title: Transcription Factor Binding Sites by ChIP-seq from ENCODE/Caltech

first: ENCODE

last: DCC

summary:
This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Barbara Wold mailto:woldb@caltech.edu, Georgi K. Marinov mailto:georgi@caltech.edu, Diane Trout mailto:diane@caltech.edu). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu).

Rationale for the Mouse ENCODE project
Our knowledge of the function of genomic DNA sequences comes from three basic approaches. Genetics uses changes in behavior or structure of a cell or organism in response to changes in DNA sequence to infer function of the altered sequence. Biochemical approaches monitor states of histone modification, binding of specific transcription factors, accessibility to DNases and other epigenetic features along genomic DNA. In general, these are associated with gene activity, but the precise relationships remain to be established. The third approach is evolutionary, using comparisons among homologous DNA sequences to find segments that are evolving more slowly or more rapidly than expected given the local rate of neutral change. These are inferred to be under negative or positive selection, respectively, and we interpret these as DNA sequences needed for a preserved (negative selection) or adaptive (positive selection) function.
The ENCODE project aims to discover all the DNA sequences associated with various epigenetic features, with the reasonable expectation that these will also be functional (best tested by genetic methods). However, it is not clear how to relate these results with those from evolutionary analyses. The mouse ENCODE project aims to make this connection explicitly and with a moderate breadth. Assays identical to those being used in the ENCODE project are performed in cell types in mouse that are similar or homologous to those studied in the human project. Thus, we will be able to discover which epigenetic features are conserved between mouse and human, and we can examine the extent to which these overlap with the DNA sequences under negative selection. The contribution of DNA that with a function preserved in mammals versus that with a function in only one species will be discovered. The results will have a significant impact on our understanding of the evolution of gene regulation.
Maps of Occupancy by Transcription Factors
Genome-wide occupancy maps of transcription factors (TFs) are generated by ChIP-seq. A ChIP-Seq experiment combines a chromatin immunoprecipitation (ChIP) experiment that enriches genomic DNA for the segments bound by specific proteins (the antigens recognized by the antibody) with high-throughput short read sequencing of the enriched DNA fragments (Wold & Myers, 2008). Proteins are crosslinked to DNA (usually with formaldehyde), chromatin is sheared and immunoprecipitated with the antibody of interest. The immunoprecipitated material is turned into a sequencing library and sequenced. The sequencing reads are then aligned to the genome. A control sample consisting of sonicated chromatin that has not been immunoprecipitated or immunoprecipitated with a non-specific immunoglobulin is also sequenced. The ChIP and the control datasets are analyzed with a variety of software packages to identify regions occupied by the target protein. The sequencing data, alignments and analysis files for these experiments are available for download.

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:
Cells were grown according to the approved ENCODE cell culture protocols (http://genome-test.cse.ucsc.edu/ENCODE/protocols/cell/mouse).
Chromatin immunoprecipitation followed published methods (Johnson & Mortazavi et al., 2007) with the exception of certain experiments for which glutaraldehyde was added to the crosslink reaction. Information on the antibodies used is available via the metadata for each subtrack. Libraries were constructed using the Illumina ChIP-seq Sample Preparation Kit or using a modified protocol that includes the addition of multiplexing tags to the fragments. DNA fragments were repaired to generate blunt ends and a single A nucleotide was added to each end. Double-stranded Illumina adaptors or Double-stranded Illumina adaptors with multiplexing tags were ligated to the fragments. Ligation products were amplified by 18 cycles of PCR, and the DNA between 150-250 bp was gel purified. Completed libraries were quantified with Quant-iT dsDNA HS Assay Kit. The DNA library was sequenced on the Illumina GAII and GAIIx sequencing systems, and more recently, for multiplexed libraries, several of them were pooled and sequenced on the HiSeq platform. Cluster generation, linearization, blocking and sequencing primer reagents were provided in the Illumina Cluster Amplification kits. Older libraries were generated using 2 rounds of PCR. Matched input samples were sequenced for each variation of fixation conditions and the number of PCR rounds. Reads of 32 bp, 36 bp or 50 bp length were generated.
Sequencing reads (fastq files) were assigned to the corresponding libraries based on the multiplexing tag for pooled libraries (all tags have been removed from reads in the fastq files available for download) or directly processed. Bowtie (Langmead et al., 2009) was used to map reads to the male or female version of the mouse genome (excluding the _random chromosomes in the assembly) depending on the cell line sex. The following parameters were used: "-v 2 -k 11 -m 10 -t --best --strata". Aligned reads were converted into rds files using the ERANGE package (Johnson & Mortazavi et al., 2007) and the findall.py program in ERANGE was used to identify enriched regions against the matching input sample. The following settings were used for point-source transcription factors: "--shift learn --ratio 3 --minimum 2 --listPeak --revbackground". For histone modifications, the settings were changed to "--notrim --nodirectionality --spacing 100 --ratio 3 --minimum 2 --listPeak --revbackground".

