann

first: Yoon-A

last: Kang

first: Kyunghee

last: Choi

first: Peggy

last: Farnham


title: Insights into GATA-1-Mediated Gene Activation versus Repression via Genome-wide Chromatin Occupancy Analysis

city: Cambridge

first: Laura

last: Riva

state: MA

address: 77 Massachusetts Avenue

department: Biological Engineering

country: USA

institute: MIT

type: Genome binding/occupancy profiling by high throughput sequencing

summary:
The transcription factor GATA-1 is required for terminal erythroid maturation and functions as an activator or repressor depending on gene context. Yet its in vivo site selectivity and ability to distinguish between activated versus repressed genes remain incompletely understood. In this study, we performed GATA-1 ChIP-seq in erythroid cells and compared it to GATA-1-induced gene expression changes. Bound and differentially expressed genes contain a greater number of GATA-binding motifs, a higher frequency of palindromic GATA sites, and closer occupancy to the transcriptional start site versus nondifferentially expressed genes. Moreover, we show that the transcription factor Zbtb7a occupies GATA-1-bound regions of some direct GATA-1 target genes, that the presence of SCL/TAL1 helps distinguish transcriptional activation versus repression, and that polycomb repressive complex 2 (PRC2) is involved in epigenetic silencing of a subset of GATA-1-repressed genes. These data provide insights into GATA-1-mediated gene regulation in vivo.

Keywords: Gene regulation

overall_design: Examination of GATA-1 occupancy in MEL cell line.

first: Ming

last: Yu

first: Huafeng

last: Xie

first: Yocheved

last: Schindler

first: Tyler

middle: B

last: Moran

first: Yong

last: Cheng

first: Duonan

first: Ross

last: Hardison

first: Mitchell

middle: J

last: Weiss

first: Stuart

middle: H

last: Orkin

first: Bradley

middle: E

last: Bernstein

first: Ernest

last: Fraenkel

first: Alan

last: Cantor


title: A Genome-Wide Characterization of MicroRNA Genes in Maize

status: Public on Nov 23 2009

city: Cold Spring Harbor

first: Jer-M

last: Chia

state: NY

address: 1, Bungtown Road

institute: Cold Spring Harbor Laboratory

type: Non-coding RNA profiling by high throughput sequencing

summary: MicroRNAs (miRNAs) are small non-coding RNAs that play essential roles in plant growth and development. We conducted a genome-wide survey of maize miRNA genes, characterizing their structure, expression, and evolution. Computational approaches based on homology and secondary structure modeling identified 150 high-confidence genes within 26 miRNA families. For 25 families, expression was verified by deep-sequencing of small RNA libraries that were prepared from an assortment of maize tissues. PCR-RACE amplification of 68 miRNA transcript precursors, representing 18 families conserved across several plant species, showed that splice variation and the use of alternative transcriptional start and stop sites is common within this class of genes. Comparison of sequence variation data from diverse maize inbred lines versus teosinte accessions suggest that the mature miRNAs are under strong purifying selection while the flanking sequences evolve equivalently to other genes. Since maize is derived from an ancient tetraploid, the effect of whole-genome duplication on miRNA evolution was examined. We found that, like protein-coding genes, duplicated miRNA genes underwent extensive gene-loss, with ~35% of duplicate homeologous miRNA genes retained. This number is higher than that observed with protein-coding genes. A search for putative miRNA targets indicated a bias towards genes in regulatory and metabolic pathways. As maize is one of the principal models for plant growth and development, this study will serve as a foundation for future research into the functional roles of miRNA genes.

overall_design: Surveying miRNA genes in 5 maize tissues (root, seedling, tassel, ear, and pollen) by sequencing small RNA libraries using the Illumina Genome Analyzer

first: Lifang

last: Zhang

first: Zhijie

last: Liu

first: Jer-Ming

first: Doreen

last: Ware


title: Identification of small RNAs in rice AGO1 complexes and their targets

city: Beijing

first: Yijun

last: Qi

address: No.7 Science Park Road,Zhongguancun Life Science Park

country: China

institute: National Institute of Biological Sciences, Beijing

type: Expression profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Sequencing of rice degradome

summary: miRNAs can regulate target gene expression by mRNA cleavage. Rice degradome sequencing was employed to validate mRNA targets of rice miRNAs.

overall_design: Two rice samples, 3-week-old seedling and young panicles were included in the study.

first: Liang

last: Wu

first: Qingqing

first: Huanyu

last: Zhou

first: Fangrui

last: Ni


title: Profiling of small RNA populations in rice total extract and purified AGO1 complexes

summary: microRNAs (miRNAs) are a class of small silencing RNAs that have regulatory roles in gene expression. miRNAs interact with Argonaute (AGO) proteins to form effector complexes that can cleave target mRNAs or repress their translation. Rice encodes four AGO1 homologs (AGO1a, AGO1b, AGO1c, and AGO1d). We used an RNAi approach to knock down the four AGO1s. The RNAi lines displayed pleiotropic developmental phenotypes and had increased accumulation of miRNA targets, suggesting the involvement of AGO1s in the miRNA pathway. Three of the AGO1s (AGO1a, AGO1b, and AGO1c complexes) were purified and further characterized. We showed that the three AGO1s all have a strong preference for binding small RNAs (sRNAs) with 5’ U and have Slicer activity. We catalogued the sRNAs in each AGO1 complex by deep sequencing, and found all three AGO1s predominantly bound known miRNAs. Most of the miRNAs were evenly distributed in the three AGO1 complexes, suggesting a redundant role for the AGO1s in the function of these miRNAs. Intriguingly, we also found a subset of miRNAs were specifically incorporated into or excluded from one of the AGO1s, suggesting that there is also functional specialization among the rice AGO1s.

overall_design: Four samples, total extract and three AGO1 complexes were analyzed


title: Genome wide maps of histone demethylase jmjd3, chromatin state and RNA pol in mouse bone marrow-derived macrophages

city: Singapore

first: Vipin

last: Narang

address: 60 Biopolis Street, #02-01, Genome

country: Singapore

institute: Genome Institute of Singapore

summary:
Inflammatory responses triggered by either microbial or endogenous stimuli rely on a complex transcriptional program that involves the differential expression of hundreds of genes. Jmjd3, a JmjC family histone demethylase (HDM), is quickly induced by the transcription factor NF-kB in response to inflammatory stimuli. Jmjd3 erases a histone mark associated with transcriptional repression and silencing, trimethylated lysine 27 in histone H3 (H3K27me3). Thus, Jmjd3-mediated demethylation of H3K27me3 links inflammation to the control of a histone modification involved in lineage determination, differentiation and tissue homeostasis. However, the specific contribution of Jmjd3 induction to innate immunity and inflammation remains unknown. Here we combined genome-wide mapping and gene knockout studies to investigate this issue. Chromatin immunoprecipitation (ChIP) coupled to ultra high-throughput sequencing (ChIP-Seq) in LPS-stimulated primary mouse macrophages demonstrated that Jmid3 is recruited to a large number of genomic targets with a strong preference for active transcription start sites (TSS). Virtually all Jmjd3-bound TSSs were characterized by high levels of H3K4me3, a marker of gene activity, and high levels of RNA polymerase II (Pol_II). Inducible genes showing a strong increase in H3K4me3 and Pol_II recruitment after endotoxin treatment (including those encoding several cytokines, chemokines and antiviral proteins) were in most cases Jmjd3-associated. In Jmjd3-knockout macrophages, initial RNA_Pol II recruitment and activation of Jmjd3 target genes was unaffected, but RNA_Pol II was prematurely released, thus resulting in non-sustained responses. Importantly, most Jmjd3 target genes were not associated with detectable levels of H3K27me3, and transcriptional effects of Jmjd3 absence in the window of time analyzed here were uncoupled from measurable effects on this histone mark. Our data indicate that Jmjd3 is the effector of an NF-kB-controlled feed-forward transcriptional loop pervasively sustaining inflammatory transcriptional responses in a manner that is independent of H3K27me3 demethylation, and suggest the possible use of anti-Jmjd3 drugs to dampen pathologic inflammation.

Keywords: Epigenetics

overall_design: Genome wide maps of histone demethylase jmjd3, the histone marks H3K4me3 and H3K27me3, and RNA-Polymerase II induction in mouse bone marrow-derived macrophages of two types: (a) untreated and (b) stimulated with lipopolysaccharide and interferon gamma to produce an inflammatory response.

first: Chia-Lin

last: Wei


title: Hierarchical rules for Argonaute loading in Drosophila

status: Public on Nov 15 2009

first: Benjamin

last: Czech

address: 1 Bungtown Rd

summary: Drosophila Argonaute-1 and Argonaute-2 differ in function and small RNA content. AGO2 binds to siRNAs, whereas AGO1 is almost exclusively occupied by microRNAs. microRNA duplexes are intrinsically asymmetric, with one strand, the miR strand, preferentially entering AGO1 to recognize and regulate the expression of target mRNAs. The other strand, miR*, has been viewed as a by-product of microRNA biogenesis. Here, we show that miR*s are often loaded as functional species into AGO2. This indicates that each microRNA precursor can potentially produce two mature small RNA strands that are differentially sorted within the RNAi pathway. miR* biogenesis depends upon the canonical microRNA pathway, but loading into AGO2 is mediated by factors traditionally dedicated to siRNAs. By inferring and validating hierarchical rules that predict differential AGO loading, we find that intrinsic determinants, including structural and thermodynamic properties of the processed duplex, regulate the fate of each RNA strand within the RNAi pathway.

overall_design: These were independently processed and sequenced using the Illumina GAII platform. In total, five libraries were analyzed.

first: Rui

first: Yaniv

last: Erlich

first: Julius

last: Brennecke

first: Richard

last: Binari

first: Christians

last: Villalta

first: Assaf

last: Gordon

first: Norbert

last: Perrimon

first: Gregory

last: Hannon


title: Sex-specific and lineage-specific alternative splicing in primates

status: Public on Dec 01 2009

city: Chicago

first: Ran

last: Blekhman

state: IL

institute: University of Chicago

summary:
Comparative studies of gene regulation suggest an important role for natural selection in shaping gene expression patterns within and between species. Most of these studies, however, estimated gene expression levels using microarray probes designed to hybridize to only a small proportion of each gene. Here we used recently-developed RNA sequencing protocols, which side-step this limitation, to assess intra- and inter-species variation in gene regulatory processes in considerably more detail than was previously possible. Specifically, we used RNAseq to study transcript levels in humans, chimpanzees, and rhesus macaques, using liver RNA samples from three males and three females from each species. Our approach allowed us to identify a large number of genes whose expression levels likely evolve under natural selection in primates. These include a subset of genes with conserved sexually dimorphic expression patterns across the three species, which we found to be enriched for genes involved in lipid metabolism. Our data also suggest that while alternative splicing is tightly regulated within and between species, sex-specific and lineage-specific changes in the expression of different splice forms are also frequent. Intriguingly, among genes in which a change in exon usage occurred exclusively in the human lineage, we found an enrichment of genes involved in anatomical structure and morphogenesis, raising the possibility that differences in the regulation of alternative splicing have been an important force in human evolution.

