title: miR-221 is required for endothelial tip cell behaviors during vascular development

status: Public on Feb 28 2012

city: Worcester

first: Abirami

last: Lakshmanan

state: Massachusetts

address: 55 Lake Avenue North

department: PGFE

institute: Umass Medical School

summary: Through deep sequencing and functional screening in zebrafish, we find that miR-221 is essential for angiogenesis. miR-221 knockdown phenocopied defects associated with loss of the tip cell-expressed Flt4 receptor.  Furthermore, miR-221 was required for tip cell proliferation and migration, as well as tip cell potential in mosaic blood vessels.  miR-221 knockdown also prevented “hyper-angiogenesis” defects associated with Notch deficiency and miR-221 expression was inhibited by Notch signaling.  Finally, miR-221 promoted tip cell behavior through repression of two targets: cyclin-dependent kinase inhibitor 1b (cdkn1b) and phosphoinositide-3-kinase regulatory subunit 1 (pik3r1).  These results identify miR-221 as an important regulatory node through which tip cell migration and proliferation are controlled during angiogenesis.

overall_design: Identification of endothelial-expressed microRNA from FACS-isolated zebrafish endothelial cells.

first: Nathan

last: Lawson

first: Stefania

last: Nicoli


title: Conserved molecular interactions within MYST-ING acetyltransferase complexes that regulate cell proliferation

status: Public on Mar 15 2012

city: Lausanne

first: Eric

middle: R

last: Paquet

state: Vaud

address: Station 15  CH - 1015

department: Life sciences

country: Switzerland

institute: EPFL

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Systematic Discovery of Structural Elements Governing Mammalian mRNA Stability

city: San Francisco

first: Hani

last: Goodarzi

state: CA

department: Biochemistry and Biophysics

country: USA

institute: UCSF

type: Non-coding RNA profiling by high throughput sequencing

type: Expression profiling by high throughput sequencing

summary:
Decoding post-transcriptional regulatory programs underlying gene expression is a crucial step toward a predictive dynamical understanding of cellular state transitions. Despite recent systematic efforts, the sequence determinants of such mechanisms remain largely uncharacterized. An important obstacle in revealing these elements stems from the contribution of local secondary structures in defining interaction partners in a variety of regulatory contexts, including but not limited to transcript stability, alternative splicing and localization. There are many documented instances where the presence of a structural regulatory element dictates alternative splicing patterns (e.g. human cardiac troponin T) or affects other aspects of RNA biology. Thus, a full characterization of post-transcriptional regulatory programs requires capturing information provided by both local secondary structures and the underlying sequence. We have developed a computational framework based on context-free grammars and mutual information that systematically explores the immense space of structural elements and reveals motifs that are significantly informative of genome-wide measurements of RNA behavior. The application of this framework to genome-wide mammalian mRNA stability data revealed eight highly significant elements with substantial structural information, for the strongest of which we showed a major role in global mRNA regulation. Through biochemistry, mass-spectrometry, and in vivo binding studies, we identified HNRPA2B1 as the key regulator that binds this element and stabilizes a large number of its target genes. Ultimately, we created a global post-transcriptional regulatory map based on the identity of the discovered linear and structural cis-regulatory elements, their regulatory interactions and their target pathways. This approach can also be employed to reveal the structural elements that modulate other aspects of RNA behavior.

This SuperSeries is composed of the SubSeries listed below.


title: The translational landscape of mTOR signaling steers cancer initiation and metastasis

first: Andrew

middle: Caleb

last: Hsieh

address: 1450 3rd Street, HD Rm 319

department: Urology

summary: The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth and cancer. However, the downstream translationally regulated nodes of gene expression that may direct cancer development are poorly characterized. Employing ribosome profiling we uncover specialized translation of the prostate cancer genome by oncogenic mTOR signaling, revealing a remarkably specific repertoire of genes involved in cell proliferation, metabolism, and invasion. We extend these findings by functionally characterizing a class of translationally controlled pro-invasion mRNAs that we show direct prostate cancer invasion and metastasis downstream of oncogenic mTOR signaling. We further develop a clinically relevant ATP site inhibitor of mTOR, INK128, which reprograms this gene expression signature with therapeutic benefit for prostate cancer metastasis, for which there is presently no cure. Together, these findings significantly extend our understanding of how the “cancerous” translation machinery steers specific cancer cell behaviors and may be therapeutically targeted.

overall_design: Examination of mRNA translation in human prostate cancer upon differential inhibition of the mTOR signaling pathway.

first: A

middle: C

first: Y

last: Liu

first: M

middle: P

last: Edlind

first: N

middle: T

last: Ingolia

last: Janes

last: Sher

first: E

middle: Y

last: Shi

first: C

last: Stumpf

last: Christensen

first: MJ

last: Bonham

first: S

last: Wang

first: P

last: Ren

last: Martin

first: K

last: Jessen

middle: E

last: Feldman

first: J

middle: S

last: Weissman

middle: M

last: Shokat

last: Rommel

first: D

last: Ruggero


title: Extrachromosomal microDNAs and chromosomal microdeletions in normal tissues

status: Public on Feb 27 2012

city: Charlottesville,

first: Anindya

last: Dutta

email: ad8q@virginia.edu, pk7z@virginia.edu

state: VA

address: Room no. 1232, Jordan Hall

department: MD-BIOC BC/Mole Genetics~School of medicine

institute: University of Virginia

summary: We have identified tens of thousands of short extrachromosomal circular DNAs (microDNA) in mouse tissues as well as mouse and human cell lines.  These microDNAs are 200-400 bp long, derived from unique non-repetitive sequence and are enriched in the 5' untranslated regions of genes, exons and CpG islands.  Chromosomal loci that are enriched sources of microDNA in adult brain are somatically mosaic for micro-deletions that appear to arise from the excision of microDNAs.  Germline microdeletions identified by the "Thousand Genomes" project may also arise from the excision of microDNAs in the germline lineage.  We have thus identified a new DNA entity in mammalian cells and provide evidence that their generation leaves behind deletions in different genomic loci.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Circular DNA profiling by high throughput sequencing

first: Pankaj

last: Kumar

first: Yoshiyuki

last: Shibata


title: GATA-1 in proliferating and differentiating murine ES cell derived erythroid progenitors

city: Bronx

first: Sandeep

middle: Nagaraj

last: Wontakal

state: NY

address: 1300 Morris Park Ave Chanin 402

department: Cell Biology

institute: Albert Einstein College of Medicine


title: Genome-wide occupancy map of GATA-1 in proliferating and differentiating murine ES cell derived erythroid progenitors (ES-EP)

summary: We find GATA-1 occupies 6,600 sites in proliferating erythroid progenitors and 10,600 sites in differentiating progenitors.  80-90% of GATA-1 binds within intragenic or intergenic regions, while <20% of GATA-1 is found within 2kb of TSS.

overall_design: Assaying GATA-1 occupancy in normal erythroid progenitors in both proliferating and differentiating conditions.

first: Arthur

last: Skoultchi


title: 4-7 Hour ORC ChIP-Seq experiment

city: Toronto

first: DCC

last: modENCODE

state: Ontario

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

country: Canada

institute: Ontario Institute for Cancer Research

summary:
modENCODE_submission_4192
This submission comes from a modENCODE project of David MacAlpine. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: We will precisely identify sequence elements that direct DNA replication by using chromatin immunoprecipitation of known replication initiation complexes.  These experiments will be conducted in multiple cell types and developmental tissues.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: Oregon-R(official name : Oregon-R-modENCODE genotype : wild type ); Developmental Stage: Embryo 4-7h; Genotype: wild type;  EXPERIMENTAL FACTORS: Strain Oregon-R(official name : Oregon-R-modENCODE genotype : wild type ); read length (read_length) ; Antibody dORC2 (target is Drosophila ORC2p); Developmental Stage Embryo 4-7h

first: David

last: MacAlpine

first: Heather

first: Matthew

last: Eaton

first: Joseph

last: Prinz


title: Integrated genome-wide analysis of transcription factor occupancy, RNA polymerase II binding and steady-state RNA levels identify differentially regulated functional gene classes

status: Public on Mar 09 2012

city: Utrecht

first: Michal

last: Mokry

address: Lundlaan 6

country: Netherlands

institute: Wilhelmina Children's Hospital, University Medical Center Utrecht

summary: Routine methods for assaying steady-state mRNA levels such as RNA-seq and micro-arrays are commonly used as readouts to study the role of transcription factors (TFs) in gene expression regulation. However, cellular RNA levels do not solely depend on activity of TFs and subsequent transcription by RNA polymerase II (Pol II), but are also affected by RNA turnover rate. Here, we demonstrate that integrated analysis of genome-wide TF occupancy, Pol II binding and steady-state RNA levels provide important insights in gene regulatory mechanisms. Pol II occupancy, as detected by Pol II ChIP-seq, was found to correlate better with TF occupancy compared to steady-state RNA levels and is thus a more precise readout for the primary transcriptional mechanisms that are triggered by signal transduction. Furthermore, analysis of differential Pol II occupancy and RNA-seq levels identified genes with high Pol II occupancy and relatively low RNA levels and vice versa. These categories are strongly enriched for genes from different functional classes. Our results demonstrate a complementary value in Pol II chip-seq and RNA-seq approaches for better understanding of gene expression regulation.

overall_design: 3 replicates of RNAPII ChIP-seq and RNA-seq under normal and Wnt suppressed conditions

first: Pantelis

last: Hatzis

first: Jurian

last: Schuijers

first: Nico

last: Lansu

first: Frans-Paul

last: Ruzius

first: Hans

last: Clevers

first: Edwin

last: Cuppen


title: Gene expression and ChIP-seq analyses of mesenchymal stem cells, osteoblasts and the U2OS osteosarcoma cell line

status: Public on Mar 02 2012

city: BALTIMORE

first: hari

last: easwaran

state: MD

address: 1650 Orleans Street, CRB1, Room 530

department: Oncology

institute: Johns Hopkins University


title: 4-7 Hour PolII ChIP-Seq experiment

summary:
modENCODE_submission_4193
This submission comes from a modENCODE project of David MacAlpine. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: We will precisely identify sequence elements that direct DNA replication by using chromatin immunoprecipitation of known replication initiation complexes.  These experiments will be conducted in multiple cell types and developmental tissues.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: Oregon-R(official name : Oregon-R-modENCODE genotype : wild type ); Developmental Stage: Embryo 4-7h; Genotype: wild type;  EXPERIMENTAL FACTORS: Strain Oregon-R(official name : Oregon-R-modENCODE genotype : wild type ); read length (read_length) ; Antibody Anti-RNA polymerase II clone CTD4H8 (target is RNA polymerase II); Developmental Stage Embryo 4-7h


title: Loss of PHF20 does not negatively affect deposition of H4K16 acetylation, but does impact the expression of MOF targets

city: Houston

first: kaifu

last: chen

address: 1 Baylor plaza, suite 450A

institute: Baylor College of Medicine

summary: Protein post-translational modifications transmit signals in part by creating binding sites for effector molecules. This is especially true in epigenetic pathways where histone tails are heavily modified, resulting in the recruitment of molecules that can affect transcription. One such molecule, plant homeodomain finger protein 20 (PHF20), uses a Tudor domain to read dimethyl-lysine residues and is a known component of the MOF histone acetyltransferase protein complex, suggesting it plays a role in the crosstalk between lysine methylation and histone acetylation. We sought to investigate the biological role of PHF20 by generating a knockout mouse. Without PHF20, mice die shortly after birth and display a wide variety of phenotypes within the skeletal and hematopoietic systems. Mechanistically, PHF20 is not required for maintaining the global H4K16 acetylation levels, but instead works downstream in transcriptional regulation of MOF target genes.