first: Millard

last: Jacobs

first: Muriel

first: Georgi

last: Marinov

first: Katherine

last: Fisher

first: Gordon

last: Kwan

first: Antony

last: Kirilusha

first: Gilberto

last: DeSalvo

first: Lorianne

last: Schaeffer

first: Diane

last: Trout

first: Igor

last: Antoschechkin

first: Lu

first: Gary

last: Schroth


title: Dynamic Transformations of Genome-wide Epigenetic Marking and Transcriptional Control Establish T Cell Identity [RNA-Seq]

status: Public on Apr 14 2012

city: Pasadena

first: Jingli

middle: A

last: Zhang

state: CA

address: 1200 E California Blvd

department: Biology

country: USA

institute: California Institute of Technology

type: Expression profiling by high throughput sequencing

summary: T cell development comprises a stepwise process of commitment from a multipotent precursor. To define molecular mechanisms controlling this progression, we probed five stages spanning the commitment process using deep sequencing RNA-seq and ChIP-seq methods to track genome-wide shifts in transcription, cohorts of active transcription factor genes, histone modifications at diverse classes of cis-regulatory elements, and binding patterns of GATA-3 and PU.1, transcription factors with complementary roles in T-cell development. The results locate potential promoter-distal cis-elements in play and reveal both activation sites and diverse mechanisms of repression that silence genes used in alternative lineages. Histone marking is dynamic and reversible, and while permissive marks anticipate, repressive marks often lag behind changes in transcription. In vivo binding of PU.1 and GATA-3 relative to epigenetic marking reveals distinctive, factor-specific rules for recruitment of these crucial transcription factors to different subsets of their potential sites, dependent on dose and developmental context.

overall_design: Genome-wide expression profiles, global distributions of three different histone modifications, and global occupancies of two transcription factors were examined in five developmentally related immature T populations. High throughput sequencing generated on average 9-30 million of mappable reads (single-read) for each ChIP-seq sample, and 10-15 million (single-read) for RNA-seq. Independent biological replicates were analyzed for individual populations. Terminology: FLDN1_RNA-seq_sample1 and FLDN1_RNA-seq_sample2 are independent biological replicates for the same cell type.

first: Ali

last: Mortazavi

first: Brian

last: Williams

first: Barbara

middle: J

last: Wold

first: Ellen

middle: V

last: Rothenberg


title: A pioneer protein encoded by required to maintain repression2 facilitates locus-specific paramutation in Zea mays

status: Public on Apr 16 2012

city: St. Louis

first: Blake

middle: C.

last: Meyers

state: MO

address: 975 N Warson Road

institute: Donald Danforth Plant Science Center

type: Non-coding RNA profiling by high throughput sequencing

summary: This research identifies a novel protein required for paramutation at the maize purple plant1 locus. This 'required to maintain repression2' (RMR2) protein represents the founding member of a plant-specific clade of hypothetical proteins. We show that RMR2 is required for transcriptional repression at the Pl1-Rhoades haplotype, for accumulation of 24 nt RNA species, and for maintenance of a 5-methylcytosine pattern distinct from that maintained by RNA polymerase IV. Genetic tests indicate that RMR2 is not required for paramutation occurring at the red1 locus. These results distinguish the paramutation-type mechanisms operating at specific haplotypes. The RMR2 clade of proteins provides a new entry point for understanding the diversity of epigenomic control operating in higher plants.

overall_design: Examination of small RNAs using Illumina's sequencing-by-synthesis (SBS) platform to deep sequence small RNA libraries made from the 4-cm cobs of rmr2 mutant and non-mutant siblings.