Keywords: Gene Regulation Study

sample_organism:
Macaca mulatta
Pan troglodytes
Homo sapiens

overall_design: Examination of gene expression levels in livers from three primate species (human, chimpanzee, and rhesus macaque), using 3 male and 3 female samples from each species.

first: John

middle: C

last: Marioni

first: Paul

last: Zumbo

first: Matthew

last: Stephens

first: Yoav

last: Gilad


title: Sequence, Biogenesis, and Function of Diverse sRNA Classes Bound to the Piwi-family Proteins of Tetrahymena thermophila

city: Boston

first: Mary

middle: T

last: Couvillion

address: 77 Avenue Louis Pasteur

department: Genetics

institute: Harvard Medical School

summary: PAZ/PIWI-domain (PPD) proteins carrying small RNAs (sRNAs) function in gene and genome regulation. The ciliate Tetrahymena thermophila encodes numerous PPD proteins exclusively of the Piwi clade. We show that the three Tetrahymena Piwi-family proteins (Twis) preferentially expressed in growing cells differ in their genetic essentiality and subcellular localization. Affinity purification of all eight distinct Twi proteins revealed unique properties of their bound sRNAs. Deep sequencing of Twi-bound and total sRNAs in strains disrupted for various silencing machinery uncovered an unanticipated diversity of 23-24 nt sRNA classes in growing cells, each with distinct genetic requirements for accumulation. Altogether Twis distinguish sRNAs derived from loci of pseudogene families, three types of DNA repeats, structured RNAs, and EST-supported loci with convergent or paralogous transcripts. Most surprisingly, Twi7 binds complementary strands of unequal length while Twi10 binds a specific permutation of the guanosine-rich telomeric repeat. These studies greatly expand the structural and functional repertoire of endogenous sRNAs and RNPs.

overall_design: 11 libraries were analyzed. 5 are total sRNA (from knockout strains or WT background strains), and 6 are Argonaute-bound sRNA

first: Kathleen

last: Collins


title: Genome-wide maps of GATA factor occupancy in K562 cells

first: Emery

last: Bresnick

summary:
GATA factors interact with simple DNA motifs (WGATAR) to regulate critical processes, including hematopoiesis, but very few WGATAR motifs are occupied in genomes. Given the rudimentary knowledge of mechanisms underlying this restriction, and how GATA factors establish genetic networks, we used ChIP-seq to define GATA-1 and GATA-2 occupancy genome-wide in erythroid cells. Coupled with genetic complementation analysis and transcriptional profiling, these studies revealed a rich collection of targets containing a characteristic binding motif of greater complexity than WGATAR. GATA factors occupied loci encoding multiple components of the Scl/TAL1 complex, a master regulator of hematopoiesis and leukemogenic target. Mechanistic analyses provided evidence for cross-regulatory and autoregulatory interactions among components of this complex, including GATA-2 induction of the hematopoietic corepressor ETO-2 and an ETO-2 negative autoregulatory loop. These results establish fundamental principles underlying GATA factor mechanisms in chromatin and illustrate a complex network of considerable importance for the control of hematopoiesis.

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: Examination of GATA1 and GATA2 occpancy in K562 cells

first: Tohru

last: Fujiwara

first: Henriette

last: O'Geen

first: Sunduz

last: Keles

first: Kimberly

last: Blahnik

first: Amelia

last: Linnemann

first: Yoon-A

last: Kang

first: Kyunghee

last: Choi

first: Peggy

last: Farnham


title: Effect of read-mapping biases on detecting allele-specific expression from RNA-sequencing data.

status: Public on Oct 22 2009

first: Jacob

middle: F

last: Degner

department: Human Genetics

summary: Next-generation sequencing has become an important tool for genome-wide quantification of DNA and RNA.  However, a major technical hurdle lies in the need to map short sequence reads back to their correct locations in a reference genome.  Here we investigate the impact of SNP variation on the reliability of read-mapping in the context of detecting allele-specific expression (ASE).We generated sixteen million 35 bp reads from mRNA of each of two HapMap Yoruba individuals.  When we mapped these reads to the human genome we found that, at heterozygous SNPs, there was a significant bias towards higher mapping rates of the allele in the reference sequence, compared to the alternative allele.  Masking known SNP positions in the genome sequence eliminated the reference bias but, surprisingly, did not lead to more reliable results overall. We find that even after masking, $\sim$5-10\% of SNPs still have an inherent bias towards more effective mapping of one allele. Filtering out inherently biased SNPs removes 40\% of the top signals of ASE.  The remaining SNPs showing ASE are enriched in genes previously known to harbor cis-regulatory variation or known to show uniparental imprinting.  Our results have implications for a variety of applications involving detection of alternate alleles from short-read sequence data. Scripts, written in Perl and R, for simulating short reads, masking SNP variation in a reference genome, and analyzing the simulation output are available upon request from JFD.

overall_design: RNA-Seq on two YRI Hapmap cell lines. Each individual sequenced on two lanes of the Illumina Genome Analyzer

first: Athma

middle: A

last: Pai

first: Joseph

last: Pickrell

first: Everlyne

last: Nkadori

first: Jonathan

last: Pritchard


status: Public on Oct 16 2009

first: Kevin

middle: P.

last: White

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

department: Institute for Genomics and Systems Biology

type: Genome binding/occupancy profiling by genome tiling array

summary:
Full title: Discrete genomic domains exhibit co-occurrence of H3K9me3 and H3K27me3 histone modifications throughout Drosophila development

Trimethylation of histone H3 on lysine 9 (H3K9me3) and lysine 27 (H3K27me3) is strongly associated with gene silencing and transcriptional repression in both Drosophila and mammals.  H3K9me3 is linked to the formation of heterochromatin, while H3K27me3 is a hallmark of repression of euchromatic genes by Polycomb group proteins.  We examined the genomic distribution of the two modifications at high resolution over nine different stages of Drosophila development, and found that they are present in large overlapping domains in euchromatin.  These dual-mark domains are stable throughout the life of the fly, and cover genomic regions greatly enriched in developmental regulatory genes with spatially restricted expression.

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:
In this study we have performed ChIP-chip in triplicate for 2 repressive histone marks in Drosophila: H3K27me3 and H3K9me3. Hybridizations on Agilent microarrays have been perfomed to compare with Input chromatin sample as a negative control to calculate enrichments.
ChIP-chip experiments have been performed for 9 developmental time points: E0-4h; E4-8h; E12-16h; E16-20h; E20-24h; L1; L2; Pupae and Adult Males.
In addition, 3 ChIP-seq experiments have been performed for validation for E-0-4h, L1 and Pupae time-points where one replicate has been analyzed on the Solexa platform.
To complement this study, total RNA (Poly-A +) has been sequenced on the Solexa platform (2 lanes each) for matching time-points, in order to survey the gene expression levels associated with the ChIP experiments.

first: Ulrich

last: Wagner

first: Nicolas

last: Nègre

first: Zirong

last: Li

first: Haruhiko

last: Ishii

first: Steven

middle: W

last: Miller

first: Parantu

last: Shah

first: Gary

last: Hon

first: Carolyn

last: Morrison

first: Elizabeth

last: Heinz

first: Nicholas

last: Bild

first: David

last: Hanley

first: Robert

last: Grossman

first: Bing

last: Ren

first: James

last: Posakony

middle: P


title: Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway

status: Public on Oct 31 2009

city: Worcester

first: Jia

last: Xu

address: 55 Lake Avenue North

department: Program in Bioinformatics and Integrative Biology

institute: UMASSMED

summary: In flies, small silencing RNAs are sorted between Argonaute1 (Ago1), the central protein component of the microRNA (miRNA) pathway, and Argonaute2 (Ago2), which mediates RNA interference. Extensive double-stranded character—as is found in small interfering RNAs (siRNAs)—directs duplexes into Ago2, whereas central mismatches, like those found in miRNA/miRNA* duplexes, direct duplexes into Ago1. Central to this sorting decision is the affinity of the small RNA duplex for the Dcr-2/R2D2 heterodimer, which loads small RNAs into Ago2. Here, we show that while most Drosophila miRNAs are bound to Ago1, miRNA* strands accumulate bound to Ago2. Like siRNA loading, efficient loading of miRNA* strands in Ago2 favors duplexes with a paired central region and requires both Dcr-2 and R2D2. Those miRNA and miRNA* sequences bound to Ago2, like siRNAs diced in vivo from long double-stranded RNA, typically begin with cytidine, whereas Ago1-bound miRNA and miRNA* disproportionately begin with uridine. Consequently, some pre-miRNA generate two or more isoforms from the same side of the stem that differentially partition between Ago1 and Ago2. Our findings provide the first genome-wide test for the idea that Drosophila small RNAs are sorted between Ago1 and Ago2 according to their duplex structure and the identity of their first nucleotide.

overall_design: Sequencing of small RNAs (either total small RNAs or Ago1-associated small RNAs) in wild-type, dcr-2 and r2d2 mutant flies. Small RNA sequencing, Small RNAs (18-29 nt long), Size selection (18 to 30 nt).

first: Megha

last: Ghildiyal

first: Hervé

last: Seitz

first: Zhiping

last: Weng

first: Phillip

middle: D

last: Zamore


title: High-throughput sequencing of small RNAs in Medicago truncatula

status: Public on Nov 09 2009

city: Norwich

first: Simon

last: Moxon

state: Norfolk

address: University of East Anglia

country: United Kingdom

institute: University of East Anglia

summary:
Medicago truncatula endogenous small RNAs

The dataset contains Medicago truncatula Gaertn. cv. Jemalong endogenous small RNA sequences in the range 18-28 nucleotides. High-throughput Solexa/Illumina sequencing was carried out at the Sainsbury Laboratory, Norwich, UK. Please see www.illumina.com for details of the technology. Small RNA sequences were mapped to Medicago truncatula genome release 2.0 (http://www.medicago.org/genome/), the number of matches to the unfinished genome, if any, is recorded in the Series supplementary file GSE13761_sequence_annotations.txt.gz.

overall_design: Size fractionated small RNA from total RNA extracts was ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to Solexa/Illumina high throughput pyrosequencing. Please see www.illumina.com for details of the sequencing technology.

first: Gyorgy

last: Szittya

first: Dulce

middle: M

last: Santos

first: Manuel

last: Fevereiro

first: Vincent

last: Moulton

first: Tamas

last: Dalmay


title: The small RNA profile in wildtype, R2D2 and loquacious mutant Drosophila

status: Public on Nov 04 2009

first: Trevis

last: Alleyne

address: 303 E. Superior Street

department: Feinberg School of Medicine

institute: Northwestern University

summary: We used SOLiD technology to deep sequence libraries prepared from 18-35 nt PAGE purfied small RNAs  derived from female Drosophila. The objective of our study was to compare the impact of R2D2 or loquacious mutantions on different small RNA populations in the fruit fly.