overall_design: ChIP sequencing of H4K16ace ChIP DNA from PHF20 knockout and wild type cells using Illumina Solexa Genome Analyzer II single end sequencing protocol.

first: Aimee

middle: I

last: Baduea

first: Kim

last: Cardenas

first: Kaifu

last: Chen

first: Donna

last: Kusewitt

first: Ellen

last: Richie

first: Wei

last: Li

first: Mark

last: Bedford


title: Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay

status: Public on Feb 26 2012

city: Cambridge

first: Tarjei

last: Mikkelsen

state: MA

address: 7 Cambridge Center

institute: Broad Institute

summary: We apply a massively parallel reporter assay (MPRA) that relies on mRNA and plasmid tag sequencing (Tag-Seq) to compare the regulatory activities of more than 27,000 distinct variants of two inducible enhancers in human cells: a synthetic cAMP-regulated enhancer and the virus-inducible interferon beta enhancer. The resulting data define accurate maps of functional transcription factor binding sites in both enhancers at single-nucleotide resolution and can be used the to train quantitative sequence-activity models (QSAMs).

sample_organism:
Escherichia coli
Homo sapiens

platform_organism:
Escherichia coli
Homo sapiens

overall_design: Reporter Tag-Seq from HEK293 cells transfected with each of six MPRA plasmid pools, with and without stimulation (forskolin or Sendai virus). The reporter mRNAs contain unique 10 nucleotide tags that facilitates quantitation of their abundances. The same tags were also sequenced from each ransfected plasmid pool to facilitate normalization to plasmid copy numbers. The reporter constructs were designed according to two different mutagenesis strategies: 'single-hit scanning' and 'multi-hit sampling'. The specific variants are included in the processed data files.

first: Alexandre

last: Melnikov

first: Anand

last: Murugan

first: Xiaolan

last: Zhang


title: Genome-wide analysis of histone modification, protein-DNA binding, cytosine methylation and transcriptome data in mouse and human ES cells and pig iPS cells

city: La Jolla

first: Pengfei

last: Yu

address: 9500 Gilman Drive MC 0419

department: Bioengineering

institute: University of California San Diego

type: Methylation profiling by high throughput sequencing

summary:
Genome-wide analysis of histone modification (H2AZ, H3K27ac, H3K27me3, H3K36me3, H3K4me1, H3K4me2, H3K4me3 and H3K9me3), protein-DNA binding (TAF1, P300, Pou5f1  and Nanog), cytosine methylation and transcriptome data in mouse and human ES cells and pig iPS cells
We generated histone modification data (H2AZ, H3K27ac, H3K27me3, H3K36me3, H3K4me1, H3K4me2, H3K4me3 and H3K9me3) and protein-DNA binding data (TAF1, P300, Pou5f1  and Nanog) using Chromatin Immunoprecipitation followed by short sequencing (ChIP-seq), cytosine methylation data using methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq) and DNA digestion by methyl-sensitive restriction enzymes followed by sequencing (MRE-seq), transcriptome data with RNA short sequencing (RNA-seq) in human embryonic stem cells, mouse embryonic stem cells, pig induced pluripotent stem cells and mouse embryonic stem cells under activin-A-induced-differentiation.

sample_organism:
Homo sapiens
Sus scrofa
Mus musculus

platform_organism:
Homo sapiens
Sus scrofa
Mus musculus

overall_design: Examination of 8 histone modifications, 4 protein-DNA binding, cytosine methylation and transcriptome in human embryonic stem cells, mouse embryonic stem cells, pig induced pluripotent stem cells and mouse embryonic stem cells under activin-A-induced-differentiation.

first: Shu

last: Xiao

first: Dan

last: Xie

first: Xiaoyi

last: Cao

first: Xiaoyun

last: Xing

first: Chieh-Chun

first: Meagan

last: Musselman

first: Mingchao

first: Franklin

middle: D

last: West

first: Harris

middle: A

last: Lewin

first: Ting

last: Wang

first: Sheng

last: Zhong


title: A molecular mechanism for compartmentalization and silencing of chromatin domains at the nuclear lamina

status: Public on Feb 24 2012

city: Chicago

first: Joseph

last: Zullo

state: IL

address: 929 E 57th Street

department: MGCB

institute: University of Chicago

type: Genome binding/occupancy profiling by genome tiling array


title: Impaired Insulin-/IGF1-Signaling Extends Life Span by Promoting Mitochondrial L-Proline Catabolism to Induce a Transient ROS-Signal

status: Public on Feb 25 2012

city: Jena

first: JenAge

last: Project

address: Beutenbergstr. 11

country: Germany

institute: Leibniz Institute for Age Research - Fritz Lipmann Institute

summary:
Transcriptome profiling of three models with impaired insulin/IGF1 signaling. 1. Deep sequencing of endogenous mRNA from Caenorhabditis elegans N2 var. Bristol (wildtype) and daf-2(e1370) mutant; 2. Deep sequencing  of endogenous mRNA from murine embryonic fibroblasts (MEF)  wildtype and irs1-/- knockout; 3. Deep sequencing of endogenous mRNA from murine embryoinic fibroblast (MEF) insr+/- -lox and insr+/- knockout

Jena Centre for Systems Biology of Ageing - JenAge (www.jenage.de)

sample_organism:
Caenorhabditis elegans
Mus musculus

platform_organism:
Caenorhabditis elegans
Mus musculus

overall_design: 14 samples examined: C. elegans N2 var. Bristol (wildtype) vs. daf-2(e1370) mutant; MEF wildtype vs. irs1-/- knockout; MEF insr+/- -lox vs. insr +/- knockout

first: Kim

last: Zarse

first: Michael

last: Ristow

first: Marco

last: Groth

first: Steffen

last: Priebe


title: HNRPA2B1 HITS-CLIP

first: Hani

last: Goodarzi

summary: Expression, crosslinking and immunoprecipitation of tagged HNRPA2B1 (variant A2) to identify bound RNA sequences

overall_design: RNA molecules that are immunoprecipitated are sequenced followed purification under denaturing conditions

first: Hamed

last: Najafabadi

first: Panos

last: Oikonomou

first: Todd

last: Greco

first: Lisa

last: Fish

first: Reza

last: Salavati

first: Ileana

last: Cristea

first: Saeed

last: Tavazoie


title: Mediator Complex Regulates Alternative mRNA Processing via the Med23 Subunit

city: Shanghai

first: yan

last: huang

address: Yueyang Road 320

country: China

institute: Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

summary: Mediator complex is an integrative hub for transcriptional regulation. Here we show that Mediator regulates alternative mRNA processing via its Med23 subunit. Combining tandem affinity purification and mass spectrometry, we identified a number of mRNA processing factors that bind to a soluble recombinant Mediator subunit MED23 but not to several other Mediator components. One of these factors, hnRNP L, specifically interacts with MED23 in vitro and in vivo. Consistently, Mediator partially colocalizes with hnRNP L and the splicing machinery in the cell. Functionally Med23 regulates a subset of hnRNP L-targeted alternative splicing (AS) and alternative cleavage and polyadenylation (APA) events as shown by minigene reporters and exon array analysis. ChIP-seq analysis revealed that Med23 can regulate hnRNP L occupancy at their co-regulated genes. Taken together, these results demonstrate a crosstalk between Mediator and the splicing machinery, suggesting a novel mechanism for coupling mRNA processing to transcription.

overall_design: Examination of hnRNP L and H3K36me3 enrichment in sictrl and si23 Hela cells

first: Yan

last: Huang

first: Xiao

last: Yao

first: Gang


title: Analysis of H3K79 methylation during reprogramming

status: Public on Feb 22 2012

city: Boston

first: Amit

last: Sinha

address: 44 Binney St

department: Pediatric Oncology

institute: Dana-Farber Cancer Institute

summary: Chromatin immunoprecipitation followed by Solexa sequencing for H3K27me3 and H3K79me2 in Fibroblasts, Embryonic stem cells, and fibroblast undergoing reprogramming

overall_design: Inhibition of the histone 3 lysine 79 (H3K79) methyltransferase increases repgoramming efficiency and genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. Dot1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state.

first: Tamer

last: Onder

first: Scott

last: Armstrong

first: George

last: Daley


title: Polycomb repressive complex 2-dependent and –independent functions of Jarid2 in transcriptional regulation in Drosophila

status: Public on Feb 23 2012

city: Kansas CIty

first: Alexander

middle: (Garrett)

last: Garruss

state: Missouri

address: 1000 East 50th Street

institute: Stowers Institute for Medical Research


title: Epigenetic profiling of WT and Ezh2-null MLL-AF9 murine leukemic cells

summary: Chromatin immunoprecipitation (ChIP) for H3K27me3 followed by Solexa sequencing in WT and Ezh2-null leukemic cells from primary and secondary recipients.

overall_design: Leukemic cells from primary and secondary recipients of MLL-AF9-transduced WT and Ezh2-null cells were sorted for GFP and YFP expression and analyzed by Chip-Seq.

first: Tobias

last: Neff


title: The Polycomb Repressive Complex 2 Is Required For MLL-AF9 Leukemia


title: Polycomb repressive complex 2-dependent and -independent functions of Jarid2 in transcriptional regulation in Drosophila [ChIP-Seq]

summary: Jarid2 was recently identified as an important component of the mammalian Polycomb Repressive Complex 2 (PRC2), where it has a major effect on PRC2 recruitment in mouse embryonic stem cells. Although Jarid2 is conserved in Drosophila, it has not previously been implicated in Polycomb (Pc) regulation. Therefore, we purified Drosophila Jarid2 and its associated proteins and find that Jarid2 associates with all of the known canonical PRC2 components, demonstrating a conserved physical interaction with PRC2 in flies and mammals. Furthermore, in vivo studies with Jarid2 mutants in flies demonstrate that among several histone modifications tested, only H3K27 methylation, the mark implemented by PRC2, was affected. Genome-wide profiling of Jarid2, Su(z)12 and H3K27me3 occupancy by ChIP-seq indicates that Jarid2 and Su(z)12 have a very similar distribution pattern on chromatin. However, Jarid2 and Su(z)12 occupancy levels at some genes are significantly different with Jarid2 being present at relatively low levels at many Pc response elements (PREs) of certain Homeobox (Hox) genes, providing a rationale for why Jarid2 was never identified in Pc screens. Gene expression analyses show that Jarid2 and E(z) (a canonical PRC2 component) are required not only for transcriptional repression but might also function in active transcription. Identification of Jarid2 as a conserved PRC2 interactor in flies provides an opportunity to begin to probe some of its novel functions in Drosophila development.