first: Joy-El

last: Barboura

first: Irene

middle: T

last: Liao

first: Jennifer

middle: L

last: Stonaker

first: Jana

middle: P

last: Lim

first: Clarissa

middle: C

last: Lee

first: Susan

middle: E

last: Parkinson

first: Jerry

last: Kermicle

first: Stacey

last: Simon

first: Rosalind

last: Williams-Carrier

first: Alice

last: Barkane

first: Jay

middle: B

last: Hollick


title: piRNA-mediated transgenerational inheritance of an acquired trait

status: Public on Apr 13 2012

city: MONTPELLIER

first: SEVERINE

last: CHAMBEYRON

address: 141, rue de la Cardonille

department: Institute of Human Genetics

country: France

institute: CNRS

summary:
The maintenance of genome integrity is an essential trait to the successful transmission of genetic information. In animal germ cells, piRNAs guide PIWI proteins to silence transposable elements (TEs) in order to maintain genome integrity. In insects, most of TE silencing in the germline is achieved by secondary piRNAs that are produced by a feed-forward loop (the ping-pong cycle), which requires the piRNA-directed cleavages of two types of RNAs: mRNAs of functional euchromatic TEs and heterochromatic transcripts that contain defective TE sequences. The first cleavage which initiates such amplification loop remains poorly understood.
Taking advantage of the existence of strains that are devoid of functional copies of the LINE-like I-element, we report that in such Drosophila ovaries, the initiation of a ping-pong cycle is achieved only by secondary I-element piRNAs that are produced in the ovary and deposited in the embryonic germline. This unusual secondary piRNA biogenesis, detected in the absence of functional I-element copies, results from the processing of sense and antisense transcripts of several different defective I-elements. Once acquired, for instance after ancestor aging, this capacity to produce heterochromatic-only secondary piRNAs is partially transmitted through generations via maternal piRNAs. Furthermore, such piRNAs acting as ping-pong initiators in a chromatin-independent manner confer to the progeny a high capacity to repress the I-element mobility. Our study explains at the molecular level the basis for epigenetic memory of maternal immunity that protects females from hybrid dysgenesis caused by transposition of paternally inherited functional I-elements.

overall_design: Comparison of Drosophila small RNA populations in ovaries and/or eggs from 3-day-old or 25-day-old females.

first: Thomas

last: Grentzinger

first: Claudia

last: Armenise

first: Christine

last: Brun

first: Bruno

last: Mugat

first: Vincent

last: Serrano

first: Alain

last: Pelisson

first: Severine

last: Chambeyron


title: Genome-wide maps of polyadenylation sites in control and PABPN1kd cells

status: Public on Apr 15 2012

city: Tel Aviv

first: Ran

last: Elkon

address: Ramat Aviv

department: Human Genetics

country: Israel

institute: Tel Aviv University

summary: We applied deep-sequencing based technique, 3'-Seq, to obtain comprehansive maps of poly-A sites in human cells. 3'-Seq was applied to two cell lines (U2OS and RPE-1), in control and PABPN1 knockdown cells

overall_design: Examination of poly-A sites in control and PABPN1kd cells (in two different cell lines)

first: Mathias

last: Jenal

first: Reuven

last: Agami


title: Spatial organisation of the X inactivation center

status: Public on Apr 11 2012

city: Paris

first: Elphege

middle: Pierre

last: Nora

address: 11-13 rue Pierre et Marie Curie

department: Mammalian developmental epigenetics

institute: Institut Curie

summary: We report the application of Chromosome Conformation Capture Carbon-copy (5C) to a 4.5 Mb stretch of the mouse X chromosome encompassing the X inactivation center locus. We uncover a series of discrete 200kb-1Mb topologically associating domains (TADs). These align with several domain-wide epigenomic features as well as co-regulated gene clusters. 5C analysis in EED and G9A mutants reveal that this segmental  organisation in TADs does not relie on the underlying H3K27me3 or H3K9me2 blocks. Deletion of a boundary between two TADs leads to ectopic chromosomal contacts between them. Analysis of mESCs, mNPCs and MEFs suggest that the positioning of TADs on the chromosome is stable during cell differentiation though their internal organisation changes. Comparison of male (XY) and female (XX) differentiated cells highlights that the long-range chromosomal contacts within TADs are dampened on the inactive X compared to the active X.

overall_design: 5C oligonucleotides were designed around HindIII restriction site following an alternative scheme

first: Bryan

middle: R

last: Lajoie

first: Job

last: Dekker

first: Edith

last: Heard


title: Whole Transcriptome analysis of the coral acropora millepora reveals complex responses to ocean acidification during the initiation of calcification

status: Public on Apr 10 2012

city: Canberra

first: Sylvain

last: Foret

state: ACT

address: Gould Building #116

department: Research School of Biology

country: Australia

institute: The Australian National University

summary: We used the Illumina RNAseq approach to study the effects of acute exposure to elevated CO2 on gene expression in primary polyps of Acropora millepora