overall_design: We deep sequenced three separate libraries derived from a wildtype reference, R2D2 and loquacious mutant Drosophila.

first: Joao

last: Marques

first: Richard

last: Carthew


title: Expression of genes and transposons in mop1 mutants and non-mutants

status: Public on Nov 20 2009

city: Ames

first: Patrick

middle: S.

last: Schnable

state: IA

address: 2035B Roy J Carver Co-Lab

institute: Iowa State University

summary: Transposable elements (TEs) comprise a substantial portion of many eukaryotic genomes and are typically transcriptionally silenced. RNA–dependent RNA polymerase 2 (RDR2) is a component of the RNA–directed DNA methylation (RdDM) silencing pathway. In maize, loss of mediator of paramutation1 (mop1) encoded RDR2 function results in reactivation of transcriptionally silenced Mu transposons and a substantial reduction in the accumulation of 24 nt short-interfering RNAs (siRNAs) that recruit RNA silencing components. An RNA–seq experiment conducted on shoot apical meristems (SAMs) revealed that, as expected based on a model in which RDR2 generates 24 nt siRNAs that suppress expression, most differentially expressed DNA TEs (78%) were up-regulated in the mop1 mutant. In contrast, most differentially expressed retrotransposons (68%) were down-regulated. This striking difference suggests that distinct silencing mechanisms are applied to different silencing templates. In addition, 6,000 genes (24% of analyzed genes), including nearly 80% (286/361) of genes in chromatin modification pathways, were differentially expressed. Overall, two-thirds of differentially regulated genes were down-regulated in the mop1 mutant. This finding suggests that RDR2 plays a significant role in regulating the expression of not only transposons, but also of genes. A re-analysis of existing small RNA data identified both RDR2–sensitive and RDR2–resistant species of 24 nt siRNAs that we hypothesize may at least partially explain the complex changes in the expression of genes and transposons observed in the mop1 mutant.

overall_design:
Single sequencing library was constructed for mop1 mutant and non-mutant. Each library was sequenced using 2 lanes on a Solexa flow cell.
Processed data file 'ZmB73_4a.53_filtered_genes.fasta' and its README file are linked below as supplementary files.  The fasta file contains the gene model ID and corresponding sequence generated from maize genome project.  This fasta file was used for the following samples: GSM418173, GSM418174, GSM420173, GSM420174, GSM422828, GSM422829.

first: Yi

last: Jia

first: Damon

middle: R

last: Lisch

first: Kazuhiro

last: Ohtsu

first: Michael

last: Scanlon

first: Dan

last: Nettleton

middle: S


title: A single-base resolution map of an archaeal transcriptome

status: Public on Oct 21 2009

city: Rehovot

first: Omri

last: Wurtzel

address: Hertzel

department: Molecular Genetics

country: Israel

institute: Weizmann Institute of Science

summary: Organisms of the third domain of life, the Archaea, share molecular characteristics both with bacteria and eukarya. These organisms attract scientific attention as research models for regulation and evolution of processes such as transcription, translation and RNA processing. We have reconstructed the primary transcriptome of Sulfolobus solfataricus P2, one of the most widely studied model archaeal organisms. Analysis of 625 million bases of sequenced cDNAs yielded a single-bp resolution map of transcription start sites and operon structures for more than 1000 transcriptional units. The analysis led to the discovery of 310 expressed non-coding RNAs, with an extensive expression of overlapping cis-antisense transcripts to a level unprecedented in any bacteria or archaea but resembling that of eukaryotes. As opposed to bacterial transcripts, most Sulfolobus transcripts completely lack 5' UTR sequences, suggesting that mRNA/ncRNA interactions differ between bacteria and archaea. The data also reveal internal hotspots for transcript cleavage linked to RNA degradation, and predict sequence motifs that promote RNA destabilization. This study emphasizes the importance of transcriptome sequencing as a key tool for understanding the mechanisms and extent of RNA-based regulation for bacteria and archaea.

overall_design: 5 samples of cDNA sequencing (2 of these are replicates), and 3 samples of RACE-cDNA sequencing (described in the samples section).

first: Rotem

last: Sorek


title: Genome-wide mapping of Eset-binding sites and H3K9me3 state in mouse embryonic stem cells

first: Mikael

last: Huss

address: 60 Biopolis Street

type: Methylation profiling by high throughput sequencing

summary:
The histone H3 lysine 9 (H3K9) methyltransferase Eset is an epigenetic regulator critical for the development of the inner cell mass (ICM). Although ICM-derived embryonic stem (ES) cells are normally unable to contribute to the trophectoderm (TE) in blastocysts, we find that depletion of Eset by shRNAs leads to differentiation with the formation of trophoblast-like cells and induction of trophoblast-associated gene expression. Using ChIP-seq analyses, we identified Eset target genes with Eset-dependent H3K9 trimethylation. We confirmed that genes that are preferentially expressed in the TE (Tcfap2a and Cdx2) are bound and repressed by Eset. Single cell PCR analysis shows that the expression of Cdx2 and Tcfap2a is also induced in Eset-depleted morula cells. Importantly, Eset-depleted cells can incorporate into the TE of a blastocyst and subsequently placental tissues. Co-immunoprecipitation and ChIP assays further demonstrates that Eset interacts with Oct4, which in turn recruits Eset to silence these trophoblast-associated genes. Our result suggests that Eset restricts the extraembryonic trophoblast lineage potential of pluripotent cells and links an epigenetic regulator to key cell fate decision through a pluripotency factor.

Keywords: Epigenetics

overall_design: Examine Eset-binding sites and compare the H3K9me3 state between Eset knockdown mouse ES cells and control knockdown mouse ES cells.

first: P

last: Yuan

first: H

last: Ng


title: TimEX from human embryonic stem cells, mesenchymal stem cells, and erythroid cells

city: The Bronx

first: Eric

last: Bouhassira

address: 1300 Morris Park avenue

institute: Albert Einstein College of Medicine

type: Genome variation profiling by genome tiling array

type: Genome variation profiling by high throughput sequencing

summary: The organization of mammalian DNA replication is poorly understood. We have produced genome-wide high-resolution dynamic maps of the timing of replication in human erythroid, mesenchymal and embryonic stem cells using TimEX, a method that relies on gaussian convolution of massive, highly redundant determinations of DNA copy number variations during S phase obtained using either high-density oligonucleotide tiling arrays or massively-parallel sequencing to produce replication timing profiles. We show that in untransformed human cells, timing of replication is highly regulated and highly synchronous, and that many genomic segments are replicated in temporal transition regions devoid of initiation where replication forks progress unidirectionally from origins that can be hundreds of kilobases away. Absence of initiation in one transition region is shown at the molecular level by SMARD analysis. Comparison of ES and erythroid cells replication patterns revealed that these cells replicate about 20% of their genome in different quarter of S phase and that ES cells replicate a larger proportion of their genome in early S phase than erythroid cells. Importantly, we detected a strong inverse relationship between timing of replication and distance to the closest expressed gene. This relationship can be used to predict tissue specific timing of replication profiles from expression data and genomic annotations. We also provide evidence that early origins of replication are preferentially located near highly expressed genes, that mid firing origins are located near moderately expressed genes and that late firing origins are located far from genes.

overall_design:
Comparison of S/G1 from ES cells, MSCs and erythroid cells

The purpose of the study was to measure replication timing by taking advantage of the fact that copy number of DNA increases from 2 two 4 during replication. Specifically, the study was designed to compare the number of copies of DNA of cells in the G1 phase of the cell cycle with the number of copies of DNA of cells in the S phase of the cell cycle. The cells in G1 and S of the cell cycle were obtained by sorting cells based on DNA content after staining with propidium iodide, a fluorescent compound that binds to DNA. We first tested the strategy with Nimblegen tiling arrays representing about 3% of the genome and found that it was indeed possible to measure such a small difference of copy number if a high-level of redundancy was used. We then extended the study genome-wide using massively parallel sequencing on either the SolID or the Illumina platform. Both platforms were tested and we found that for that application, both platforms were equivalent and provided sufficient number of reads to measure timing of replication genome-wide. We then attempted to better understand the correlation between timing of replication and transcription. We therefore obtained RNA from hESC and from erythroblast and hybridized them to standard Affymetrix U133plus2 arrays. This analysis revealed that it was possible to predict the replication timing patterns with relatively good accuracy (r=0.8) using expression data and distance of every genomic windows to express genes.

first: Romain

last: Desprat

first: Nathalie

last: Lailler

first: Julien

last: Lajugie

first: Danielle

last: Thierry-Mieg

first: Jean

first: Carl

last: Schildkraut


title: MicroRNA identity and abundance in porcine skeletal muscles determined by deep sequencing

status: Public on Nov 24 2009

city: Tjele

first: Jakob

last: Hedegaard

address: PO-box 50

department: Department of Genetics and Biotechnology

country: Denmark

institute: Aarhus University, Faculty of Agricultural Sciences

summary: MicroRNAs (miRNA) are short single-stranded RNA molecules that regulate gene expression post-transcriptionally by binding to complementary sequences in the 3' untranslated region (3' UTR) of target mRNAs. MiRNAs participate in the regulation of myogenesis, and identification of the complete set of miRNAs expressed in muscles is likely to significantly increase our understanding of muscle growth and development. To determine the identity and abundance of miRNA in porcine skeletal muscle, we applied a deep sequencing approach. This allowed us to identify the sequences and relative expression levels of 212 annotated miRNA genes, thereby providing a thorough account of the miRNA transcriptome in porcine muscle tissue. The expression levels displayed a very large range, as reflected by the number of sequence reads, which varied from single counts for rare miRNAs to several million reads for the most abundant miRNAs. Moreover, we identified numerous examples of mature miRNAs that were derived from opposite sides of the same predicted precursor stem-loop structures, and also observed length and sequence heterogeneity at the 5' and 3' ends. Furthermore, KEGG pathway analysis suggested that highly expressed miRNAs are involved in skeletal muscle development and regeneration, signal transduction, cell-cell and cell-extracellular matrix communication and neural development and function.

overall_design:
Examination of small RNA profiles in 7 isolates of porcine muscle

The raw sequences were trimmed to 30 nucleotides, and it was set as a requirement that any sequence must appear at least three times and be present in at least two of the seven libraries. All identical reads within a library were grouped and converted into unique sequences. Reads containing Ns or long tracks (¡Ý8) of As were removed and the sequences were trimmed for adaptor-sequences. To annotate the unique sequences, a Decypher Tera-BLASTN Search was performed against a database of mature miRNAs obtained from miRBase (release 12.0). Hits with a match of 16 or more nucleotides to a miRNA from the database were gathered, and the count of each miRNA was normalized to the total number of sequence reads per lane. The outcome of this procedure can be seen in the Aarhus_University_GBI_FC208D2AAXX_blast_mirbase table below.