overall_design: Examination of Jarid2, Su(z)12, and H3K27me3 profiles in fly larvae and Su(z)12 in eye imaginal discs under wild-type and Jarid2 mutant conditions.

last: Garrett

first: Hans-Martin

last: Herz

first: Ali

last: Shilatifard


title: ChipSeq of MLL1 in mouse leukemic blast cells

summary: We performed a chip-seq to determine the binding of MLL1 in mouse leukemic blast cells transduces with MLL-AF9 fusion

overall_design: MLL1 chip-seq in mouse leukemic blast cells

last: Zhu


title: Renormalization of GSE32442 and GSE33128 plus 1 control sample

status: Public on Feb 21 2012

city: St Louis

first: Tiandao

last: Li

state: MO

address: 4444 Forest Park Ave

institute: Washington University

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

overall_design: Refer to individual Series

first: David

middle: H

last: Price


title: bedbug RNA-seq

city: Wooster

first: Asela

middle: J

last: Wijeratne

state: Ohio

address: 1680 Madison Av

department: Molecular and Cellular Imaging Center

institute: Ohio State University

summary: A comparison of mRNA profiles of insecticide resistant and susceptible genotypes.

overall_design: Compare two biological replicates from resistant and susceptible genotypes

first: Praveen

last: Mamidala

first: Saranga

first: Karl

last: Kornacker

first: Babu

last: Suddamalla

first: Loren

last: Vega

first: Andrew

last: Hoelmer

first: Tea

last: Meulia

first: Susan

last: Jones

first: Omprakash

last: Mittapallia


title: Intrinsic Coupling of Lagging Strand Synthesis to Chromatin Assembly

city: New York

first: Iestyn

last: Whitehouse

state: New York

address: 1275 York Avenue

department: Molecular Biology

institute: Sloan-Kettering Institute

summary: We show that ligation-competent Okazaki fragments in Saccharomyces cerevisiae are sized according to the chromatin repeat.  Using deep sequencing, we demonstrate that ligation junctions preferentially occur around nucleosome midpoints rather than in internucleosomal linker regions.  Disrupting chromatin assembly or lagging strand polymerase processivity impacts both the size and the distribution of Okazaki fragments, suggesting a role for nascent chromatin, assembled immediately after the passage of the replication fork, in the termination of lagging strand synthesis.  Our studies represent the first high-resolution analysis of eukaryotic Okazaki fragments in vivo, and establish a mechanistic link between the fundamental processes of DNA replication and chromatin assembly.

overall_design: 4 samples: replicate samples of wild-type and pol32 knockout

first: Duncan

last: Smith

first: Sean

last: McGuffee


title: Sequencing-based ssDNA detection

city: Bethesda

first: Pavel

last: Khil

address: 5 Memorial Dr.

institute: NIDDK

summary: Meiotic DNA double stranded breaks (DSBs) initiate genetic recombination in discrete areas of the genome called recombination hotspots. Although DSBs can be directly mapped using ChIP-Seq and antibody against ssDNA-associated proteins, genome-wide mapping of recombination hotspots in mammals is still a challenge due to the low frequency of recombination, high heterogeneity of the germ cell population and the relatively low efficiency of ChIP. To overcome these limitations we have developed a novel method, single-stranded DNA (ssDNA) sequencing (SSDS), that specifically detects protein-bound single-stranded DNA at DSB ends. SSDS consists of a computational framework for the specific detection of ssDNA-derived reads in a sequencing library and a new library preparation procedure for the enrichment of fragments originating from ssDNA.  When applied to mapping meiotic DSBs, the use of SSDS reduces the non-specific dsDNA background more than ten-fold. Our method can be extended to other systems where the identification of ssDNA or DSBs is desired.

overall_design: Development and validation of the method, SSDS, for the specific detection of ssDNA-derived and dsDNA-derived fragments in sequencing libraries and enrichment of ssDNA-derived fragments. SSDS was used to detect meiotic DSBs in 9R/13R mice.

first: Fatima

middle: O

last: Smagulova

first: Kevin

last: Brick

first: Ivan

middle: V

last: Gregoretti

first: Rafael

last: Camerini-Otero

first: Galina

last: Petukhova


title: Whole Cell Extract

first: Tiandao

last: Li

summary: Most human genes are loaded with promoter proximally paused RNA polymerase II (RNAP II) molecules that are poised for release into productive elongation by P-TEFb. Gdown1, a protein that renders RNAP II responsive to mediator, is involved in RNAP II elongation control. During in vitro transcription assays Gdown1 specifically blocked elongation stimulation by TFIIF, inhibited the termination activity of TTF2, and influenced pausing factors NELF and DSIF, but did not affect the function of TFIIS or the mRNA capping enzyme. Without P-TEFb, Gdown1 led to the production of stably paused polymerases in the presence of nuclear extract. ChIP-Seq data demonstrated that Gdown1 mapped over essentially all poised polymerases across the human genome. Gdown1 increases the stability of poised polymerases while maintaining their responsiveness to P-TEFb, and mediator overcomes a Gdown1-mediated block of initiation by allowing TFIIF function.

overall_design: 1 whole cell extract sample in human HeLa cell. See associated publication.

first: David

last: Price


title: Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins (RNA-Seq)

status: Public on Feb 20 2012

first: Gene

last: Yeo

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

institute: UCSD

summary: Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, cross-linking and immunoprecipitation coupled with high-throughput sequencing, and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins, and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and auto-regulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells.

overall_design: RNAseq for control, hnRNP A1, hnRNP A2/B1, hnRNP H1, hnRNP F, hnRNP M, and hnRNP U siRNA treated human 293T cells

first: Stephanie

last: Huelga

middle: W


title: Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins


title: Select microRNAs are essential for early development in the sea urchin.

status: Public on Feb 17 2012

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: microRNAs (miRNAs) are small noncoding RNAs that mediate post-transcriptional gene regulation and have emerged as essential regulators of many developmental events. The transcriptional network during early embryogenesis of the purple sea urchin, Strongylocentrotus purpuratus, is well described and would serve as an excellent model to test functional contributions of miRNAs in embryogenesis. We examined the loss of function phenotypes of the major components of the miRNA biogenesis pathway. Inhibition of de novo synthesis of Drosha and Dicer in the embryo led to consistent developmental defects, a failure to gastrulate, and embryonic lethality, including changes in the steady state levels of transcription factors and signaling molecules involved in germ layer specification. We annotated and profiled small RNA expression from the ovary and several early embryonic stages by deep sequencing followed by computational analysis. All miRNAs have dynamic accumulation profiles through early development as do a large population of putative piRNAs (piwi-interacting RNAs). Defects in morphogenesis caused by loss of Drosha can be rescued with four miRNAs which permits a strong miRNA functional assay. Taken together our results indicate that post-transcriptional gene regulation directed by miRNAs is functionally important for early embryogenesis and is an integral part of the early embryonic gene regulatory network in S. purpuratus.

overall_design: Small RNA expression profiling in early sea urchin development

first: Jia

middle: L

last: Song

first: Marlon

last: Stoeckius

first: Marc

last: Friedländer

first: Nadezda

last: Stepicheva

first: Celina

last: Juliano

first: Svetlana

last: Lebedeva

first: William

last: Thompson

first: Nikolaus

last: Rajewsky

first: Gary

last: Wessel


title: An East-Asian polymorphism underlies BCR-ABL mutation-independent resistance to tyrosine kinase inhibitors in chronic myelogenous leukemia

city: Cologne

first: Axel

last: HILLMER

address: Kerpener Str. 62

department: Institute of Pathology

institute: University of Cologne

type: Genome variation profiling by high throughput sequencing

summary: We used massively parallel DNA sequencing of paired-end ditags (DNA-PET) to identify structural genetic factors associated with disease progression and drug-resistance in representative samples from four CML patients and one CML cell line.  The functional consequences of our genetic findings were evaluated in primary CML cells and cell lines, and validated in a larger CML cohort. We discovered a novel intronic deletion that correlated with imatinib-resistance, and was subsequently confirmed to be a polymorphism in normal East-Asian, but not African or Caucasian, populations. We found that the polymorphism favored expression of  transcripts lacking a pro-apoptotic domain, which is critical for imatinib-induced cell death. A CML cell line containing the polymorphism also exhibited BCR-ABL-independent imatinib-resistance.

overall_design: Structural variations of 5 human CML samples were identified by long span paired-end sequencing

first: Yijun

last: Ruan

last: Hillmer


title: A genome-wide view of the expression and processing patterns of Thermus thermophilus HB8 CRISPR RNAs

city: Bonn

first: Stefan

middle: Andreas

last: Juranek

state: NRW

address: Venusber-Campus 1

department: Medizinische Klinik III

institute: UK Bonn

summary: The CRISPR-Cas system represents an RNA-based adaptive immune response system in prokaryotes. CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) consist of arrays of short repeat sequences interspaced by non-repetitive short spacers, some of which show sequence similarity to foreign phage genetic elements. Their cistronic transcripts are processed to produce the mature CRISPR RNAs (crRNAs), the elements that confer immunity by base-pairing with exogenous nucleic acids. We characterized the expression and processing patterns of Thermus thermophilus HB8 CRISPRs using differential deep-sequencing, which differentiates between 5’ monophosphate and 5’ non-monophosphate-containing RNAs, and/or between 3’ hydroxyl and 3’ non-hydroxyl-containing RNAs. The genome of T. thermophilus HB8 encodes 11 CRISPRs, classified into three distinct repeat sequence types, all of which were constitutively expressed without deliberately infecting the bacteria with phage. Analysis of the differential deep sequencing data suggested that crRNAs are generated by endonucleolytic cleavage, leaving fragments with 5’ hydroxyl and 3’ phosphate or 2’,3’-cyclic phosphate termini. The 5’ ends of all crRNAs are generated by site-specific cleavage eight nucleotides upstream of the spacer start position, however, the 3’ ends, are generated by two alternative, repeat-sequence-type-dependent mechanisms. These observations are consistent with the operation of multiple crRNA processing systems within a bacterial strain.

overall_design: 6 cDNA libraries, 3 untreated, 3 treated with phosphatase and T4 PNK in size ranges of 19-30 nt, 35-50 nt, and  50-100 nt.