overall_design: Examination of transcriptome in Acropora millepora primary polyps at 380, 750 and 1000 ppm CO2 after 3 days exposure

first: Aurelie

last: Moya

first: David

last: Hayward

first: Eldon

last: Ball

last: Miller


title: Uncovering small RNA-mediated responses to cold stress in a wheat thermosensitive genic male sterile line by deep sequencing (Degradome-Seq)

city: Wuhan

first: Zhonghui

last: Tang

address: Huazhong Agricultural University

country: China

institute: Huazhong Agricultural University

summary: The male sterility of thermosensitive genic male sterile (TGMS) lines of wheat (Triticum aestivum) is strictly controlled by temperature. The early phase of anther development is especially susceptible to cold stress. MicroRNAs (miRNA) play an important role in plant development and in responses to environmental stress. In this study, deep sequencing of small RNA (smRNA) libraries obtained from spike tissues of the TGMS line under cold and control conditions identified a total of 81 unique miRNA sequences from 30 families, and trans-acting small interfering RNAs (tasiRNAs) derived from two TAS3 genes. To identify smRNA targets in the wheat TGMS line, we applied the degradome sequencing method, which globally and directly identifies the remnants of smRNA-directed target cleavage. We identified 26 targets of 16 miRNA families and three targets of tasiRNAs. Comparing smRNA sequencing datasets and TaqMan qPCR results, we identified six miRNAs and one tasiRNA (tasiRNA-ARF) as cold stress-responsive smRNAs in spike tissues of the TGMS line. We also determined the expression profiles of target genes that encode transcription factors in response to cold stress. Interestingly, expressions of cold-stress responsive smRNAs integrated in the auxin-signaling pathway and their target genes were largely anticorrelated. We investigated tissue-specific expression of smRNAs using a tissue microarray approach. Our data indicated that miR167 and tasiRNA-ARF play roles in regulating the auxin-signaling pathway, and possibly in the developmental response to cold stress. These data provide evidence that smRNA regulatory pathways are linked with male sterility in the TGMS line during cold stress.

overall_design: Examination of 2 mRNA degradome libraries in spike tissues during cold and control condition

first: Z

first: C

last: Zhao

first: Y

last: Zheng


title: ChIP-Seq for Nkx3-1 in whole mouse prostate

status: Public on Apr 09 2012

city: Nashville

first: Philip

middle: D

last: Anderson

state: TN

department: Pathology

institute: Vanderbilt University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We report the genome-wide binding sites of the NK homeodomain transcription factor, Nkx3-1, in mouse prostate. Three different mouse genotypes were used: Nkx3-1 wild-type (WT), Nkx3-1 heterozygote (HET), and Nkx3-1 knock-out (KO). Two biological replicates were performed for WT and HET, and one replicate for KO. The mice used were a recombinant inbred C57BL6/J x 129 strain. The KO dataset was used as a control in lieu of an IgG.

overall_design: Examination of Nkx3-1 binding sites in Nkx3-1+/+ (WT), +/- (HET) and -/- (KO) mouse prostates

first: Sydika

last: McKissic

first: Sarki

last: Abdulkadir


title: The Histone Methyltransferase Wbp7 Controls Macrophage Function through GPI Glycolipid Anchor Synthesis

city: Vienna

first: Iros

last: Barozzi

address: Borschkegasse 8a

country: Austria

institute: Medical University Vienna

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

overall_design: Refer to individual Series

first: Liv

last: Austenaa

first: Agniezska

last: Chronowska

first: Alberto

last: Termanini

first: Renato

last: Ostuni

first: Elena

last: Prosperini

first: Francis

last: Stewart

first: Giuseppe

last: Testa

first: Gioacchino

last: Natoli


title: Lai_S3cell_V074/BS23

status: Public on Apr 18 2012

city: Toronto

first: DCC

last: modENCODE

state: Ontario

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

country: Canada

institute: Ontario Institute for Cancer Research

summary:
modENCODE_submission_4771
This submission comes from a modENCODE project of Eric Lai. For full list of modENCODE projects, see http://www.genome.gov/26524648
Project Goal: We plan to generate a comprehensive catalog of expressed and functional microRNAs, and generate biological evidence for their regulatory activity. We plan also to delineate the primary transcription units of microRNA genes. Finally, we plan to annotate other classes of non-miRNA expressed small RNAs, as least some of which may define novel classes of small RNA genes.
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: RNA-seq. BIOLOGICAL SOURCE: Cell Line: S3; Tissue: embryo-derived cell-line; Replicate type: biological; Developmental Stage: late embryonic stage; Sex: Unknown;  EXPERIMENTAL FACTORS: Cell Line S3