first: Mathilde

last: Nielsen

first: Jeanette

last: Hansen

first: Rasmus

middle: O

first: Frank

last: Panitz

first: Christian

last: Bendixen

first: Bo

last: Thomsen


title: SetDB1 Contributes to Repression of Genes Encoding Developmental Regulators and Maintenance of ES Cell State

status: Public on Nov 01 2009

first: Garrett

last: Frampton

address: 9 Cambridge Center

department: Biology

institute: Whitehead Instute - MIT

summary: Transcription factors that play key roles in regulating embryonic stem (ES) cell state have been identified, but the chromatin regulators that help maintain ES cells are less well understood.  A high-throughput shRNA screen was used to identify novel chromatin regulators that influence ES cell state.  Loss of histone H3K9 methyltransferases, particularly SetDB1, had the most profound effects on ES cells.  ChIP-Seq and functional analysis revealed that SetDB1 and histone H3K9 methylated nucleosomes occupy and repress genes encoding developmental regulators.  These SetDB1-occupied genes are a subset of the “bivalent” genes, which contain nucleosomes with H3K4me3 and H3K27me3 modifications catalyzed by trithorax and polycomb group proteins, respectively. These genes are subjected to repression by both polycomb group proteins and SetDB1, and loss of either regulator can destabilize ES cell state.

overall_design: ChIP-seq data for SetDB1 and H3K9me3 in mouse ES cells.

first: Steve

last: Bilodeau

first: Michael

last: Kagey

first: Peter

middle: B

last: Rahl

first: Richard

last: Young


title: Comprehensive mapping of long-range interactions reveals folding principles of the human genome.

status: Public on Oct 09 2009

first: Erez

last: Lieberman-Aiden

address: 7 Cambridge Circle

institute: Harvard/MIT/Broad

summary:
We describe Hi-C, a method that probes the three-dimensional architecture of whole genomes by coupling proximity-based ligation with massively parallel sequencing. We constructed spatial proximity maps of the human genome with Hi-C at a resolution of 1Mb. These maps confirm the presence of chromosome territories and the spatial proximity of small, gene-rich chromosomes.  We identified an additional level of genome organization that is characterized by the spatial segregation of open and closed chromatin to form two genome-wide compartments.  At the megabase scale, the chromatin conformation is consistent with a fractal globule, a knot-free conformation that enables maximally dense packing while preserving the ability to easily fold and unfold any genomic locus. The fractal globule is distinct from the more commonly used globular equilibrium model. Our results demonstrate the power of Hi-C to map the dynamic conformations of whole genomes.

The processed heatmap data, showing the number of read pairs corresponding to particular locus pairs in the genome, are linked to the Series record below.

The processed eigenvector data used to generate the heatmaps are linked to the Series record below.

Additional DNAseI and ChIP-Seq data tracks were used in the paper, and are available at the UCSC browser (http://genome.ucsc.edu/)

DNAseI:
wgEncodeUwDnaseSeqRawSignalRep2K562
wgEncodeUwDnaseSeqRawSignalRep2Gm06990

ChIP-Seq:
wgEncodeBroadChipSeqSignalGm12878H3k36me3
wgEncodeBroadChipSeqSignalGm12878H3k27me3

overall_design: Interaction maps in 2 different cell types.

first: Nynke

last: Van Berkum


title: C. elegans small RNAs

status: Public on Oct 07 2009

city: Groningen

first: Eugene

last: Berezikov

address: Antonius Deusinglaan 1

department: European Research Institute for the Biology of Ageing

country: Netherlands

institute: University Medical Center Groningen

summary: High throughput sequencing to derive function of cde-1 in endogenous RNAi in C. elegans

overall_design: Small RNAs were cloned from C. elegans adults, following removal of tri-phosphate groups from 5' end. Sequencing was performed using the Illumina 1G platform.


title: Regulation of Gene Expression and Parent-of-Origin Effects in Maize Hybrids

first: Patrick

last: Schnable

summary:
Heterosis (hybrid vigor) refers to the superior performance of hybrid progeny relative to their parents.  Although widely exploited in agriculture, the mechanisms responsible for heterosis are not well understood.  As a monoecious organism, a given maize plant can be used as both male and female parents of crosses.  Regardless of the cross direction, the maize inbred lines B73 and Mo17 produce hybrids that substantially out-perform their parents.  These reciprocal hybrids differ phenotypically from each other despite having identical nuclear genomes.  Consistent with these phenotypic observations, 30-50% of genes were differentially expressed between these reciprocal hybrids.  An eQTL experiment conducted to better understand the regulation of gene expression in inbred and hybrid lines detected ~4,000 eQTL associations.  The majority of these eQTL act in trans to regulate expression of genes on other chromosomes.  Surprisingly, many of the trans-eQTL, when heterozygous, differentially regulated transcript accumulation in a manner consistent with gene expression in the hybrid being regulated exclusively by the paternally transmitted allele.  The design of the eQTL experiment controlled for cytoplasmic and maternal effects, suggesting that widespread paternal genomic imprinting contributes to the regulation of gene expression in maize hybrids.

Keywords: eQTL, parent-of-origin

overall_design:
GPL4521 - SAM1.2 (Reciprocal Hybrid Comparison):
Six replications of B73xMo17 and Mo17xB73 were grown in growth chambers to tightly control environmental variation.  Seeds from each genotype were taken from a single source (ear) for all six replications.  Within each replication, genotypes were randomly assigned growth locations.  Six healthy seedlings for each genotype and replication were harvested at two weeks of age.  For each replication, B73xMo17 and Mo17xB73 were hybridized to the SAM1.2 microarray (GPL4521) using a randomized, alternate dye assignment.

GPL3333 - SAM1.1 and GPL3538 - SAM3.0 (eQTL Experiment):
Four biological replications of the RIL, B73xRIL, and Mo17xRIL cross-types were planted in growth chambers using seed from a single source for each genotype.   Each RIL and its crosses onto B73 and Mo17 were planted using a split-plot design with RIL group (RIL and its cross onto B73 and Mo17) as the whole-plot treatment factor and cross-type (RIL, B73xRIL, and Mo17xRIL) as the split-plot treatment factor.  The whole-plot portion of the experiment was designed as a randomized complete block design with four replications carried out on four separate occasions in the same environment.  For the split-plot portion of the design, twelve seedlings of each RIL and its crosses were randomized within two adjacent flats in a growth chamber (six healthy seedlings per genotype were randomly chosen and pooled at harvest).  For each replication, RIL, B73xRIL, and Mo17xRIL cross-types were hybridized to custom cDNA microarrays using a loop design such that each loop included all pairwise comparisons between the RIL and its crosses with B73 and Mo17.  Four biological replications were hybridized to the SAM1.1 (GPL3333) array and two of the four biological replications were hybridized to SAM3.0 (GPL3538).  RNA samples were alternately labeled to provide dye balance within each loop and replication.

GPL8734  - Gene Expression between two maize reciprocal hybrids
Heterosis refers to the enhanced agronomic performance of a hybrid relative to its (usually) inbred parents. We have previously documented widespread differences in gene expression in the B73xMo17 hybrid relative to its inbred parents B73 and Mo17 (Swanson, et al., 2006, PNAS). The reciprocal B73xMo17 and Mo17xB73 hybrids are both highly heterotic, but despite having identical nuclear genomes exhibit statistically significant differences in multiple traits. RNA-seq experiment was conducted to compare the gene expression globally between the two reciprocal hybrids.
1 samples from B73XMo17 and Mo17XB73
RNAs were extracted from a single replication of 14-day-old B73xMo17 and Mo17xB73 seedlings. RNAs were purified using DNaseI treatment followed by cleanup with the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) as per manufacturer instructions. Sequencing library construction was completed using the Illumina mRNA-Seq sample preparation kit.
Processed data file 'ZmB73_4a.53_filtered_genes.fasta' and its README file are linked below as supplementary files.  The fasta file contains the gene model ID and corresponding sequence generated from maize genome project.  This fasta file was used for the following samples: GSM418173, GSM418174, GSM420173, GSM420174, GSM422828, GSM422829.

first: Ruth

last: Swanson-Wagner

first: Rhonda

last: DeCook

first: Yi

last: Jia

first: Tim

last: Bancroft

first: Tieming

last: Ji

first: Xuefeng

last: Zhao

first: Dan

last: Nettleton


title: GEMINI (Gastric Encyclopedia of Molecular Interactions and Nodes for Intervention) Phases A-C, normal skin fibroblasts

status: Public on Oct 06 2009

city: Basel

first: Chia-Huey

last: Ooi

address: Grenzacherstrasse 124

country: Switzerland

institute: F. Hoffmann-La Roche Ltd.

summary:
Genome-wide mRNA expression profiles of normal skin fibroblasts, used as one of the (normal) references in the study. Gastric cancer (GC) is the second leading cause of global cancer mortality, with individual gastric tumors displaying significant heterogeneity in their deregulation of various oncogenic pathways. We aim to identify major oncogenic pathways in GC that robustly impact patient survival and treatment response. We used an in silico strategy based on gene expression signatures and connectivity analytics to map patterns of oncogenic pathway activation in 301 primary gastric cancers from three independent patient cohorts. Of 11 oncogenic pathways previously implicated in GC, we identified three predominant pathways (proliferation/stem cell, NF-kB, and Wnt/b-catenin) deregulated in the majority (>70%) of gastric tumors. Using a variety of proliferative, Wnt, and NF-kB-related assays, we experimentally validated the pathway predictions in multiple GC cell lines showing similar pathway activation patterns in vitro. Patients stratified at the level of individual pathways did not exhibit consistent differences in clinical outcome. However, patients grouped by oncogenic pathway combinations demonstrated robust and significant survival differences (e.g., high proliferation/high NF-kB vs. low proliferation/low NF-kB), suggesting that tumor behavior in GC is likely influenced by the combined effects of multiple oncogenic pathways. Our results demonstrate that GCs can be successfully taxonomized by oncogenic pathway activity into biologically and clinically relevant subgroups.
Keywords: normal skin fibroblasts, cell culture

overall_design: Profiling A Normal Skin Fibroblast Cell Line on Affymetrix GeneChip Human Genome U133 Plus 2.0 Array

last: Tan


title: High-Resolution Nucleosome Mapping Reveals Transcription-Dependent Promoter Packaging

status: Public on Oct 15 2009

city: Jerusalem

first: Assaf

last: Weiner

address: Givat Ram

department: Computer Science

institute: The Hebrew University of Jerusalem

summary:
Genome-wide mapping of nucleosomes has revealed a great deal about the relationships between chromatin structure and control of gene expression, and has led to mechanistic hypotheses regarding the rules by which chromatin structure is established. High-throughput sequencing has recently become the technology of choice for chromatin mapping studies, yet analysis of these experiments is still in its infancy. Here, we introduce a pipeline for analyzing deep sequencing maps of chromatin structure and apply it to data from S. cerevisiae. We analyze digestion series where nucleosomes are isolated from under- and over-digested chromatin. We find that certain classes of nucleosomes are unusually susceptible or resistant to overdigestion, with promoter nucleosomes easily digested and mid-coding region nucleosomes being quite stable. We find evidence for highly sensitive nucleosomes located within “nucleosome-free regions,” suggesting that these regions are not always completely naked but instead are likely associated with easily-digested nucleosomes. Finally, since RNA polymerase is the dominant energy-consuming machine that operates on the chromatin template, we analyze changes in chromatin structure when RNA polymerase is inactivated via a temperature-sensitive mutation. We find evidence that RNA polymerase plays a role in nucleosome eviction at promoters, and is also responsible for retrograde shifts in nucleosomes during transcription. Loss of RNA polymerase results in a relaxation of chromatin structure to more closely match in vitro nucleosome positioning preferences. Together, these results provide analytical tools and experimental guidance for nucleosome mapping experiments, and help disentangle the interlinked processes of transcription and chromatin packaging.