first: Tali

last: Eban

first: Yael

last: Altuvia

first: Miguel

last: Brown

first: Pavel

last: Morozov

first: Thomas

last: Tuschl

first: Hanah

last: Margalit


title: Zebrafish Globin Locus

first: Anthony

middle: Dominc

last: DiBiase

address: KFRB, 1 Blackfan St, Suite 8216

department: Hem/Onc

institute: Children's Hospital Boston


title: Zebrafish Globin Locus (DNAse)

summary: Genome-wide analysis of  H3K4me3 modifications, Gata1 binding, and DNase I hypersensitivity sites in zebrafish adult red blood cells

overall_design: Zebrafish peripheral blood nuclei were isolated for DNase I hypersensitivity assays. Total DNA were purified using the Proteinase K digestion and Phenol-Chloroform extraction.  Small DNase I fragments from the DNaseI treatement were enriched using a sucrose gradient.  These ends were then labeled and amplified during library construction and for Solexa sequencing

first: Jared

last: Ganis

first: Nelson

last: Hsia

first: Eirini

last: Trompouki

first: Jill

last: de Jong

first: Zhiying

last: Jia

first: Peter

last: Sabo

first: Molly

last: Weaver

first: Richard

last: Sandstrom

first: John

last: Stamatoyannopoulos

first: Yi

last: Zhou

first: Leonard

last: Zon


title: Zebrafish Globin Locus (ChIP-seq)

overall_design: Zebrafish red cells from 10 adults were isolated for each ChIP-seq reaction. The red cells were cross-linked with formaldehyde for 20 min. DNA fragements bound by specific proteins were enriched by chromatin immunoprecipitation (ChIP) and analyzed by Solexa sequencing. A sample of whole cell extract (WCE) was sequenced and used as the background to determine enrichment. ChIP was performed using an antibody against total Gata1 and  H3K4me3 (Millipore Cat. No. 17-614) as previously described in Lee et al 2006.


title: Transcriptome of double-positive thymocyte precursors transduced with a retrovirus encoding Lin28a

first: Cuong

middle: Kieu

last: Nguyen

address: 9000 Rockville Pike

department: NIAID

institute: NIH

summary: We performed paired-end RNA-seq to compare the transcriptome of DP thymocytes that ectopically express Lin28 in vivo versus untransduced (GFP-ve) DP thymocytes.

overall_design: Transduced (GFP+) and untransduced (GFP-) CD4+ CD8+ CD3- thymocytes were sorted and pooled from three recipients of hematopoietic stem and progenitor cells transduced with Lin28-RV six weeks post-reconstitution.  Total RNA was extracted and paired-end library construction and sequencing was performed on oligo-dT purified RNA.

first: Joan

last: Yuan

first: Patrick

last: Burr

middle: K

first: Stefan

last: Muljo


title: Alternative Splicing Networks Regulated by Signaling in Human T Cells

city: Toronto

first: Qun

last: Pan

address: 160 College St.

institute: University of Toronto

summary: The formation and execution of a productive immune response requires, among many things, the maturation of competent T cells and a robust change in cellular activity upon antigen challenge. Such changes in cellular function require regulated alterations of protein expression. Much work has previously gone into defining the transcriptional changes that regulate protein expression during T cell development and antigen stimulation. Here we describe a parallel pathway of gene regulation that occurs during T cell stimulation, namely alternative splicing. Specifically, we use RNA-Seq to identify 178 exons in 168 genes that exhibit robust changes in inclusion in response to a stimulation of a human T cell line. Interestingly, these signal-responsive genes are enriched for functions related to immune response including, cell trafficking, inflammatory and immune response, immunologic disease and several cell signaling pathways. The vast majority of these genes also exhibit different isoform expression in naive and activated primary T cells. Comparison of the responsiveness of splicing to various stimuli in the cultured and primary T cells reveal important insight into the diversity of signaling pathways that control splicing. Using this data we are able to classify signal-responsive exons into at least three distinct networks. Importantly, we find that each regulatory network is characterized by distinct sequence hallmarks, further suggesting independent regulatory mechanisms.

overall_design: We utilize high-throughput RNA sequencing (RNA-Seq) to investigate global changes in alternative splicing in a cultured T cell line and in primary human T cells. We identify 178 genes that are predicted to exhibit robust signal-induced changes in isoform expression in cultured T cells.

first: Nicole

last: Martinez

first: Brian

middle: S

last: Cole

first: Christopher

last: Yarosh

first: Grace

last: Babcock

first: Florian

last: Heyd

first: William

last: Zhu

first: Sandya

last: Ajith

first: Benjamin

last: Blencowe

first: Kristen

last: Lynch


title: Identification of Transcription Factor FOS-1::GFP Binding Regions in L1

status: Public on Feb 16 2012

first: DCC

last: modENCODE

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

institute: Ontario Institute for Cancer Research

summary:
modENCODE_submission_3387
This submission comes from a modENCODE project of Michael Snyder. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: We are identifying the DNA binding sites for 300 transcription factors in C. elegans. Each transcription factor gene is tagged with the same GFP fusion protein, permitting validation of the gene's correct spatio-temporal expression pattern in transgenic animals.  Chromatin immunoprecipitation on each strain is peformed using an anti-GFP antibody, and any bound DNA is deep-sequenced using Solexa GA2 technology.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: OP304(official name : OP304 genotype : unc119(ed3);wgIs304(fos-1::TY1 EGFP FLAG;unc119) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline.  The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The FOS-1::EGFP fusion protein is expressed in the correct fos-1 spatio-temporal expression pattern.  This strain was used for ChIP-seq experiments to map the in vivo binding sites for the FOS-1 transcription factor. made_by : R Waterston ); Developmental Stage: fed L1; Genotype: unc119(ed3);wgIs304(fos-1::TY1 EGFP FLAG;unc119); Sex: Hermaphrodite;  EXPERIMENTAL FACTORS: Developmental Stage fed L1; Target gene fos-1; Strain OP304(official name : OP304 genotype : unc119(ed3);wgIs304(fos-1::TY1 EGFP FLAG;unc119) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline.  The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The FOS-1::EGFP fusion protein is expressed in the correct fos-1 spatio-temporal expression pattern.  This strain was used for ChIP-seq experiments to map the in vivo binding sites for the FOS-1 transcription factor. made_by : R Waterston ); temp (temperature) 20 degree celsius

first: Mei

last: Zhong

first: Mike

last: Snyder

first: Cindie

last: Slightam

first: Stuart

last: Kim

first: John

last: Murray

first: Robert

last: Waterston

first: Mark

last: Gerstein

first: Wei

last: Niu

first: Judi

last: Janette

first: Debasish

last: Raha

first: Ashish

last: Agarwal

first: Valerie

last: Reinke

first: Mihail

last: Sarov

first: Anthony

last: Hyman


title: Chromosomal translocations are guided by the spatial organization of the genome

city: Worcester

first: Rachel

middle: Patton

last: McCord

address: 364 Plantation St. LRB 570M

department: Program in Gene Function and Expression

institute: University of Massachusetts Medical School

summary: The extent to which the three-dimensional organization of the genome contributes to chromosomal translocations is an important question in cancer genomics.  We now have generated a high-resolution Hi-C spatial organization map of the G1-arrested mouse pro-B cell genome and mapped translocations from target DNA double-strand breaks (DSBs) within it via high-throughput genome-wide translocation sequencing.  RAG endonuclease-cleaved antigen-receptor loci are dominant translocation partners for target DSBs regardless of genomic position, reflecting high frequency DSBs at these loci and their co-localization in a fraction of cells.  To directly assess spatial proximity contributions, we normalized genomic DSBs via ionizing-radiation.  Under these conditions, translocations were highly enriched in cis along single chromosomes containing target DSBs and within other chromosomes and sub-chromosomal domains in a manner directly related to pre-existing spatial proximity.  Our studies reveal the power of combining two high-throughput genomic methods to address long-standing questions in cancer biology.

overall_design: Hi-C interaction maps for WT and ATM -/- G1-arrested AMuLV-transformed pro-B cell lines.

middle: P

first: Yu

last: Zhang

first: Bryan

middle: R

last: Lajoie

first: Yu-Jui

last: Ho

first: Frederick

last: Alt

first: Job

last: Dekker


title: The effects on transcriptome from absence of Spt4/Supt4h by RNA-seq

status: Public on Feb 15 2012

city: Stanford

first: Ning

last: Deng

address: 300 Pasteur Drive, RmL-314

department: Genetics Department

institute: Stanford University

summary: We have done RNAseq analyses in yeast and in rat and mouse striatal neurons.  The experiments in yeast examine globally the physiological effects of spt4 deletion on the transcriptome.  The experiments in neurons examine the global effects of supt4 knockdown to a cellular level (i.e., 50% knockdown) that we have shown  prevents toxicity of variant Htt protein by interfering with transcription of extended CAG repeats.  The results of both sets of experiments indicate that absent spt4 function has limited effects on overall transcription.  Moreover, the mammalian cell experiments indicate that supt4h knockdown to a cellular level that alters production of transcripts containing long CAG repeats has minimal effects on normal genes, supporting the notion that anti-supt4h measures may prove useful in combating trinucleotide repeat diseases.

sample_organism:
Saccharomyces cerevisiae
Mus musculus
Rattus norvegicus

overall_design: RNA-seq was done for two yeast WT strain W303 and two spt4 deletion strain spt4∆ as biological repeats, and rat ST14A cells and mouse HdhQ7/Q7 cells after transfection with NC control siRNA or Supt4h siRNA

first: Richard

last: Lin


title: Base-resolution analyses of parent-of-origin and sequence dependent allele specific DNA methylation in the mouse genome (ChIP-seq and Methyl-seq)

city: San Diego

last: Xie

address: 9500 Gilman Dr. CMM East, Room 2071

summary: Allele specific DNA methylation (ASM) is crucial for genomic imprinting and mammalian development. Here we present a base-resolution, genome-wide allelic DNA methylation map for both CG and non-CG sites in the mouse brain. We found parent-of-origin dependent (imprinted) ASM at 1,952 CGs which form 55 discrete clusters. This uncovers 31 reported differentially methylated regions (DMRs), including virtually all known germline DMRs, and 24 novel candidate DMRs with some occurring at microRNA genes. In the same adult tissue we also report a surprising presence of non-CG methylation with some showing evidence of imprinting. Finally, we identified sequence dependent ASM at 131,765 CGs. Interestingly, methylation at these sites exhibits a strong dependence on the immediate adjacent bases, allowing us to define a conserved sequence preference for the mammalian DNA methylation machinery. Our genome-wide ASM map should help with understanding the epigenetic differences between two parental genomes in mammals.

overall_design:
The crosses of the two mouse strains 129x1/SvJ (129) and Cast/EiJ (Cast) were performed at Jackson Laboratories (http://jaxmice.jax.org/) and the male mice F1 offspring and males of each of the two parental strains were shipped to investigator laboratories at 8 to 9 weeks of age.

MethylC-Seq
Five ug of genomic DNA was extracted from the frontal cortex of the F1 crosses or the parental strains, and was spiked in with 25 ng unmethylated cl857 Sam7 Lambda DNA (Promega, Madison, WI). The DNA was fragmented by sonication to 100-600 bp. Purified DNA fragments were end-repaired and ligated to paired-end cytosine- methylated adapters provided by Illumina. Size-selected adapter-ligated DNA was treated with sodium bisulfite using the EZ DNA methylation-Gold Kit (Zymo Research). The resulting DNA molecules were enriched by PCR, purified and sequenced following standard protocols from Illumina.