first: Eric

last: Lai


title: ERα and PolII ChIP seq from Mouse Uterus

city: Research Triangle Park

first: Sylvia

last: Hewitt

state: NC

address: 111 Alexander Dr

department: RDBL

institute: NIEHS

summary: To advance understanding of mechanisms leading to biological and transcriptional endpoints related to estrogen action in the mouse uterus, we have mapped ERα and RNA polymerase II binding sites using chromatin immunoprecipitation (ChIP) followed by sequencing of enriched chromatin fragments (ChIP-seq).   In the absence of hormone, 5184 ERα binding sites were apparent in the vehicle treated ovariectomized uterine chromatin, while 17240 were seen one hour after estrogen (E2) treatment, indicating that some sites are occupied by unliganded ERα, and that ERα binding is increased by E2.  Approximately 15% of the uterine ERα binding sites were adjacent to (<10 KB) annotated transcription start sites and many sites are found within genes or are found more than 100 KB distal from mapped genes; however, the density (sites per bp) of ERα binding sites is significantly greater adjacent to promoters.  An increase in quantity of sites but no significant positional differences were seen between vehicle and E2 treated samples in the overall locations of ERα binding sites either distal from, adjacent to or within genes.  Analysis of the PolII data revealed the presence of poised promoter proximal PolII on some highly upregulated genes. Additionally, co-recruitment of PolII and ERα to some distal enhancer regions was observed. A de novo motif analysis of sequences in the ERα bound chromatin confirmed that estrogen response elements (EREs) were significantly enriched. Interestingly, in areas of ERα binding without predicted ERE motifs, homeodomain transcription factor (Hox) binding motifs were significantly enriched. The integration of the ERα and PolII binding sites from our uterine ChIP-seq data with transcriptional responses revealed in our uterine microarrays has the potential to greatly enhance our understanding of mechanisms governing estrogen response in uterine and other estrogen target tissues.

overall_design: one sample each, vehicle ER-alpha ChIP seq,1 hour estradiol ER-alpha ChIP seq, vehicle RNA polymerase II ChIP seq,1 hour estradiol RNA polymerase II ChIP seq, input DNA

first: Leping

last: Li

first: Sara

last: Grimm

first: Yu

last: Chen

first: Liwen

last: Liu

first: Yin

first: Pierre

last: Bushel

first: David

last: Fargo

first: Kenneth

middle: S

last: Korach


title: The Caenorhabditis elegans RDE-10/RDE-11 complex regulates RNAi by promoting secondary siRNA amplification

status: Public on Apr 07 2012

city: Boston

first: Taiowa

last: Montgomery

state: MA

address: 185 Cambridge Street, CPZN-7250

department: Molecular Biology

institute: Massachusetts General Hospital

summary: From a forward genetic screen for C. elegans genes required for RNAi, we identified rde-10 and through proteomic analysis of RDE-10-interacting proteins, we identified a protein complex containing the new RNAi factor RDE-11, the known RNAi factors RSD-2 and ERGO-1, as well as other candidate RNAi factors. The newly identified RNAi defective genes rde-10 and rde-11 encode a novel protein and a RING-type zinc finger domain protein, respectively. Mutations in rde-10 and rde-11 genes cause dosage-sensitive RNAi deficiencies: these mutants are resistant to low dosage, but sensitive to high dosage of double-stranded RNAs. We assessed the roles of rde-10, rde-11, and the dosage-sensitive RNAi defective genes rsd-2, rsd-6 and haf-6 in both exogenous and endogenous small RNA pathways using high-throughput sequencing and qRT-PCR. These genes are required for the accumulation of secondary siRNAs in both exogenous and endogenous RNAi pathways.

overall_design: Small RNA analysis by deep sequencing in various wild type and mutant C. elegans strains.

first: Chi

last: Zhang


title: The Histone Methyltransferase Wbp7 Controls Macrophage Function through GPI Glycolipid Anchor Synthesis. [ChIP_seq]

first: Iros

last: Barozzi

summary:
Histone methyltransferases catalyze site-specific deposition of methyl groups, enabling recruitment of transcriptional regulators. In mammals, trimethylation of lysine 4 in histone H3, a modification localized at the transcription start sites of active genes, is catalyzed by six enzymes (SET1a and SET1b, MLL1–MLL4) whose specific functions are largely unknown. By using a genomic approach, we found that in macrophages, MLL4 (also known as Wbp7) was required for the expression of Pigp, an essential component of the GPI-GlcNAc transferase, the enzyme catalyzing the first step of glycosylphosphatidylinositol (GPI) anchor synthesis. Impaired Pigp expression in Wbp7-/- macrophages abolished GPI anchor-dependent loading of proteins on the cell membrane. Consistently, loss of GPI-anchored CD14, the coreceptor for lipopolysaccharide (LPS)
and other bacterial molecules, markedly attenuated LPS-triggered intracellular signals and gene expression changes. These data link a histone-modifying enzyme to a biosynthetic pathway and indicate
a specialized biological role for Wbp7 in macrophage function and antimicrobial response.