Keywords: Genomic

overall_design: Solexa/Illumina sequencing of mononucleosomes. Titration series: gel-purified mononucleosomal DNA from three different titration levels – underdigested, typical digestion, and overdigested. RP021 series: MNase-seq at 0, 20, and 120 minutes after shifting these cells from 25 C to 37 C.

first: Amanda

last: Hughes

first: Moran

last: Yassour

first: Oliver

middle: J

last: Rando

first: Nir

last: Friedman


title: Gene expression study of anaplastic large cell lymphomas: cellular origin, pathogenesis and relation to Hodgkin lymphoma

status: Public on Nov 16 2009

city: Essen

first: Ralf

last: Küppers

address: Virchowstr. 173

department: Institute for Cell Biology (Tumor Research)

country: Germany

institute: University of Duisburg-Essen, Medical School

summary: Anaplastic large cell lymphoma (ALCL) is a main type of T cell lymphomas and comprises three distinct entities: systemic ALK+, systemic ALK- and cutaneous ALK- ALCL. Little is known about their pathogenesis and their cellular origin, and morphological and immunophenotypical overlap exists between ALK- ALCL and classical Hodgkin lymphoma (cHL). We conducted gene expression profiling of microdissected lymphoma cells of ALK+ and ALK- systemic ALCL, cutaneous ALCL and cHL, and of eight subsets of normal T and NK cells. The analysis supports a derivation of ALCL from activated T cells, but the lymphoma cells acquired a gene expression pattern hampering an assignment to a CD4+, CD8+ or CD30+ T cell origin. Indeed, ALCL display a general down-modulation of T cell characteristic molecules. All ALCL types show significant expression of NFκB target genes and upregulation of genes involved in oncogenesis (e.g. EZH2). Surprisingly few genes are differentially expressed between systemic and cutaneous ALK- ALCL despite their different clinical behaviour, and between ALK- ALCL and cHL despite their different cellular origin. ALK+ ALCL are characterized by expression of genes regulated by pathways constitutively activated by ALK. This study provides multiple novel insights into the molecular biology and pathogenesis of ALCL.

overall_design: Analysis of differential gene expression in primary human lymphoma cells of ALK-positive, ALK- negative and primary cutaneous anaplastic large cell lymphoma (ALK+ ALCL, ALK- ALCL and cALCL) in comparison to primary lymphoma cells of (tumor cell rich) classical Hodgkin lymphoma ((tcr)-cHL) and subsets of non-neoplastic T- and NK-lymphocytes isolated from tonsils or blood of healthy donors . 64 gene expression profiles were analysed.

first: Susan

last: Eckerle

first: Verena

last: Brune

first: Claudia

last: Döring

first: Enrico

last: Tiacci

first: Christer

last: Sundström

first: Roman

last: Kodet

first: Marco

last: Paulli

first: Brunangelo

last: Falini

first: Wolfram

last: Klapper

first: Audrey

last: Baur Chaubert

first: Klaus

last: Willenbrock

first: Dirk

last: Metzler

first: Andreas

last: Bräuninger

first: Martin-Leo

last: Hansmann


title: Primary Gastric Cancer Expression Profiles (UK Patient Cohort)

first: Chia-Huey

last: Ooi

summary:
Genome-wide mRNA expression profiles of 31 primary gastric tumors from the UK patient cohort. Gastric cancer (GC) is the second leading cause of global cancer mortality, with individual gastric tumors displaying significant heterogeneity in their deregulation of various oncogenic pathways. We aim to identify major oncogenic pathways in GC that robustly impact patient survival and treatment response. We used an in silico strategy based on gene expression signatures and connectivity analytics to map patterns of oncogenic pathway activation in 25 unique GC cell lines, and in 301 primary gastric cancers from three independent patient cohorts. Of 11 oncogenic pathways previously implicated in GC, we identified three predominant pathways (proliferation/stem cell, NF-kB, and Wnt/b-catenin) deregulated in the majority (>70%) of gastric tumors. Using a variety of proliferative, Wnt, and NF-kB-related assays, we experimentally validated the pathway predictions in multiple GC cell lines showing similar pathway activation patterns in vitro. Patients stratified at the level of individual pathways did not exhibit consistent differences in clinical outcome. However, patients grouped by oncogenic pathway combinations demonstrated robust and significant survival differences (e.g., high proliferation/high NF-kB vs. low proliferation/low NF-kB), suggesting that tumor behavior in GC is likely influenced by the combined effects of multiple oncogenic pathways. Our results demonstrate that GCs can be successfully taxonomized by oncogenic pathway activity into biologically and clinically relevant subgroups.
Keywords: gastric cancer, cell culture

overall_design: Profiling of 31 primary gastric tumors on Affymetrix GeneChip Human Genome U133 Array Set HG-U133A. All tumors were collected with approvals from the St James's University Hospital, United Kingdom and the Research Ethics Review Committee.

first: Patrick

last: Tan


title: Oncogenic Pathway Combinations Predict Clinical Prognosis in Gastric Cancer

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

overall_design: Refer to individual Series


title: Gene expression in csr-1 mutants and sequencing of sRNAs from CSR-1 IP complexes and csr-1 and ego-1 mutants

status: Public on Oct 02 2009

first: Julie

middle: M.

last: Claycomb

address: 373 Plantation Street Biotech2 Suite 219

department: Program in Molecular Medicine

institute: University of Massachusetts Medical School

type: Expression profiling by genome tiling array


summary:
High-throughput pyrosequencing of endogenous small RNAs from CSR-1 IP complexes and csr-1(tm892) and ego-1(om97) mutants with corresponding controls.
RNAi-related pathways regulate diverse processes, from developmental timing to transposon silencing. Here, we show that in C. elegans the Argonaute CSR-1, the RNA-dependent RNA polymerase EGO-1, the Dicer-related helicase DRH-3, and the Tudor-domain protein EKL-1 localize to chromosomes and are required for proper chromosome segregation. In the absence of these factors chromosomes fail to align at the metaphase plate and kinetochores do not orient to opposing spindle poles. Surprisingly, the CSR-1 interacting small RNAs (22G-RNAs) are antisense to thousands of germline-expressed protein-coding genes. Nematodes assemble holocentric chromosomes in which continuous kinetochores must span the expressed domains of the genome. We show that CSR-1 interacts with chromatin at target loci, but does not down-regulate target mRNA or protein levels.  Instead, our findings support a model in which CSR-1 complexes target protein-coding domains to promote their proper organization within the holocentric chromosomes of C. elegans.

overall_design: 5 samples examined. Small RNAs that co-immunopercipitate with CSR-1 protein and input sample. Small RNAs from csr-1(tm892) and ego-1(om97) mutants and corresponding congenic wild type strain.

first: Craig

last: Mello


title: The Physcomitrella Degradome

status: Public on Oct 01 2009

city: University Park

first: Michael

last: Axtell

state: PA

address: 280 Mueller Lab

institute: Pennsylvania State University

summary: Deep sequencing of the 5' ends of uncapped, polyA-enriched mRNA from the moss Physcomitrella patens was performed (e.g. "degradome sequencing").  This GEO record contains all genome-matched and/or transcriptome-matched processed 24nt tags, representing the 5' ends of uncapped messages.  These data were used to 1) discover microRNA targets and 2) to examine patterns of MIRNA hairpin cleavage.

overall_design: A sample of wild-type polyA+ RNA from P. patens protonemata was used to construct a degradome library, which was subsequently sequenced using an ABI SOLiD instrument.  Computational processing to trim 5' and 3' adapter sequences left 24nt tags, the 5' ends of which represented the 5' ends of uncapped mRNAs.

first: Charles

last: Addo-Quaye

first: JoAnn

last: Snyder


title: FLX sequencing of Piwi-associated small RNAs extracted from Drosophila ovarian somatic cells (OSC)

city: Koto-ku

first: Toutai

last: MITUYAMA

state: Tokyo

department: Artificial Intelligence Research Team

country: Japan

institute: National Institute of Advanced Industrial Science and Technology (AIST)

summary:
piRNAs function in silencing retrotransposons by associating with the PIWI proteins, AGO3, Aub, and Piwi, in Drosophila germlines. Bioinformatics analyses of piRNAs in Drosophila ovaries suggested that piRNAs are produced by two systems, the primary processing pathway and the amplification loop, from repetitive genes and piRNA clusters in the genome. The amplification loop occurs in a Dicer-independent, PIWI-Slicer-dependent manner. However, the primary processing pathway remains largely conceptual. Here we show that in ovarian somatic cells, which lack Aub and AGO3 but express Piwi, the primary processing pathway for piRNAs indeed exists.