ChIP-Seq
The crosses of the two mouse strains 129x1/SvJ (129) and Cast/EiJ (Cast) were performed at Jackson Laboratories (http://jaxmice.jax.org/) and the male mice F1 offspring and males of each of the two parental strains were shipped to investigator laboratories at 8 to 9 weeks of age.  Frozen mouse frontal cortex from the F1 crosses was thawed on ice and processed with a razor blade into small pieces. Tissue was then crosslinked with formaldehyde, washed, homogenized, and proceeded for ChIP. To isolate chromatin, formaldehyde-cross-linked nuclei were sonicated using a Branson 450 Sonifier (Branson, Danbury, CT). ChIP DNA was subjected to end repair, A-tailing, adaptor ligation, and gel purification. Following PCR amplification for 18 cycles, the final libraries were subjected to sequencing.

first: Cathy

last: Barr

first: Audrey

last: Kim

first: Feng

last: Yue

first: AhYoung

last: Lee

first: James

last: Eubanks

first: Emma

last: Dempster

first: Bing

last: Ren


title: Base-resolution analyses of parent-of-origin and sequence dependent allele specific DNA methylation in the mouse genome (MRE-seq)

first: Wei

overall_design: The crosses of the two mouse strains 129x1/SvJ (129) and Cast/EiJ (Cast) were performed at Jackson Laboratories (http://jaxmice.jax.org/) and the male mice F1 offspring and males of each of the two parental strains were shipped to investigator laboratories at 8 to 9 weeks of age.  A total of 500ng genomic DNA isolated from IMR90, MEF, and the frontal cortex of F1i and F1r was digested in parallel by the DNA methylation dependent restriction enzyme FspEI. FspEI recognizes the CmC site (the second cytosine is methylated and can be in the context of CG, CHG or CHH) and creates a 5  protruding end 17 bases downstream of the methylcytosine. A similar experiment was performed by incubating the F1i genomic DNA with a DNA methylation independent restriction enzyme BstNI. The digested DNA was gel purified, size selected for fragments within 100-600bp. The resulting DNA was then prepared as genomic DNA libraries for high-throughput sequencing (Illumina).

first: Ah

middle: Y


title: Base-resolution analyses of parent-of-origin and sequence dependent allele specific DNA methylation in the mouse genome (RNA-seq)

overall_design: The crosses of the two mouse strains 129x1/SvJ (129) and Cast/EiJ (Cast) were performed at Jackson Laboratories (http://jaxmice.jax.org/) and the male mice F1 offspring and males of each of the two parental strains were shipped to investigator laboratories at 8 to 9 weeks of age.  The frontal cortex from the F1 crosses of the two mouse lines was dissected and the RNA was isolated followed by two DNAseI treatments to remove DNA contaminant. The presence of DNA in the RNA was tested using quantitative PCR and probes designed to span exons.  The quality of the RNA was determined by the Agilent 2100 Bioanalyzer prior to the construction of the libraries.  RNA was treated with RiboMinus (Invitrogen, Carlsbad CA) to remove the ribosomal RNA.  The methods for the preparation of the libraries are outlined in the Whole Transcriptome library preparation for SOLiD sequencing protocol (https://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_065852.pdf). Briefly, RNA depleted of ribosomal RNA was fragmented using RNAseIII. Purified RNA was hybridized and ligated to primers then converted to cDNA using reverse transcriptase.  The cDNA was size selected to contain 50 to 150 bp inserts then purified and amplified prior to sequencing. Libraries were constructed with RNA from three independent mice (3 biological replicates) for each of the experimental crosses. Sequencing was performed at EdgeBio (http://www.edgebio.com/) and the data from the 3 mice for each experimental cross line were combined given the high correlation between each of the libraries (R >0.98).

first: A


title: Base-resolution analyses of sequence and parent-of-origin dependent DNA methylation

overall_design: Refer to individual Series


title: Exploring the DNA-Recognition Potential of Homeodomains

first: Scot

last: Wolfe

address: 364 Plantation Street, LRB 619

department: MCCB

institute: UMass Medical School

summary: We utilized a bacterial selection system to isolate Engrailed variants from a randomized library that are compatible with each of the 64 possible 3’ triplet sites (i.e. TAANNN).  The DNA-binding specificity of 151 representative HD variants from these populations was subsequently characterized.   Many of these variants contain novel combinations of specificity determinants that are uncommon or absent in extant HDs.  These novel determinants, when grafted into different HD backbones, produce a corresponding alteration in their specificity.  The identified determinates expand our understanding of HD recognition.

overall_design: Homeodomains where selected against each of the 64 possible 3' triplet sites (ie: TAANNN).  The DNA-binding specificity of 151 representative HD variants from these populations was subsequently characterized.  7 novel determinants, when grafted into 3 different HD backbones.  5' speicifity was changed for 3 novel determinants.

first: Stephanie

last: Chu

first: Marcus

middle: B

last: Noyes

first: Ryan

middle: G

last: Christensen

first: Brian

last: Pierce

first: Zhiping

last: Weng

first: Gary

middle: D

last: Stormo

middle: A


title: Genome-wide analysis of STAT3-binding and gene expression changes in peritoneal macrophages upon interleukin-10 stimulation

status: Public on Feb 13 2012

city: Shenzhen

first: Andrew

last: Hutchins

address: 1088 Xueyuan Rd

department: Bioinformatics

institute: Southern University of Science and Technology

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

first: Diego

last: Miranda-Saavedra


title: Nuclear Matrix Factor hnRNP U/SAF-A Exerts a Global Control of Alternative Splicing by Regulating U2 snRNP Maturation

city: La Jolla

first: Xiang-Dong

last: Fu

address: 9500 Gilman Drive, Room 231

department: CMM

institute: UC San Diego

summary: The nuclear matrix associated hnRNP U/SAF-A protein has been implicated in diverse  pathways from transcriptional regulation to telomere length control to X inactivation, but the precise  mechanism underlying each of these processes has remained elusive. Here, we report hnRNP U as a  regulator of SMN2 splicing from a custom RNAi screen. Genome-wide analysis by CLIP-seq reveals that  hnRNP U binds virtually to all classes of regulatory non-coding RNAs, including all snRNAs required for  splicing of both major and minor classes of introns, leading to the discovery that hnRNP U regulates U2  snRNP maturation and Cajal body morphology in the nucleus. Global analysis of hnRNP U-dependent  splicing by RNA-seq coupled with bioinformatic analysis of associated splicing signals suggests a  general rule for splice site selection through modulating the core splicing machinery. These findings  exemplify hnRNP U/SAF-A as a potent regulator of nuclear ribonucleoprotein particles in diverse gene  expression pathways.

overall_design: Examination of hnRNP U regulated splicing in Hela cells with CLIP-seq (two biological replicates) and paired-end RNA-seq (control and hnRNP U knockdown)

first: Rui

last: Xiao

first: Peng

last: Tang

first: Bo

last: Yang

first: Jie

last: Huang

first: Yu

last: Zhou

first: Changwei

last: Shao

first: Hairi

last: Li

first: Hui

last: Sun

first: Yi

last: Zhang


title: Non-coding small RNA profiling by high throughput sequencing of bovine primary retinal microvascular endothelial cells

city: Belfast

first: David

middle: Arthur

last: Simpson

state: N. Ireland

address: QUB Institue of clinical Science, Block A, RVH

department: Centre for Experimental Medicine

country: United Kingdom

institute: Queen's University Belfast

summary: The aim of this study was to identify and quantify microRNAs and other small regulatory RNAs expressed in primary retinal microvascular endothelial cells (RMECs) using deep sequencing. RMECs were isolated, RNA extracted, a small RNA library prepared and deep sequencing performed. A total of 6.8 million reads were mapped to 250 known microRNAs in miRBase (release 16). Several novel microRNAs and multiple new members of the miR-2284/2285 family were detected. Several ~30 nucleotide sno-miRNAs were identified, with the most highly expressed being derived from snoRNA U78. Highly expressed microRNAs previously associated with endothelial cells included miR-126 and miR-378, but the most highly expressed was miR-21, comprising more than one third of all mapped reads. The independence from prior sequence knowledge provided by deep sequencing facilitates analysis of novel microRNAs and other small RNAs. This approach also enables quantitative evaluation of microRNA expression, which has highlighted the predominance of a small number of microRNAs in RMECs. Further characterisation of the functions of the highly expressed microRNAs will provide insights into endothelial biology.

overall_design: Single sample of primary cell culture


title: Genome-wide binding of STAT3 in peritoneal macrophages

summary: To try to identify the mechanism of STAT3âs indirect action we have used a genomic approach to map the binding sites of STAT3 within the genome and also used RNA-seq technology to map the changes in RNA expression and transcript isoforms in response to IL-10.

overall_design: Examination of STAT3 binding by ChIP-seq in Unstimulated and IL-10 treated peritoneal exudate macrophages purified from mice. We sequenced anti-STAT3 ChIP-seq as well as corresponding control (Input) libraries


title: Comparison of transcript abundance in mycelium from the rice blast fungus Magnaporthe oryzae grown in rich medium and in minimal medium using RNA-Seq

city: Exeter

first: Darren

middle: Mark

last: Soanes

address: RILD Building, RD&E Hospital Wonford, Barrack Road

department: College of Medicine and Health

institute: University of Exeter

summary: Mycelium from the rice blast fungus Magnaporthe oryzae was grown in both rich medium and under nutrient limiting conditions. Genes were identified that were more highly expressed in one condition as compared to the other.

overall_design: Samples were taken from mycelium grown in both complete medium and in glucose minimal medium. One replicate for each sample.

middle: M

first: Apratim

last: Chakrabarti

first: Konrad

middle: H

last: Paszkiewicz

first: Angus

last: Dawe

first: Nicholas

middle: J

last: Talbot


title: Expression data from CD4 single positive thymocyte subsets from C57BL/6 mice of 6-8 wks of age

city: Beijing

first: Qing

last: Ge

address: 38 Xueyuan Road

department: Immunology

institute: Peking University Health Science Center

summary:
After positive selection in the thymus, the newly generated single positive (SP) thymocytes are phenotypically and functionally immature and undergo apoptosis upon antigen stimulation.  In the thymic medullary microenvironment, SP cells progressively acquire immunocompetence. Negative selection to remove autoreactive T cells also occur at this stage.  We have defined four subsets of CD4 SP, namely, SP1, SP2, SP3, and SP4 that follow a functional maturation program and a sequential emergence during mouse ontogeny.
We used microarray to detail the global programm of gene expression during the maturation of murine CD4 single positive thymocytes

overall_design: Four subsets of CD4+CD8-CD25-NK1.1- thymocytes from C57BL/6 mice of 6-8 wks of age were purified for RNA extraction and hybridization on Affymetrix microarrays, namely, SP1 (Qa-2-6C10+CD69+), SP2 (Qa-2-6C10-CD69+), SP3 (Qa-2-6C10-CD69-), SP4 (Qa-2+6C10-CD69-).