overall_design: Chromatin immuno-precipitations of H3 histone try-methylated on lysine 4 followed by multiparallel sequencing performed in murine bone marrow-derive macrophages (BMDM). Experiments carried out in untreated cells as well as in cells treated for 4hrs with lipopolysaccharide (LPS), for both Wbp7+/- (HET) and Wbp7-/- (KO) mice.

first: Liv

last: Austenaa

first: Agniezska

last: Chronowska

first: Alberto

last: Termanini

first: Renato

last: Ostuni


title: Sex-biased expression in Drosophila willistoni

status: Public on Apr 08 2012

city: Houston

first: Richard

last: Meisel

state: TX

address: 3455 Cullen Blvd

department: Biology and Biochemistry

institute: University of Houston

summary: We performed mRNA sequencing of samples isolated from the heads, thoraxes, and abdomens of males and females of Drosophila willistoni to identify genes that are differentially expressed between the sexes.

overall_design: Comparison of expression levels in females and males

first: Andrew

middle: G

last: Clark


title: RNA-sequencing analysis of 32Dclone3 cells overexpressing miR-125b

status: Public on Apr 05 2012

city: Cambridge

first: Marina

last: Bousquet

address: 9 Cambridge Center

institute: Whitehead Institute for Biomedical Research

summary: To better understand the mechanisms of blockage of myeloid differentiation and apoptosis and induction of proliferation by miR-125b, we preceded to identify miR-125b target genes involved in these pathways. We analyzed the total cellular gene expression pattern by RNA-sequencing of the parental 32Dclone3 myeloid cell line and that ectopically expressing miR-125b. We generated four cDNA libraries corresponding to duplicates of miR-125b and control cells.

overall_design: Compare the gene expression level in vector transduced 32Dclone3 cells with that in miR-125b transduced 32Dclone3 cells.

first: Harvey

middle: F

last: Lodish


title: RNA-sequencing analysis of NB4 cells overexpressing miR-125b

status: Public on Apr 06 2012

city: Oxford

first: Diu

last: Nguyen

address: Headley way

department: Clinical Laboratory Sciences

country: United Kingdom

institute: University of Oxford

summary: To better understand the mechanisms of blockage of myeloid differentiation and apoptosis and induction of proliferation by miR-125b, we proceeded to identify miR-125b target genes involved in these pathways. We analyzed the total cellular gene expression pattern by RNA-sequencing of the parental NB4 myeloid cell line and that transiently transfected with miR-125b. We generated four cDNA libraries corresponding to duplicates of miR-125b and control cells.

overall_design: Compare the gene expression levels in miR control transfected cells with that in miR-125b transfected NB4 cells.


title: Penetrance of biallelic SMARCAL1 mutations is associated with environmental and genetic disturbances of gene expression

city: Vancouver

first: Alireza

last: Baradaran-Heravi

state: BC

address: 950 West 28th Avenue, Room 218

department: Medical Genetics

institute: UBC

sample_organism:
Drosophila melanogaster
Homo sapiens
Mus musculus


title: Reorganization of the host epigenome by a viral oncogene

city: Los Angeles

first: Siavash

middle: K

last: Kurdistani

address: 615 Charles E Young Dr South

department: Biological Chemistry

institute: UCLA

summary: Adenovirus small e1a causes ~70% reduction in cellular levels of histone H3 lysine 18 acetylation (H3K18ac). It is unclear, however, where this dramatic reduction occurs genome-wide. ChIP-seq revealed that e1a erases 95% of H3K18ac peaks in normal fibroblasts and replaces them with one-third as many at new genomic locations. H3K18ac at promoters and intergenic regions of genes with fibroblast-related functions are relocalized after infection to promoters of highly-induced genes that regulate cell cycling and to new putative enhancers. Strikingly, a significant fraction of the post-infection H3K18ac peaks occurs precisely at regions bound by RB1 in uninfected cells, but not by p107 or p130 without RB1. In contrast, over half of H3K9ac peaks are similarly distributed before and after infection, independently of RB1. The strategic redistribution of H3K18ac by e1a highlights the importance of this modification for transcriptional activation and cellular transformation.