Keywords: Small RNA profiling by high throughput sequencing

overall_design: Piwi-associated small RNAs were extracted from Drosophila ovarian somatic cells and their deep sequencing was carried out.

first: Kuniaki

last: Saito

first: Sachi

last: Inagaki

last: Mituyama

first: Yukiteru

last: Ono

first: Eri

last: Sakota

first: Hazuki

last: Kotani

first: Kiyoshi

last: Asai

first: Haruhiko

last: Siomi

first: Mikiko


title: Genome-wide maps of GATA1 occupany in induced G1E ER4 cell line.

status: Public on Sep 19 2009

first: Ross

last: Hardison

address: 303 Wartik Lab

summary: we mapped the locations of DNA segments occupied by GATA1 using chromatin immunoprecipitation (ChIP). We have  produced  genome-wide GATA1 ChIP datasets after restoration and activation in G1E-ER4 cells. we employed the sequence census methodology of ChIP-seq , using Illumina GA2 technology to produce 23 million reads (36 nucleotides long) uniquely mapped to the mouse genome (mm8 assembly) for the GATA1 ChIP DNA and 15 million mapped reads for the input DNA

overall_design: Examination of transcription factor GATA1 occupancy

first: Yong

last: Cheng


title: High-throughput sequencing of small RNAs associated with Chalcone Synthase A (CHS-A) sense cosuppression in petunia

status: Public on Sep 28 2009

city: Udine

first: Emanuele

last: De Paoli

state: UDINE

address: via delle Scienze 206

department: Agricultural, Food, Environmental and Animal Sciences

country: Italy

institute: University of Udine

summary: We report the application of sequencing-by-synthesis technology for high-throughput profiling of small RNAs involved in Chalcone synthase A (CHS-A) sense cosuppression in petunia. Sense cosuppression is a classical form of eukaryotic post-transcriptional gene silencing. It was first reported in transgenic petunia, where a transgene overexpressing the host Chalcone Synthase-A (CHS-A) gene caused the degradation of the homologous transcripts and the loss of flower pigmentation. Though sense cosuppression is recognized as an RNA silencing mechanism, little evidence has been yet provided demonstrating its association with the generation of individual small interfering RNAs (siRNAs) that are the assumed determinants. In this work, the deep sequencing of small RNAs in cosuppressed transgenic petunia and WT petunia respectively allowed for the identication of siRNAs that vastly predominate in the silenced flower and guide prominent cleavage events in CHS-A mRNA.

overall_design: Examination of 2 small RNA populations from WT and cosuppressed petunia petals respectively

first: Ana

last: Dorantes-Acosta

first: Jixian

last: Zhai

first: Monica

last: Accerbi

first: Dong-Hoon

last: Jeong

first: Blake

last: Meyers

first: Richard

last: Jorgensen

first: Pamela

last: Green


title: A Hierarchy of H3K4me3 and H3K27me3 Acquisition in Spatial Gene Regulation in Xenopus Embryos

status: Public on Sep 15 2009

city: Nijmegen

first: Gert Jan

last: Veenstra

address: Geert Grooteplein 28

department: Molecular Developmental Biology

institute: Radboud University

summary: Epigenetic mechanisms set apart the active and inactive regions in the genome of multicellular organisms to produce distinct cell fates during embryogenesis. Here, we report on the epigenetic and transcriptome genome-wide maps of gastrula-stage Xenopus tropicalis embryos using massive parallel sequencing of cDNA (RNA-seq) and DNA obtained by chromatin immunoprecipitation (ChIP-seq) of histone H3 K4 and K27 trimethylation and RNA Polymerase II (RNAPII). These maps identify promoters and transcribed regions. Strikingly, genomic regions featuring opposing histone modifications are mostly transcribed, reflecting spatially regulated expression rather than bivalency as determined by expression profile analyses, sequential ChIP, and ChIP-seq on dissected embryos. Spatial differences in H3K27me3 deposition are predictive of localized gene expression. Moreover, the appearance of H3K4me3 coincides with zygotic gene activation, whereas H3K27me3 is predominantly deposited upon subsequent spatial restriction or repression of transcriptional regulators. These results reveal a hierarchy in the spatial control of zygotic gene activation.

overall_design: ChIP-seq profiles of two histone modifications (H3K4me3 and H3K27me3) and RNA Polymerase II, and a RNA-seq profile, of gastrula stage Xenopus tropicalis embryos

first: Robert

last: Akkers

first: Simon

last: van Heeringen

first: Ulrike

middle: G

last: Jacobi

first: Eva

last: Janssen-Megens

first: Kees-Jan

last: Françoijs

first: Hendrik

last: Stunnenberg

first: Gert


title: Illumina mRNA-Seq of control and hnRNP H knockdown in 293T cells

status: Public on Sep 13 2009

city: Cambridge

first: Xinshu

last: Xiao

address: 77 Mass Ave. MIT, 68-211

institute: MIT

summary: Pre-mRNA splicing is regulated through combinatorial activity of RNA motifs including splice sites and splicing regulatory elements (SREs).  Here, we show that the activity of a major class of mammalian SREs is highly sensitive to the strength of the adjacent 5' splice site (5'ss) sequence, and that this has important functional and evolutionary implications.  Activity of G-run SREs was higher for intermediate strength 5'ss by ~4-fold relative to weak 5'ss, and by ~1.3-fold relative to strong 5'ss.  The dependence on 5'ss strength was supported both by comparative genomics and by microarray and Illumina mRNA-Seq analyses of splicing changes following RNAi against the splicing factor heterogeneous nuclear ribonucleoprotein (hnRNP) H, which binds G-runs.  This dependence implies that the responsiveness of exons to changes in hnRNP H levels is a bivariate function of both SRE abundance and 5'ss strength; this relationship may hold also for other splicing factors.  This pattern of activity enables G-runs and hnRNP H to buffer the effects of 5'ss mutations, augmenting both the frequency of 5'ss polymorphism and the evolution of new splicing patterns.

overall_design: Examine mRNA expression in 293T cells following hnRNP H or control siRNA knockdown

first: Zefeng

last: Wang

first: Minyoung

last: Jang

first: Razvan

last: Nutiu

first: Eric

first: Christopher

last: Burge


title: In vivo RNAi screening identifies regulators of actin dynamics as key determinants of lymphoma progression

status: Public on Sep 23 2009

first: Corbin

middle: Elizabeth

last: Meacham

address: 40 Ames Street

department: Koch Institute for Integrative Cancer Research

summary: Mouse models have dramatically improved our understanding of cancer development and tumor biology.  However, these models have shown limited efficacy as tractable systems for unbiased genetic experimentation.  Here, we report the adaptation of loss of function screening to mouse models of cancer.  Using this approach, we have been able to screen nearly 1000 genetic alterations in the context of an individual tumor-bearing mouse.  Results from these experiments have identified a central role for regulators of actin dynamics and cell motility in lymphoma cell homeostasis in vivo, and validation experiments confirmed that these proteins represent bona fide lymphoma drug targets.  Additionally, suppression of one of these targets, Rac2, potentiated the action of the front-line chemotherapeutic vincristine in vivo, suggesting a critical relationship between tumor cell motility and tumor relapse in hematopoietic malignancies.

sample_organism:
Mus musculus
synthetic construct

platform_organism:
Mus musculus
synthetic construct

overall_design: Eu-myc Arf-/- lymphoma cells were transduced with a GFP-tagged, pooled library of 2250 shRNAs targeting 1000 genes with known or putative roles in cancer. Infected cells were GFP sorted. Acutely after sorting, lymphoma cells were tail-vein injected into three syngeneic recipient mice, maintained in three separate culture dishes for two weeks, or collected immediately to serve as a reference sample. At the time of lymphoma presentation, approximately two weeks after injection, tumors were harvested and genomic DNA was extracted from primary tumors and cultured cells. shRNAs were PCR amplified from genomic DNA using common primers, and hairpin representation was analyzed by high throughput sequencing (454 sequencing was used to determine the relative abundance of shRNAs before and after a period of cancer cell growth.). 8 samples were examined - the retroviral plasmid library, cells that were collected immediately after retroviral infection and subsequent GFP-sorting, the three samples that were maintained in vitro for two weeks, and tumors from three individual mice.

middle: E

first: Emily

last: Ho

first: Esther

last: Dubrovsky

first: Frank

last: Gertler

first: Michael

last: Hemann


title: modENCODE RNA-Seq of Drosophila S2-DRSC small RNAs

status: Public on Sep 10 2009

city: Woodbury

first: Chris

last: Zaleski

address: 500 Sunnyside Blvd

institute: Cold Spring Harbor Labs

summary: Deep Sequencing of S2-DRSC small RNAs. 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: Seq of small RNAs from D. melanogaster S2-DRSC

first: Carrie

last: Davis

first: Tom

last: Gingeras

first: Sue

last: Celniker


title: modENCODE RNA-Seq of Drosophila Kc167 small RNAs

summary: Deep Sequencing of Kc167 small RNAs. 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: Seq of small RNAs from D. melanogaster Kc167


title: modENCODE RNA-Seq of Drosophila Cl.8 small RNAs

summary: Deep Sequencing of Cl.8 small RNAs. 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: Seq of small RNAs from D. melanogaster Cl.8


title: modENCODE RNA-Seq of Drosophila Kc167

summary: Deep Sequencing of Kc167 mRNA. 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: Seq of Poly-A+ RNA from D. melanogaster Kc167


title: Uncovering early response of gene regulatory networks in ES cells by systematic induction of transcription factors

city: Baltimore

first: Minoru

last: Ko

state: MD

address: 251 Bayview Blvd, Suite 100, 10C

department: National Institute on Aging

institute: NIH


title: Gastric Cancer Project '08 (Singapore Patient Cohort)

first: Chia-Huey

last: Ooi

summary:
Genome-wide mRNA expression profiles of 200 primary gastric tumors from the Singapore patient cohort. Gastric cancer (GC) is the second leading cause of global cancer mortality, with individual gastric tumors displaying significant heterogeneity in their deregulation of various oncogenic pathways. We aim to identify major oncogenic pathways in GC that robustly impact patient survival and treatment response. We used an in silico strategy based on gene expression signatures and connectivity analytics to map patterns of oncogenic pathway activation in 25 unique GC cell lines, and in 301 primary gastric cancers from three independent patient cohorts. Of 11 oncogenic pathways previously implicated in GC, we identified three predominant pathways (proliferation/stem cell, NF-kB, and Wnt/b-catenin) deregulated in the majority (>70%) of gastric tumors. Using a variety of proliferative, Wnt, and NF-kB-related assays, we experimentally validated the pathway predictions in multiple GC cell lines showing similar pathway activation patterns in vitro. Patients stratified at the level of individual pathways did not exhibit consistent differences in clinical outcome. However, patients grouped by oncogenic pathway combinations demonstrated robust and significant survival differences (e.g., high proliferation/high NF-kB vs. low proliferation/low NF-kB), suggesting that tumor behavior in GC is likely influenced by the combined effects of multiple oncogenic pathways. Our results demonstrate that GCs can be successfully taxonomized by oncogenic pathway activity into biologically and clinically relevant subgroups.
Keywords: gastric cancer, primary tumor

overall_design:
Profiling of 200 primary gastric tumors on Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. All tumors were collected with approvals from the National Cancer Centre, Singapore; the Research Ethics Review Committee; and signed patient informed consent.