first: Fei

last: Teng

first: Yubin

first: Wei-Feng

last: Chen


title: Comparison of transcript abundance in mycelium from the rice blast fungus Magnaporthe oryzae grown in rich medium and in minimal medium using HT-SuperSAGE.

overall_design: Samples were taken from mycelium grown in both complete medium and in  glucose minimal medium. Three replicates were taken for each condition.

middle: L


title: Transcriptome changes in IL-10 treated peritoneal macrophages

first: Andrew

last: Hutchins

summary: To try to identify the mechanism of STAT3s indirect action we have used a genomic approach to map the binding sites of STAT3 within the genome and also used RNA-seq technology to map the changes in RNA expression and transcript isoform abundance in response to IL-10.

overall_design: Examination of transcriptome changes in peritoneal macrophages when treated with IL-10 for 4 hours. RNA was extracted and sequenced.

first: Diego

last: Miranda-Saavedra


title: Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types

status: Public on Feb 10 2012

first: Julia

last: Salzman

address: 279 West Campus Drive, MC: 5307

summary: Most human pre-mRNAs are spliced into linear molecules that retain the exon order defined by the genomic sequence.  By deep sequencing of RNA from a variety of normal and malignant human cells, we found RNA transcripts from many human genes in which the exons were arranged in a non-canonical order.  Statistical estimates and biochemical assays provided strong evidence that a substantial fraction of the spliced transcripts from hundreds of genes are circular RNAs.  Our results suggest that a non-canonical mode of RNA splicing, resulting in a circular RNA isoform, is a widespread and perhaps general feature of the gene expression program in human cells.

overall_design: 3 samples of non-malignant primary human leukocytes, one replicate each


title: Genome-wide profiling of gene expression of normal and Tbx20 knockout adult mouse whole heart

city: Chicago

first: Noboru

middle: Jo

last: Sakabe

address: 920 E 58th st

department: Human Genetics

institute: University of Chicago

summary: Tbx20 is a transcription factor important for heart development. To assess the role of Tbx20 in the adult heart, we generated a conditional knockout for this gene, specifically in cardiomyocytes. We profiled gene expression levels using RNA-seq in both normal and knockout adult mouse hearts to identify genes and pathways regulated by Tbx20. The article describing the Tbx20 knockout mouse is under review, a reference will be added when published.

overall_design: Analysis of triplicate mRNA samples of adult mouse, comparing normal and knockout

first: Ivy

last: Aneas

first: Marcelo

last: Nobrega

first: Tao

last: Shen

first: Sylvia

last: Evans


title: Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins  (CLIP-Seq)

first: Gene

last: Yeo

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

institute: UCSD

summary: Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, cross-linking and immunoprecipitation coupled with high-throughput sequencing, and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins, and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and auto-regulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells.

overall_design: CLIPseq for hnRNP A1, hnRNP A2/B1, hnRNP F, hnRNP M, and hnRNP U in human 293T cells

first: Stephanie

middle: C

last: Huelga

middle: W


title: Analysis of transcript abundance during appressorium formation in the rice blast fungus Magnaporthe oryzae using HT-SuperSAGE.

first: Darren

last: Soanes

summary: The rice-blast fungus Magnaporthe oryzae is disseminated using a three-celled spore or conidium. Upon landing on the surface of a rice leaf, the conidium germinates and forms a specialised structure for entry into the host plant - the appressorium. In this study we have followed gene expression thoughout germination of the conidium and formation of the appressorium to identify genes that may be important for appressorium function. We have also compared gene expression in the wild-type germinating conidium at four hours to a mutant strain deleted for the MAP kinase pmk1. The pmk1 mutant is unable to form appressoria.

overall_design: Samples were taken from five time-points in wild-type germinating conidia and one time-point in the pmk1 mutant. Two replicates were taken at each time-point.

first: Apratim

last: Chakrabarti

first: Konrad


title: Transcription-associated Loading and Translocation of Condensin II on Chromosome Arms in Embryonic Stem Cells (flavopiridol ChIP-Seq)

status: Public on Feb 09 2012

city: Cambridge

first: Richard

last: Young

address: 9 Cambridge Center

institute: Whitehead Institute for Biomedical Research

summary: Condensin molecules are loaded onto the genome to mediate essential changes in chromosome condensation during mitosis, but it is not clear why there are two forms of vertebrate condensin that become differentially distributed on chromosomes.  We report here that condensin II, the form of condensin present in the nucleus throughout the cell cycle, functions specifically at active genes.  Condensin II is loaded at transcriptionally active promoters in embryonic stem cells (ESCs), migrates through these genes in a transcription-dependent fashion and accumulates in transcription termination regions.  Unlike cohesin, which is also loaded at active promoters, condensin II has little influence on transcription.  We conclude that condensin II is loaded and distributed across actively transcribed chromatin and thus serves to specifically condense this euchromatic portion of chromosomes during the cell division cycle.

overall_design: ChIP-Seq data for Condensin II and Cohesin in v6.5 ESCs treated or not with the RNA polymerase II elongation inhibitor flavopiridol, as well as data for the histone modifications H3K4me3 and H3K27me3.

first: Jill

last: Dowen

first: Steve

last: Bilodeau


title: MacroH2A1 regulates the balance between self-renewal and differentiation commitment in embryonic and adult stem cells

city: Badalona

first: Roberto

last: Malinverni

state: Barcelona

address: Ctra. de Can Ruti, camí de les escoles, s/n

country: Spain

institute: IMPPC Institut de Medicina Predictiva i Personalitzada del Càncer

summary: One of the most striking epigenetic alterations that occurs at the level of the nucleosome is the complete exchange of the canonical H2A histones for the macroH2A variant. Here, we provide insight in the function of this unique histone variant in embryonic and adult stem cells. Knockdown of macroH2A1 in mouse embryonic stem (mES) cells limited their capacity to differentiate but not their self-renewal. The loss of macroH2A1 interfered with the proper activation of differentiation genes, most of which are direct target genes of macroH2A. Additionally, macroH2A1-deficient mES cells displayed incomplete inactivation of pluripotency genes and formed defective embryoid bodies. In vivo, macroH2A1-deficient teratomas contained a massive expansion of malignant, undifferentiated carcinoma tissue. In the heterogeneous culture of primary human keratinocytes, macroH2A1 levels negatively correlated with the self-renewal capacity of the pluripotent compartment. Together these results establish macroH2A1 as a critical chromatin component that regulates the delicate balance between self-renewal and differentiation of embryonic and adult stem cells.

overall_design: Examination of the histone variant macroH2A1 in mouse Embryonic stem cells

first: Catherine

last: Creppe

first: Peggy

last: Janich

first: Neus

last: Cantariño

first: Marc

last: Noguera

first: Vanesa

last: Valero

first: Eva

last: Musulén

first: Julien

last: Douet

first: Melanija

last: Posavec

first: Juan

last: Caballero

first: Lauro

last: Sumoy

first: Luciano

last: Di Croce

first: Salvador

last: Benitah

first: Marcus

last: Buschbeck


title: Direct Recruitment of Polycomb Repressive Complex 1 (PRC1) to Chromatin by Core Binding Transcription Factors

first: Tali

last: Mazor

address: 77 Massachusetts Ave

institute: MIT

first: Ming

last: Yu

first: Hui

last: Huang

first: Hsuan-Ting

first: Katie

last: Kathrein

first: Andrew

last: Woo

first: Candace

last: Chouinard

first: Adam

last: Labadorf

first: Thomas

middle: E

last: Akie

first: Tyler

last: Moran

first: Huafeng

last: Xie

first: Sima

last: Zacharek

first: Ichiro

last: Taniuchi

first: Robert

last: Roeder

first: Carla

middle: F

last: Kim

first: Leonard

middle: I

last: Zon

first: Ernest

last: Fraenkel

first: Alan

last: Cantor


title: ChIP-Seq of TAL1 and its regulatory partners in T-ALL cells

first: Richard

last: Young

summary: The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of T-cell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1, LMO2, GATA3 and RUNX1 in T-ALL cells. We show that TAL1 forms an interconnected auto-regulatory loop with its partners, and that the TAL1 complex directly activates the MYB oncogene, forming a feed-forward positive regulatory loop that further promotes the TAL1-regulated oncogenic program. one of the cirtical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and HEB/E2A, and is essential for the survival of T-ALL cells.

overall_design: Human T-ALL cells were cross-linked with formaldehyde for 20 min. DNA was enriched by chromatin immunoprecipitation (ChIP) and analyzed by Solexa sequencing. A sample of whole cell extract (WCE) was sequenced and used as the background to determine enrichment. ChIP was performed using an antibody against total TAL1 (Santa Cruz SC-12984), TCF12/HEB (Santa Cruz SC-357),TCF3/E2A (Santa Cruz SC-349X), LMO1 (Santa Cruz SC-10494), LMO2 (R&D AF2726),GATA3 (Santa Cruz SC-22206) and RUNX1 (Santa Cruz SC-8563). This represents the ChIP-seq portion of this dataset.

first: Lee

middle: N

last: Lawton

first: Zi-Peng

last: Fan


title: HIGHLY DIVERSE TCRalpha CHAIN REPERTOIRE OF PRE-IMMUNE CD8+ T CELLS REVEALS NEW INSIGHTS IN GENE RECOMBINATION

city: Lausanne

first: Brian

middle: James

last: Stevenson

address: Genopode, UNIL-Sorge

institute: Swiss Institute of Bioinformatics

summary: By sequencing tens of millions of TCRα chain transcripts from naïve mouse CD8+ T cells, we observed a hugely diverse repertoire, comprising nearly all possible TRAV-TRAJ combinations. Our findings are not compatible with sequential coordinate gene recombination, but rather with a model in which contraction and DNA looping in the TCRαδ locus provide equal access to TRAV and TRAJ gene segments, similar to that demonstrated for IgH gene recombination

overall_design: High-throughput sequencing of entire TCRa repertoire from C57Bl/6 mice

first: Raphael

last: Genolet

first: Brain

first: Laurent

last: Farinelli

first: Magne

last: Osteras

first: Immanuel

last: Luescher


title: Estrogen-independent genomic ER binding analysis

city: Nashville

first: Todd

last: Miller

state: TN

address: 2220 Pierce Ave, 777 PRB

department: Medicine

institute: Vanderbilt Univ

summary: Hormone-independent breast cancer cells (MCF-7/LTED and HCC-1428/LTED) were analyzed by ChIP-seq for estrogen receptor a to identify ER-DNA binding events that occur in the absence of estrogens.

overall_design: MCF-7/LTED and HCC-1428/LTED cells were maintained in IMEM + 10% DCC-FBS (dextran-charcoal-treated serum). ChIP for ERa was performed, following by high-throughput DNA sequencing.