overall_design:
Examination of two histone acetylations and RB family members binding.
mRNA-Seq RPKM file linked as supplementary file on Series record.

first: Ferrari

last: Roberto

first: Trent

last: Su

first: Bing

last: Li

first: Giancarlo

last: Bonora

first: Amit

last: Oberai

first: Rajkumar

last: Sasidharan

first: Yvonne

last: Chan

first: Arnold

last: Berk

first: Matteo

last: Pellegrini


title: Penetrance of biallelic SMARCAL1 mutations is associated with environmental and genetic disturbances of gene expression (3)

summary: Biallelic mutations of the DNA annealing helicase SMARCAL1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin, subfamily a-like 1) cause Schimke immuno-osseous dysplasia (SIOD, MIM 242900), an incompletely penetrant autosomal recessive disorder. Using human, Drosophila, and mouse models, we show that the proteins encoded by SMARCAL1 orthologues localize to transcriptionally active chromatin and modulate gene expression. We also show that similar to SIOD patients, deficiency of the SMARCAL1 orthologues alone is insufficient to cause disease in fruit flies and mice although such deficiency causes modest diffuse alterations in gene expression. Rather, disease manifests when SMARCAL1 deficiency interacts with genetic and environmental factors that further alter gene expression. We conclude that the SMARCAL1 annealing helicase buffers fluctuations in gene expression and that alterations in gene expression contribute to the penetrance of SIOD.

overall_design: The RNA sequencing libraries were constructed from the liver RNA of 3-4-month Smarcal1del/del and wt female mice (n=3/group) at 20°C and after 1 hour at 39.5°C. These libraries were sequenced using the whole transcriptome shotgun sequencing procedure.

first: Cornelius

last: Boerkoel


title: mRNA decapping factors and the exonuclease Xrn2 function in widespread premature termination of RNA polymerase II transcription

city: Aurora

first: David

middle: L.

last: Bentley

state: CO

department: Biochemistry and Mol. Genetics

institute: U. Colorado School of Medicine

summary: We report a function of human mRNA decapping factors in control of transcription by RNA polymerase II. Decapping proteins Edc3, Dcp1a and Dcp2 and the termination factor TTF2 co-immunoprecipitate with Xrn2, the nuclear 5'-3' exonuclease  torpedo that facilitates transcription termination at the 3' ends of genes. Dcp1a, Xrn2 and TTF2 localize near transcription start sites (TSSs) by ChIP-Seq. At genes with 5' peaks of paused pol II, knockdown of decapping or termination factors, Xrn2 and TTF2, shifted polymerase away from the TSS toward upstream and downstream distal positions. This re-distribution of pol II is similar in magnitude to that caused by depletion of the elongation factor Spt5. We propose that coupled decapping of nascent transcripts and premature termination by the  torpedo  mechanism is a widespread mechanism that limits bidirectional pol II elongation. Regulated co-transcriptional decapping near promoter-proximal pause sites followed by premature termination could control productive pol II elongation.

overall_design: RNA pol II (GSE30895: GSM766171), Xrn2, TTF2 and Dcp1a were localized by ChIP-Seq in HeLa cells. RNA pol II was localized in control HEK293 cells and cells infected with lentiviruses expressing a scrambled control shRNA (scr), and shRNAs targeting the following proteins: Xrn2, TTF2, Xrn2+TTF2, Edc3, Dcp1a, and Dcp2.

first: Kris

last: Brannan

first: Hyunmin

last: Kim

first: Benjamin

last: Erickson

first: Kira

last: Glover-Cutter

first: Soojin

first: Nova

last: Fong

first: Lauren

last: Kiemele

first: Kirk

last: Hansen

first: Richard

last: Davis

first: Jens

last: Lykke-Andersen


title: Induction of lignocellulose-degrading enzymes in Neurospora crassa by cellodextrins

city: Berkeley

first: N

middle: Louise

last: Glass

address: 341 Koshland Hall, Univerisity of California

department: Plant and Microbial Biology

institute: UC Berkeley

summary: Transcriptional profiling with next-generation sequencing methods demonstrated that a Neurospora crassa mutant with the three most highly expressed beta-glucosidase genes deleted had a transcriptional response to cellobiose similair to that of wild type N. crassa exposed to cellulose.