FINAL PUBLICATION ANALYSIS INCLUDED 192 GASTRIC ADENOCARCINOMA SAMPLES [LIU ET AL. (2013) GASTROENTEROLOGY]

FAILED QUALITY CONTROL:
GC-011LGE-T, GC-021LAH-T, GC-035PCC-T, GC-038LYC-T

NOT GASTRIC ADENOCARCINOMA:
GC-026-GJK-T, GC-039-TSC-T, GC-2000619T, GC-980327T

first: Patrick

last: Tan


title: GEMINI (Gastric Encyclopedia of Molecular Interactions and Nodes for Intervention) Phases A-C

summary:
Genome-wide mRNA expression profiles of 25 unique gastric cancer cell lines (GCCLs). Gastric cancer (GC) is the second leading cause of global cancer mortality, with individual gastric tumors displaying significant heterogeneity in their deregulation of various oncogenic pathways. We aim to identify major oncogenic pathways in GC that robustly impact patient survival and treatment response. We used an in silico strategy based on gene expression signatures and connectivity analytics to map patterns of oncogenic pathway activation in 25 unique GCCLs, and in 301 primary gastric cancers from three independent patient cohorts. Of 11 oncogenic pathways previously implicated in GC, we identified three predominant pathways (proliferation/stem cell, NF-kB, and Wnt/b-catenin) deregulated in the majority (>70%) of gastric tumors. Using a variety of proliferative, Wnt, and NF-kB-related assays, we experimentally validated the pathway predictions in multiple GC cell lines showing similar pathway activation patterns in vitro. Patients stratified at the level of individual pathways did not exhibit consistent differences in clinical outcome. However, patients grouped by oncogenic pathway combinations demonstrated robust and significant survival differences (e.g., high proliferation/high NF-kB vs. low proliferation/low NF-kB), suggesting that tumor behavior in GC is likely influenced by the combined effects of multiple oncogenic pathways. Our results demonstrate that GCs can be successfully taxonomized by oncogenic pathway activity into biologically and clinically relevant subgroups.
Keywords: gastric cancer, cell culture

overall_design: Profiling of 25 unique Gastric Cancer Cell Lines on Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. Replicates are included for a total of 33 arrays.


title: RNAi in budding yeast

city: San Francisco

first: David

middle: Eric

last: Weinberg

state: CA

address: 600 16th Street, GH S572F

institute: University of California, San Francisco

sample_organism:
Saccharomyces bayanus
Saccharomyces cerevisiae
Candida albicans
Naumovozyma castellii
Vanderwaltozyma polyspora


title: Small RNAs in budding yeast

summary: RNAi, a gene-silencing pathway triggered by double-stranded RNA, is conserved in diverse eukaryotic species but has been lost in the model budding yeast, Saccharomyces cerevisiae.  We report that RNAi is present in other budding-yeast species, including Saccharomyces castellii and Candida albicans.  These species use noncanonical Dicer proteins to generate siRNAs, which mostly correspond to transposable elements and Y´ subtelomeric repeats.  In S. castellii, RNAi mutants are viable but have excess Y´ mRNA levels.  In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons.  These results identify a novel class of Dicer proteins, bring the tool of RNAi to the study of budding yeasts, and bring the tools of budding yeast to the study of RNAi.

platform_organism:
Saccharomyces bayanus
Saccharomyces cerevisiae
Candida albicans
Naumovozyma castellii
Vanderwaltozyma polyspora

overall_design: Employ high-throughput sequencing of endogenous small RNAs from the budding yeasts Saccharomyces castellii, Kluyveromyces polysporus, Candida albicans, Saccharomyces cerevisiae, and Saccharomyces bayanus.

first: Ines

middle: A

last: Drinnenberg

first: Kathleen

middle: T

last: Xie

first: Jeffrey

last: Mower

first: Kenneth

middle: H

last: Wolfe

first: Gerald

last: Fink

last: Bartel


title: modENCODE RNA-Seq of Drosophila BG3 small RNAs

summary: Deep Sequencing of BG3 small RNAs. 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: Seq of small RNAs from D. melanogaster BG3


title: The non-coding RNA of the multidrug resistance-linked vault particle encodes multiple regulatory small RNAs

status: Public on Sep 08 2009

city: Lund

first: Helena

last: Persson

address: Scheelevagen 2

department: Dept of Clinical Sciences, Oncology

country: Sweden

institute: Lund University

summary:
Vault particles are conserved organelles that have been implicated in multidrug resistance and intracellular transport. They are formed by three different proteins and the non-coding vault RNAs (vRNAs). Here we show that human vRNAs produce several small RNAs (svRNAs) by mechanisms different from the canonical microRNA (miRNA) pathway. At least one of these svRNAs, we named svRNAb, associates with Argonaute proteins to guide sequence-specific cleavage and regulate gene expression in a manner similar to miRNAs. We demonstrate that svRNAb downregulates CYP3A4, a key enzyme in drug metabolism. Our findings expand the repertoire of small regulatory RNAs and assign, for the first time, a function to vRNAs that may help explain the observed association between vaults and drug resistance.

Keywords: Transcriptome analysis

overall_design: Small RNAs 18-35 nt were isolated from MCF7 cell total RNA and sequenced on the Illumina Genome Analyzer

first: Anders

last: Kvist

first: Johan

last: Vallon-Christersson

first: P

last: Medstrand

first: A

last: Borg

first: C

last: Rovira


title: Low oxygen responsive small RNAs in Arabidopsis

city: Canberra

first: Andrew

last: Spriggs

state: ACT

address: GPO Box 1600

department: Bioinformatics Group

country: Australia

institute: CSIRO Plant Industry

summary:
Low-oxygen stress associated with natural phenomena such as waterlogging, results in widespread transcriptome changes and a metabolic switch from aerobic respiration to anaerobic fermentation. High-throughput sequencing of small RNA libraries obtained from low-oxygen stressed and control root tissue identified a total of 65 unique microRNA (miRNA) sequences from 46 families, and 14 trans-acting small interfering RNA (tasiRNA) from 3 families. Low-oxygen stress resulted in changes to the abundance of 46 miRNAs from 19 families, and all 3 tasiRNA families. Chemical inhibition of mitochondrial respiration caused similar changes in expression in a majority of the low-oxygen responsive small RNAs analysed. Our data indicate that miRNAs and tasiRNAs play a role in gene regulation and possibly developmental responses to low oxygen, and that a major signal for these responses is likely to be dependent on mitochondrial function.

Keywords: Small RNA transcriptome analysis

overall_design: Examination of root tissue under 2 different environments, control and low oxygen

first: Iain

middle: W

last: Wilson

first: Dov

last: Moldovan

first: Jun

last: Yang

first: Elizabeth

last: Dennis


title: Cdx2 Binding Sites On Cdx2 Expressing ES Cells

first: Minoru

last: Ko

summary:
A) Chromatins were prepared from Cdx2-inducible ES cells cultured for 48 - 60 hours in the Dox+ and Dox- conditions. Chromatin immunoprecipitation (ChIP) was carried out by using anti-FLAG M2 affinity gel. ChIP product was tested by Western blotting using anti-FLAG antibody. Nuclear extract from ES cells cultured for 48 - 60 hours in Dox+ and Dox- condition was used for the Western blot.
B) CDX2 ChIP-Seq peaks in the Hoxa7 gene region. UCSC Mouse Mm9 browser view of Hoxa7 gene locus after mapping CDX2 ChIP-Seq tags locations in the wiggle format. CDX2 ChIP-Seq peaks are shown in red color.
C) Cdx2 ChIP-Seq result was verified by qPCR. Target genes were indicated in (G). Primers flanking a promoter region of Hbb-b1 and Pou5f1 as well as a gene desert region in chromosome 3 were used as negative controls. Primers flanking of Actb gene promoter were used for normalization. The relative enrichment of CDX2 binding was indicated as fold change.
(D) CDX2-binding motifs identified with CisFinder using 200 bp sequences centered at ChIP sites.
(F) Potential CDX2-direct target genes based on ChIP-Seq and the alteration of expression by Cdx2-overexpression.
(G) Identification of CDX2 target genes by combining information on binding sites with gene expression response to Cdx2 over-expression

overall_design:
Chromatin IP against CDX2-Flag fusion protein. MC1 ES cells were genetically modified for ROSA26 locus to have Tet-Off expression cassette for C-terminal FLAG tagged Cdx2.
The peaks are obtained from the Eland Multi Alignment file.
The number of tags in peaks was compared with the number of tags in the control sample for the same region corrected by the total coverage of tags.
See supplemental file of the paper for details.

first: Alexei

last: Sharov

first: Marshall

middle: P

last: Thomas

first: Md

middle: N

last: Islam

first: Dan

middle: L

last: Longo


title: Illumina sequencing of small RNAs from C. elegans embryos

status: Public on Sep 06 2009

city: Berlin

first: Jonas

last: Maaskola

address: Robert-Roessle Str. 10

department: Systems Biology of Gene Regulatory Elements

institute: Max-Delbrueck Centrum Berlin-Buch

summary: Caenorhabditis elegans is one of the most prominent model systems to study embryogenesis. However, it has been impractical to collect large amounts of precisely staged embryos. Thus, early C. elegans embryogenesis has not been amenable to most modern high-throughput genomics or biochemistry assays. To overcome this problem, we devised a method to collect large amounts of cleanly staged C. elegans embryos by Fluorescent Activated Cell Sorting (termed eFACS). eFACS can in principle be applied to all embryonic developmental stages up to hatching. As a proof of principle we show that a single eFACS run routinely yields tens of thousands of almost perfectly staged one-cell embryos. Since in animals the earliest embryonic events are driven by post-transcriptional regulation, we combined eFACS with next-generation sequencing technology to systematically profile the embryonic expression of small, non-coding RNAs. We discovered a wealth of complex and orchestrated changes in the expression between and within almost all classes of small RNAs, including miRNAs, during embryogenesis. Our data indicate that half of all known miRNAs are already expressed in the one-cell stage embryo and we also shed light on the expression and genomic organization of the previously under-appreciated 26G-RNAs. Together, our eFACS data suggest that the complexity of small RNA expression dynamics in animals is comparable to the expression dynamics of protein encoding genes.

overall_design: Various C. elegans embryo samples were generated: mixed embryos by traditional bleaching (Brenner, 1974), early embryos by eFACS (Stoeckius et al., in press). RNA was extracted and length fractionated. Small RNA was subjected to a 5'-dependent ligation protocol to add sequencing adapters. The small RNA samples were sequenced using the Illumina GA I & II.

first: Marlon

last: Stoeckius

first: Teresa

last: Colombo

first: Hans-Peter

last: Rahn

first: Marc

middle: R

last: Friedländer

last: Li

first: Wei

last: Chen

first: Fabio

last: Piano

first: Nikolaus

last: Rajewsky


title: The impact of RNAi on the Saccharomyces castellii transcriptome

first: David

last: Weinberg

overall_design: Examine mRNA abundance in two biological replicates of wild-type (DPB005) and RNAi deletion strains (DPB007, DPB009) of S. castellii.

first: Ines

last: Drinnenberg

first: Kathleen

last: Xie

first: Jeffrey

last: Mower

first: Kenneth

last: Wolfe

first: Gerald

last: Fink

last: Bartel


title: miRNA expression of human cancer and mouse cell lines

status: Public on Aug 25 2009

first: Christine

last: Mayr

address: 9 Cambridge Center

institute: Whitehead Institute

summary:
miRNA expression profile

Keywords: small RNA profiling by high throughput sequencing

overall_design: The small RNA fraction (18-30nt) was cloned and sequenced from total RNA of each cell line


title: Expression data from kidney of 36d old PKD/Mhm rats.

status: Public on Sep 30 2009

city: Mannheim

first: Li

address: Theodor-Kutzer-Ufer

institute: University Heidelberg

summary:
Gender has strong impact on kidney performance. Female gender tends to be renal protective. PKD/Mhm is rat model for human polycystic kidney disease (PKD) caused by a mutation in gene Anks6. PKD progresses faster in male rats compared with females.
We used microarrays to detect gender-dependent gene expression in kidney of 36d old PKD/Mhm rats.