first: Carlos

last: Arteaga


title: Short RNAs expression profiling in SET2 cells.

status: Public on Feb 08 2012

city: Padova

first: Stefania

last: Bortoluzzi

address: via G. Colombo 3

department: Department of Molecular Medicine

country: Italy

institute: University of Padova

summary: We report the application of Illumina short RNA sequencing for characterization and discovery of miRNAs and moRNAs in SET2 cells carrying JAK2 V617F mutation.

overall_design: Short RNAs sequencing for miRNA quantification, discovery and characterisation.

first: Alessandro

last: Vannucchi


title: MicroRNAs induced during response to stress in C. elegans

status: Public on Feb 04 2012

city: New Haven

first: Frank

last: Slack

state: CT

address: 266 Whitney Ave

department: MCDB

institute: Yale University

summary: To understand how microRNAs are involved in stress response, we examined their expression changes in C. elegans animals that were exposed to stress conditions, including heat shock, oxidation, hypoxia and starvation.

overall_design: Total RNAs were purified from young adult animals that were exposed to each stress, and used for cDNA library preparation for small RNAs. In this experiment, spe-9(hc88), a temperature sensitive sterile mutant, that were cultured at 23dC, was used in order to avoid the effect from developing embryos. Stress conditions we examined include: Heat shock (32°C, 6 hrs), Recovery from heat shock (6 hrs recovery at 23°C after heat shock treatment at 32°C for 6 hrs), Hypoxia (0.01%, 6 hrs), Oxidation (Juglone 750 μM, 6 hrs), Starvation (complete food deprivation, 12 hrs). In addition to these stress conditions, RNAs were prepared from normally cultured, untreated animals at three time points, 0 hr (baseline), 6 hrs (as controls for heat shock, hypoxia and oxidation) and 12 hrs (as controls for heat shock recovery and starvation) after starting stress exposure. These cDNA libraries established were sequenced with Illumina Genome Analyzer II.

first: Masaomi

last: Kato


title: Protein/DNA in vitro Binding seq (PB-seq) for Drosophila HSF and genomic Drosophila DNA

status: Public on Feb 06 2012

city: Farmington

first: Michael

middle: Joseph

last: Guertin

address: 400 Farmington Ave

department: Center for Cell Analysis and Modeling

institute: University of Connecticut

summary: DNA sequence and local chromatin landscape act jointly to determine transcription factor (TF) binding intensity profiles. To disentangle these influences, we developed an experimental approach, called protein/DNA binding and high-throughput sequencing (PB-seq), that allows the binding energy landscape to be characterized genome-wide in the absence of chromatin.  We applied our methods to the Drosophila Heat Shock Factor (HSF), which inducibly binds a target DNA sequence element (HSE) following heat shock stress. PB-seq involves incubating sheared naked genomic DNA with recombinant HSF, partitioning the HSF-bound and HSF-free DNA, and then detecting HSF-bound DNA by high throughput sequencing. We compared PB-seq binding profiles with ones observed in vivo by ChIP-seq, and developed statistical models to predict the observed departures from idealized binding patterns based on covariates describing the local chromatin environment. We found that DNase I hypersensitivity and tetra-acetylation of H4 were the most influential covariates in predicting changes in HSF binding affinity.  We also investigated the extent to which DNA accessibility, as measured by digital DNase I footprinting data, could be predicted from MNase-seq data and the ChIP-chip profiles for many histone modifications and TFs, and found GAGA element associated factor (GAF), tetra-acetylation of H4, and H4K16 acetylation to be the most predictive covariates. Lastly, we generated an unbiased model of HSF binding sequences, which revealed distinct biophysical properties of the HSF/HSE interaction and a previously unrecognized substructure within the HSE.  These findings provide new insights into the interplay between the genomic sequence and the chromatin landscape in determining transcription factor binding intensity.

overall_design: We performed an in vitro binding experiment with purified HSF and naked, sheared genomic Drosophila S2 DNA (PB-seq), to derive an accurate set of potential HSF binding sites in the Drosophila genome.  HSF-bound DNA was specifically eluted and detected by high throughput sequencing.  Drosophila HSF was N-terminally tagged with glutathione s-transferase and a tobacco etch virus (TEV) protease cleavage site.  The C-terminus of the recombinant HSF was fused to the 3xFLAG epitope.  Recombinant HSF was purified from E. coli with glutathione resin as previously described (PMID: 20078429) , with the following modifications:  HSF-3xFLAG elution was achieved by addition of 6xHistidine tagged TEV protease and TEV protease was cleared from the HSF preparation using a Nickel-NTA column.  We incubated 600pM HSF and 2500ng genomic DNA (sonicated to 100-600bp fragment size as previously described in PMID: 20844575) in 1500μl final volume of 1xHSF binding buffer and let it come to equilibrium for an hour at room temperature.  We added 20μl ANTI-FLAG M2 affinity gel for 10 minutes, washed 8 times with 1xHSF binding buffer to remove unbound DNA; 3xFLAG peptide was added to a final concentration of 200ng/μl to specifically elute HSF and HSF-bound DNA.  The mock IP was done in the absence of recombinant HSF.

first: M

first: A

last: Martin

last: Siepel

first: J

middle: T

last: Lis


title: Trans Chromosomal Methylation in Arabidopsis hybrids

status: Public on Feb 05 2012

city: Canberra

first: Jennifer

last: Taylor

address: Clunies Ross St

department: Plant Industry

country: Australia

institute: CSIRO

summary: The heterotic hybrid offspring of Arabidopsis accessions C24 and Landsberg erecta have altered methylomes.  Changes occur most frequently at loci where parental methylation levels are different.  There are context-specific biases in the non-additive methylation patterns with mCG generally increased and mCHH decreased relative to the parents.  These changes are a result of two main mechanisms, Trans Chromosomal Methylation (TCM) and Trans Chromosomal deMethylation (TCdM), where the methylation level of one parental allele alters to resemble that of the other parent.  Regions of altered methylation are enriched around genic regions and are often correlated with changes in siRNA levels.  We identified examples of genes with altered expression likely to be due to methylation changes and suggest that in crosses between the C24 and Ler accessions, epigenetic controls can be important in the generation of altered transcription levels which may contribute to the increased biomass of the hybrids.

overall_design: C24, Ler, and C24 x Ler

first: Ian

middle: K

last: Greaves


title: Analysis of 22G siRNA triggered siRNA amplification and 3' 2'-O-methylated small RNAs in Caenorhabditis elegans

status: Public on Feb 03 2012

city: Boston

first: Taiowa

last: Montgomery

address: 185 Cambridge Street, CPZN-7250

department: Molecular Biology

institute: Massachusetts General Hospital

summary:
Small RNAs, including piRNAs, miRNAs and endogenous siRNAs, bind Argonaute proteins to form RNA-silencing complexes that target coding genes, transposons and aberrant RNAs. To assess the requirements for endogenous siRNA formation and activity in C. elegans, we developed a GFP-based sensor for the endogenous siRNA 22G siR-1, one of a set of abundant siRNAs processed from a precursor RNA mapping to the X chromosome, the X-cluster.  Silencing of the sensor is also dependent on the partially complementary, unlinked 26G siR-O7 siRNA. We show that 26G siR-O7 acts in trans to initiate 22G siRNA formation from the X-cluster. The presence of several mispairs between 26G siR-O7 and the X-cluster mRNA, as well as mutagenesis of the siRNA sensor, indicates that siRNA target recognition is permissive to a degree of mispairing. From a candidate reverse genetic screen, we identified several factors required for 22G siR-1 activity, including the Argonaute ergo-1 and the 3' methyltransferase henn-1. Quantitative RT-PCR of small RNAs in a henn-1 mutant and deep sequencing of methylated small RNAs indicate that siRNAs and piRNAs that associate with PIWI clade Argonautes are methylated by HENN-1, while siRNAs and miRNAs that associate with non-PIWI clade Argonautes are not. Thus, PIWI-class Argonaute proteins are specifically adapted to associate with methylated small RNAs in C. elegans.

This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Genome-wide maps of Egr2 transcription factor binding sites in NKT and anti TCRb injected thymocytes.

middle: Paul

last: Seiler

address: 929 E 57th Street

department: Committee on Immunology

summary: We report the application of single-molecule-based sequencing technology for mapping the Egr2 transcriptional program in developing thymic NKT. We found that Egr2 controls the induction of genes required for NKT development.

overall_design: Examination of developing NKT cells and thymocytes receiving a strong TCR signal in vivo by injecting 500ug anti-TCRb antibody.

middle: P

first: R

last: Mathew

last: Liszewski

first: C

last: Sponer

first: K

last: Barr

first: F

last: Meng

first: H

last: Singh

last: Bendelac


title: Genome-wide mapping of HIF-2 binding in 786-O clear cell renal carcinoma cells

status: Public on Feb 01 2012

city: Oxford

first: David

middle: Robert

last: Mole

address: Roosevelt Drive, Headington

department: Nuffield Department of Medicine

institute: University of Oxford

summary: Kidney cancer accounts for more than 100,000 deaths per year world-wide. More than 80% are clear cell tumors (ccRCCs) and the majority are associated with loss of function of the von Hippel Lindau (pVHL) tumor suppressor resulting in upregulation of HIF-{alpha} subunits, and activation of HIF-dependent transcriptional pathways. Recent GWAS studies have discovered RCC-susceptibility loci both within EPAS1 (HIF-2{alpha}) and in an intergenic region of unknown function on 11q13.3. As part of an ongoing study to define the direct transcriptional targets of HIF-2 in renal cancer, we undertook a genome-wide analysis of HIF-2-binding sites in pVHL-defective 786-O cells (that lack functional HIF-1{alpha} due to a truncated transcript) using chromatin immunoprecipitation with antibodies directed against HIF-2{alpha} and its dimerization partner HIF-1{beta}, coupled to high-throughput sequencing (ChIP-seq). Amongst approximately 600 pangenomic HIF-2{beta} ChIP signals, we observed strong binding (ranked 12th by peak height) almost precisely coinciding with the RCC predisposition SNP rs7105934 on 11q13.3.

overall_design:
We report binding of HIF-2{alpha} and HIF-1{beta} in 786-O clear cell renal carcinoma cells.
3 samples examined, HIF-2{alpha}, HIF-1{beta} and pre-immune control ChIP.

middle: R

first: Johannes

last: Schoedel


title: Adenosine deaminases that act on RNA induce reproducible changes in abundance and sequence of embryonic miRNAs

status: Public on Feb 02 2012

city: Vienna

first: Stefanie

last: Tauber

address: Dr. Bohr-Gasse 9

department: CIBIV

country: Austria

institute: MFPL

summary: We used transgenic mouse embryos that are deficient in the two enzymatically active RNA editing enzymes ADAR1 and ADAR2 to compare relative frequencies but also sequence composition of mature miRNAs in these genetically modified backgrounds to wild-type mice by Illumina next gen sequencing. Deficiency of ADAR2 leads to a reproducible change in abundance of specific miRNAs and their predicted targets. Changes in miRNA abundance seem unrelated to editing events. Additional deletion of ADAR1 has surprisingly little impact on the mature miRNA repertoire, indicating that miRNA expression is primarily dependent on ADAR2. A to G transitions reflecting A to I editing events can be detected at few sites and at low frequency during the early embryonic stage investigated. Again, most editing events are ADAR2 dependent with only few editing sites being specifically edited by ADAR1. Besides known editing events in miRNAs a few novel, previously unknown editing events were identified. Some editing events are located to the seed region of miRNAs opening the possibility that editing leads to their retargeting.

overall_design:
GSM852140-8: sequencing of mature miRNAs of wt, ADAR2-/- and ADAR1-/-/ADAR2-/- female mouse embryos at E11.5
GSM863778-81: Gene expression was measured in wiltype, ADAR2-/- and ADAR1-/-/ADAR2-/- E11.5 whole female mouse embryos using Agilent Whole Mouse Genome Oligo Microarrays 8x60K.

first: Cornelia

last: Vesely

first: Fritz

middle: J

last: Sedlazeck

first: Arndt

last: von Haeseler

last: Jantsch


title: Comparing next-generation sequencing and microarray technologies in a toxicological study of the effects of aristolochic Acid on rat kidneys

first: Zhenqiang

last: Su

address: 22 Thomson Place

department: IP & Science

institute: Thomson Reuters

summary: RNA-Seq has been increasingly used for the quantification and characterization of transcriptomes. The ongoing development of the technology promises the more accurate measurement of gene expression. However, its benefits over widely accepted microarray technologies have not been adequately assessed, especially in toxicogenomics studies. The goal of this study is to enhance the scientific community's understanding of the advantages and challenges of RNA-Seq in the quantification of gene expression by comparing analysis results from RNA-Seq and microarray data on a toxicogenomics study. A typical toxicogenomics study design was used to compare the performance of an RNA-Seq approach (Illumina Genome Analyzer II) to a microarray-based approach (Affymetrix Rat Genome 230 2.0 arrays) for detecting differentially expressed genes (DEGs) in the kidneys of rats treated with aristolochic acid (AA), a carcinogenic and nephrotoxic chemical most notably used for weight loss. We studied the comparability of the RNA-Seq and microarray data in terms of absolute gene expression, gene expression patterns, differentially expressed genes, and biological interpretation. We found that RNA-Seq was more sensitive in detecting genes with low expression levels, while similar gene expression patterns were observed for both platforms. Moreover, although the overlap of the DEGs was only 40-50%, the biological interpretation was largely consistent between the RNA-Seq and microarray data. RNA-Seq maintained a consistent biological interpretation with time-tested microarray platforms while generating more sensitive results. However, there is clearly a need for future investigations to better understand the advantages and limitations of RNA-Seq in toxicogenomics studies and environmental health research.

overall_design: Eight rats were randomly divided into two groups: four rats were administered with aristolochic acid (AA), and four rats were treated with the control vehicle. RNA samples were extracted from the kidney tissue of each rat and were independently assayed with both the NGS (Illumina Genome Analyzer II) and the microarray (Affymetrix Rat Genome 230 2.0) platforms. The RNA-Seq and microarray data were compared in terms of absolute gene expression, gene expression patterns, differentially expressed genes, and biological interpretation.

first: Zhiguang

last: Li

first: Tao

last: Chen

first: Quan-Zhen

first: Hong

last: Fang

first: Don

last: Ding

first: Weigong

last: Ge

first: Baitang

last: Ning

first: Huixiao

last: Hong

first: Roger

middle: G

last: Perkins

first: Weida

last: Tong

first: Leming

last: Shi


title: Direct Recruitment of Polycomb Repressive Complex 1 (PRC1) to Chromatin by Core Binding Transcription Factors (ChIP-Seq)

first: Tali

last: Mazor

summary: Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study, we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFbeta transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate considerable chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFbeta and functional regulation of a significant fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFbeta deficiency generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via Polycomb Repressive Complex 2 (PRC2) independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.

overall_design: ChIP-seq against Runx1, CBFb and Ring1b in L8057 cells (induced & uninduced with biological replicates) and thymocytes from control and Runx1 KO mice

first: Ming

last: Yu

first: Hui

last: Huang

first: Hsuan-Ting

first: Katie

middle: L

last: Kathrein

first: Andrew

last: Woo

first: Candace

last: Chouinard

first: Adam

last: Labadorf

first: Thomas

last: Akie

first: Tyler

middle: B

last: Moran

first: Huafeng

last: Xie

first: Sima

last: Zacharek

first: Ichiro

last: Taniuchi

first: Robert

last: Roeder

first: Carla

last: Kim

first: Leonard

last: Zon

first: Ernest

last: Fraenkel

first: Alan

last: Cantor


title: mRNA-seq and expression profile of mouse ES OS25 cells

city: London

first: Ines

middle: de

last: Santiago

address: Hammersmith Hospital Campus

department: Epigenetics

institute: MRC Clinical Sciences Centre

overall_design: Gene expression (mRNA-Seq) in mouse ES cells

last: de Santiago

first: Kelly

last: Morris

first: Ana

last: Pombo


title: Genome-wide landscape of liver X receptor chromatin binding and gene regulation

city: Olsztyn

first: Carsten

last: Carlberg

address: ul. Trylińskiego 18

department: Food Research

country: Poland

institute: Polish Academy of Sciences

summary:
Gene expression: Identification of primary target genes of liver X receptor (LXR) in an immune-related cellular model (THP-1 cells) to study, in conjunction with LXR binding data from ChIP-seq, the genome-wide mechanisms of transcriptional regulation by LXR.

ChIP-Seq: We performed ChIP-seq in macrophage-type PMA-differentiated THP-1 cells after stimulation with the potent synthetic LXR ligand T0901317 (T09). As a reference we performed microarray gene expression analysis in the same cellular model. We identified in total 1357 LXR binding locations on chromatin (FDR < 1%), of which 526 were observed after T09 treatment. De novo analysis of LXR site sequences identified DR4-type binding sites as major motif.

overall_design:
gene expression: THP-1 cells were treated for 4 h with 1 µM T09 or vehicle (DMSO)

ChIP-Seq: PMA-differentiated THP-1 cells were treated for 60 min with 1 µM T09 or vehicle (DMSO)

first: Petri

last: Pehkonen

first: Lynn

last: Welter-Stahl

first: Janine

last: Diwo

first: Anke

last: Wiencke-Baldacchino

first: Sami

last: Heikkinen

first: Eckardt

last: Treuter

first: Knut

last: Steffensen


title: Polycomb associates genome-wide with a specific RNA polymerase II variant, and regulates metabolic genes in ES cells

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


title: Deep-sequencing of endogenous microRNAs from human endothelial cells exposed to hypoxia and normoxia

status: Public on Feb 07 2012

city: Milan

first: Fabio

last: Martelli

address: via morandi 30

institute: IRCCS-Policlinico San Donato

summary: Human Umbelical Vein Endothelial Cells (HUVEC) were cultured in vitro and exposed to 1% oxygen or normoxia for 24 hours. Then, small RNA libraries were prepared and sequenced using sequencing-by-ligation technique (Solid3 Plus Sequencer platform). Alignment versus miRBase v14.0 identified >400 different microRNA/microRNA* species expressed in HUVEC. A complex repertoire of isomiR was also observed. For novel miRNA discovery, reads were mapped against the human genome. We identified 511 candidate new microRNAs and we validated 18 of them with independent techniques.

overall_design: 4 samples:  2 independent HUVEC populations were analyzed, each both  normoxic and hypoxic

first: Jeroen

last: van Rooij

first: Christine

last: Voellenkle

first: Alessandro

last: Guffanti


title: Polycomb associates genome-wide with a specific RNA polymerase II variant, and regulates metabolic genes in ES cells (ChIP-Seq)

summary:
Polycomb repressor complexes (PRCs) are important chromatin modifiers fundamentally implicated in pluripotency and cancer. Polycomb silencing in embryonic stem cells (ESCs) can be accompanied by active chromatin and primed RNA polymerase II (RNAPII), but the relationship between PRCs and RNAPII remains unclear at the genome-wide level. We mapped PRC repression markers and four RNAPII states in ESCs using ChIP-seq, and found that PRC-targets exhibit a range of RNAPII variants. Firstly, developmental PRC-targets are bound by unproductive RNAPII (S5p+S7p- S2p-) genome-wide. Sequential ChIP, Ring1B-depletion and genome-wide correlations show that PRCs and RNAPII-S5p physically bind to the same chromatin and functionally synergize. Secondly, we identify a cohort of genes marked by PRC and elongating RNAPII (S5p+S7p+S2p+); they produce mRNA and protein, and their expression increases upon PRC1 knockdown. We show that this novel group of PRC-targets switch between active and PRC-repressed states within the ESC population, and that many have roles in metabolism.

**Correction (Mar 14, 2012):
Due to curation error, runs for SRX112177 and SRX112179 switched, specifically,
Run SRR391034.1 was removed from experiment SRX112177 (GSM850468) and added to experiment SRX112179 (GSM850470) as SRR391034.3
Run SRR391040.1 was removed from experiment SRX112179 (GSM850470) and added to experiment SRX112177 (GSM850468) as SRR391040.3

overall_design: Examination of 4 different RNAPII modifications (S5p, S7p, 8WG16, S2p), and the histone modifications H2Aub1 and H3K36me3 in mouse ES cells

first: Emily

last: Brookes


title: Analysis of the Arabidopsis shoot meristem transcriptome during floral transition identifies distinct regulatory patterns and an LRR protein that promotes flowering

city: Köln

first: George

last: Coupland

address: Carl-von-Linne-Weg 10

department: Plant Developmental Biology

institute: Max Planck Institute for Plant Breeding Research

summary: We sequenced cDNA synthesized from mRNA from 3 time points of a time course of Arabidopsis thaliana shoot apical meristems. Plants were grown two weeks in short days (time point +0LD) and then exposed to long days for 1 day (time point +1LD) and 3 days (time point +3LD). Meristem cells were collected using laser capture microdissection, to generate a meristem-specific time course of gene expression in the early stages of floral induction. The experiment was done in three biological replicates (called A, B, and C). Some repetitions of sequencing on a few samples (technical replicates) were also done

overall_design: Examination of mRNA levels in shoot apical meristems at 3 time points from the stage of vegetative meristem to the stage of a transition meristem, before the formation of floral meristems.

first: Stefano

last: Torti


title: Expression profiling of Drosophila mushroom bodies

first: Sidi

last: Chen

department: Biology

summary: We performed mRNA-seq of dissected Drosophila mushroom bodies, comparing to whole brain and testis

overall_design: mRNA seq of MB, brain and testis

first: Manyuan

last: Long

first: Liqun

last: Luo

first: Maria

last: Spletter

first: Kevin

last: White

first: Xiaochun

last: Ni