Keywords: gender, genotype

overall_design: Gender-dependent gene expression was examined both in heterozygous PKD/Mhm rats affected with PKD and in their wild type littermates. Three microarrays were hybridized for each combination of gender and genotype.

first: Fluder

last: Kerstin

first: Bott

last: Andrea

first: Kranzlin

last: Bettina

first: Shahi

last: Priyanka

first: Mangold

last: Sonja

first: Gretz

last: Norbert


title: Genome-wide transcriptome sequencing at different stages of Drosophila development, RNA-seq

status: Public on Sep 12 2009

city: Chicago

first: Kevin

middle: P.

last: White

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

department: Institute for Genomics and Systems Biology

institute: University of Chicago

summary:
This is a dataset generated by the Drosophila Regulatory Elements modENCODE Project led by Kevin P. White at the University of Chicago. It contains total RNA sequencing data generated on Solexa Genome Analyzer for whole Drosophila animals at 12 different time-points of development.

Keywords: Epigenetics

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: For each time-point (E-0-4h, E-4-8h, E-8-12h, E-12-16h, E-16-20h, E20-24h, L1, L2, L3, Pupae, Adult Males and Adult Females) a total RNA extraction has been performed. After conversion into double stranded DNA, the samples have been sequenced in duplicate on Solexa Genome Analyzer following Solexa sequencing procedure.

first: Nicolas

last: Negre

first: Nick

last: Bild


title: Partitioning the C. elegans genome by nucleosome modification, occupancy, and positioning

status: Public on Aug 31 2009

city: Piscataway

first: Sam

middle: Guoping

last: Gu

state: NJ

address: Nelson Labs A125, 604 Allison Road

department: Molecular Biology and Biochemistry

institute: Rutgers University

summary:
We have characterized two post-translational histone modifications in C. elegans on a genomic scale.  Micrococcal nuclease digestion and immunoprecipitation were used to obtain distinct populations of single nucleosome cores, which were analyzed using massively parallel DNA sequencing  to obtain positional and coverage maps.  Two methylated histone H3 populations were chosen for comparison:  H3K4 histone methylation (associated with active chromosomal regions) and H3K9 histone methylation (associated with inactivity).  From analysis of the sequence data, we found nucleosome cores with these modifications to be enriched in two distinct partitions of the genome; H3K4 methylation was particularly prevalent in promoter regions of widely-expressed genes while H3K9 methylation was enriched on specific chromosomal arms.  For each of the six chromosomes, the highest level of H3K9 methylation corresponds to the pairing center responsible for chromosome alignment during meiosis.  H3K9 enrichment at pairing centers appears to be an early mark in meiotic chromosome sorting, occurring in the absence of components required for proper pairing of homologous chromosomes.  H3K9 methylation shows an intricate pattern within the chromosome arms, with a particular anti-correlation to regions that display a strong ~10 bp periodicity of AA/TT dinucleotides that is known to associate with germline trancription. By contrast to the global features observed with H3K9 methylation, H3K4 methylation profiles were most striking in their local characteristics around promoters, providing a unique promoter-central landmark for 3903 C. elegans genes and allowing a precise analysis of nucleosome positioning in the context of transcriptional initiation.

Keywords: epigenetics

overall_design: Examination of total nucleosome and nucleosomes with 2 different histone modifications in  C. elegans.  5 samples are analyzed:  1. total mononucleosome core DNA from C. elegans wild type strain N2, 2. anti-H3K4me2/3 precipitated mononucleosome core DNA from C. elegans wild type strain N2, 3.  anti-H3K9me3 precipitated mononucleosome core DNA from C. elegans wild type strain N2, 4.  total mononucleosome core DNA from C. elegans mutant  strain zim-2(tm-574), 5. anti-H3K9me3 precipitated mononucleosome core DNA from C. elegans mutant  strain zim-2(tm-574)

first: Andrew

middle: Z

last: Fire


title: Long range spreading of dosage compensation in Drosophila captures transcribed autosomal genes inserted on X

status: Public on Aug 20 2009

city: Boston

first: Peter

last: Kharchenko

address: 10 Shattuck St.

department: DBMI

institute: Harvard Medical School

summary:
Dosage compensation in D. melanogaster males is achieved via targeting of the MSL complex to X chromosomal genes. This is proposed to involve initial sequence-specific recognition of the X at ~150-300 chromatin entry sites, and subsequent spreading to nearby active genes. Here we test a model in which the spreading step requires transcription and is sequence-independent. We ask whether, in the native context of the X chromosome, MSL complex will target genes of autosomal origin. We find that MSL complex does bind such genes, but only if transcriptionally active. Targeting is accompanied by acetylation of the histone H4K16 residue and two-fold transcriptional up-regulation. We conclude that the presence of a long-sought specific DNA sequence within X-linked genes is not obligatory for MSL complex binding. Instead, physical linkage and transcription play the pivotal roles in the identification of MSL targets irrespective of their origin and DNA sequence.

Keywords: Epigenetics

overall_design: ChIP-seq measurements of MSL complex binding and input control. Chromatin was prepared from third instar male larvae of a genotype y w TrojanElephant; MSL3-TAP; msl3. TrojanElephant is a mini-white- and yellow-marked transposition of genomic region spanning 65 kb from cg13773 to snRNP70K. MSL3-TAP is a mini-white-marked TAP-tagged genomic msl3 transgene. IgG beads were used for pull-down.

first: Andrey

last: Gorchakov

first: Artyom

last: Alekseyenko

middle: V

middle: J

last: Park

first: Mitzi

middle: I

last: Kuroda


title: The maize leaf transcriptome

status: Public on Aug 15 2009

city: Ithaca

first: Lalit

last: Ponnala

address: 616 Rhodes Hall

institute: Cornell University

summary:
We have generated over 80 million 32 nt reads generated from RNA samples isolated from the tip and base of a developing Mo17 leaf.  A comparision of these data with the maize AGP resulted in the confirmation of approximately 88% of the maize filtered gene set

Keywords: Transcriptome analysis

overall_design: Examination of two different RNA samples from two different segments of a developing 3rd leaf

first: Pinghua

last: Li

first: Qi

last: Sun

first: Thomas

last: Brutnell


status: Public on Aug 03 2009

first: UCSF-UBC

last: CENTER

address: UCSF-UBC

institute: UCSF-UBC

summary:
The epigenome is the dynamic interface between our changing environment and the static genome, and understanding it is a goal of immense importance to human health. We will map reference cell epigenomes of the brain, breast, blood and approved embryonic stem cells, inclusive of males and females and different racial groups. This cooperative work will transform our understanding of the short and long-lasting consequences of environment impact on human health and disease.

We are working cooperatively with other Mapping Centers and the Data Coordination Center (EDACC) to comprehensively map epigenomes of select human cells with significant relevance to complex human disease. Our group, consisting of scientists at UCSF, UC Davis, UCSC and the British Columbia Genome Sciences Centre will focus on cells relevant to human health and complex disease including cells from the blood, brain, breast and U.S. Government-approved lines of human embryonic stem cells. We will incorporate high quality, homogeneous cells from males and females, and two predominant racial groups, and biological replicates of each cell type. Production of comprehensive maps will include 6 histone modifications selected for their opposing roles in regulating active and inactive chromatin, DNA methylation and miRNA and gene expression. This epigenetic data, along with genetic and expression data will be integrated using advanced informatics to address fundamental roles of epigenetics in differentiation, maintenance of cell-type identity and gene expression. Our cell and data production pipeline will incorporate verification and data validation with independent methods, and will operate under a model motivated by increased data production and decrease cost. We summarize the analysis capacity of our instruments and our explicit strategy for data sharing of our proposed REMC-generated resources including biological specimens, protocols, data, software tools and intellectual resources. We envision that our group in conjunction with the other REMC teams, the EDACC, ENCODE, future EHHD (Epigenetics of Human Health and Disease) centers and the NIH Roadmap program will develop methods, tools and reference epigenome maps for the research community that will make the promise of epigenetics in understand and treating human complex disease a reality. Our reference epigenomes will enable new disciplines including human population epigenetics, comparative epigenomics, neuroepigenetics, and therapeutic epigenetics for tissue regeneration and reversal of disease.

Contributor: BCCA Genome Sciences Centre

****************
For data usage terms and conditions, please refer to:
http://www.drugabuse.gov/funding/funding-opportunities/nih-common-fund/epigenomics-data-access-policies
****************

overall_design:
UCSF-UBC Human Reference Epigenome Mapping Project - samples, experiments and processed data files will continue to be added to this study as the project proceeds.

EDACC Genboree Page:
http://genboree.org/java-bin/project.jsp?projectName=XML%20Submissions%2FUCSF-UBC%2FSTUDY%2FEDACC.822


title: Identification of transcription factor EOR-1 binding sites in C.elegans

status: Public on Jul 31 2009

city: New Haven

first: Flora

last: Vaccarino

state: CT

address: 230 South Frontage Road

department: Child Study Center

institute: Yale University

summary:
We, being members of modENCODE consortium, have established an experimetal pipeline in C.elegans that allows global identification of the binding sites for transcription factors using chromatin immunoprecipitation followed by illumina high-throughput sequencing (ChIP-seq). In current study, we identified the binding sites for EOR-1 at L3 stage, which plays important roles in regulating RAS/RAF-mediated signaling during excretory system development and RAS/RAF- and WNT-mediated signaling during P12 fate specification.

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: EOR-1 was tagged with a dual GFP:3xFLAG tag at the carboxy terminus, whose expression pattern was confirmed through both fluorescence imaging and immunoblot analysis. The binding sites were determined using ChIP-seq. Two biological replicates were used in this study and non-immunoprecipitated chromatin (input) from the same sample served as a control. Concordance between two replicates is about 95% when p-value is 0.05

first: Wei

last: Niu


title: Nucleosome deposition and DNA methylation at coding region boundaries

status: Public on Aug 11 2009

city: Singapore

first: Jung Kyoon

last: Choi

address: 60 Biopolis Street

institute: Genome Institute of Singapore

summary:
We profiled CpG methylation for human T cells and compared this pattern with public T cell nucleosome (H2A.Z) and polymerase II profiles (SRA000234). Focusing on DNA regions surrounding the start codon and stop codon, instead of the transcription start and end sites, we discovered a very intriguing pattern, namely methylation and nucleosomal peaks at those regions, more prominent than peaks near transcript boundaries. Elongating polymerases tend to pause near both coding ends immediately upstream of the epigenetic peaks, causing a significant reduction in elongation efficiency, suggesting their role in control of RNA splicing. Conserved features in underlying protein coding sequences seem to dictate their evolutionary conservation across multiple species. The nucleosomal and methylation marks are commonly associated with high sequence-encoded DNA-bending propensity but differentially with CpG density.

Keywords: Epigenetics

overall_design: Sample submission examines CpG methylation from human T cells.

first: Jung

middle: K

first: Young

last: Kim