overall_design: N. crassa was pregrown in Sucrose and transferred to Avicel (cellulose), Cellobiose, Sucrose or media with no carbon added.  Biological triplicates used to identify differentially expressed genes in WT on Avicel.  Single libraries for mutant strains identify which genes show similair expression on cellobiose as in the WT on cellulose.

first: Elizabeth

middle: A

last: Znameroski

first: Samuel

last: Coradetti

first: Jamie

middle: H

last: Cate


title: Effects of Cardiac Glycosides on RNA Expression in Prostate Cancer LNCaP-abl Cells

status: Public on Apr 04 2012

city: La Jolla

first: Hai-Ri

last: Li

address: 9500 Gilman Dr.

department: Medicine

institute: University of California, San Diego

summary: Prostate cancer is the most common cancer in men and cardiac glycosides inhibit prostate cancer cell proliferation. In order to investigate the mechanism by which cardiac glycosides inhibit  prostate cancer cells, we observed genome-wide RNA expression in prostate cancer LNCaP-abl cells, hormone resistant cells, after the cardiac glycoside treatment using RNA-Seq. In addition, we profiled LNCaP-abl cells after androgen receptor (AR) knockdown to observe whether cardiac glycoside effect on RNA expression is similar to that of  AR knockdown.

overall_design: Observation of three cardioglycosides, Digoxin, Peruvoside and Strophanthidin,  and AR knockdown regulated RNA expression in LNCaP-abl with RNA-Seq (each triplicates)

first: Hairi

first: Xiang-Dong

last: Fu


title: Effects of ADARs on small RNA processing pathways in C. elegans

city: Salt Lake City

first: Michael

middle: Bryan

last: Warf

state: UT

address: 15 N Medical Dr East, room 4800

department: Biochemistry

institute: University of Utah

summary: Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that convert adenosine to inosine in double-stranded RNA (dsRNA). To evaluate effects of ADARs on small RNAs that derive from dsRNA precursors, we performed deep-sequencing, comparing small RNAs from wildtype and ADAR mutant C. elegans. While editing in small RNAs was rare, at least 40% of microRNAs had altered levels in at least one ADAR mutant strain, and miRNAs with significantly altered levels had mRNA targets with correspondingly affected levels. About 40% of siRNAs derived from endogenous genes (endo-siRNAs) also had altered levels in at least one mutant strain, including 63% of Dicer-dependent endo-siRNAs. The 26G class of endo-siRNAs was significantly affected by ADARs, and many altered 26G loci had intronic reads, and histone modifications associated with transcriptional silencing. Our data indicate ADARs, through both direct and indirect mechanisms, are important for maintaining wildtype levels of many small RNAs in C. elegans.

overall_design: Deep sequencing of small RNAs in wild-type (N2), adr-1 null, adr-2 null and adr-1;adr-2 null mixed stage C. elegans

first: M

first: W

last: Johnson

first: Brenda

last: Bass


title: Genome-wide distribution of AdpA, a global regulator for secondary metabolism and morphological differentiation in Streptomyces [liquid]

city: Bunkyo-ku, Tokyo

first: Yasuo

last: Ohnishi

department: Department of Biotechnology

country: Japan

institute: Graduate School of Agriculture and Life Science, The University of Tokyo

summary: AdpA is a global transcriptional activator triggering morphological differentiation and secondary metabolism in Streptomyces griseus. AdpA influences expression of >1,000 genes, but the overall picture of AdpA regulon has been obscure. Here, we took snapshots of distribution of AdpA across the chromosome in living S. griseus cells by ChIP/ChAP-seq analysis. In both liquid and solid cultures, AdpA bound to similar >1,200 sites, which were located on not only putative regulatory regions (65 %) but also regions (35 %) that appeared not to affect transcription. Transcriptome analysis indicated that approximately 40% of the AdpA binding sites in putative regulatory regions were involved in gene regulation. AdpA was indicated to act as a transcriptional repressor as well as an activator. Expression profiles of AdpA-target genes were very different between liquid and solid cultures in spite of similar AdpA-binding profiles. We therefore concluded that AdpA directly controls >500 genes in cooperation with other regulatory proteins in many cases. In vitro AdpA binding to the selected 304 AdpA-binding sites was examined, revealing several unique characteristics of AdpA regarding its DNA-binding property. This study gave the first experimental insight into the extent of AdpA regulon, indicating many genes under the direct control of AdpA.

overall_design: Analysis of genome-wide distribution of AdpA on liquid culture with two different methods to isolate AdpA-DNA complex

first: Akiyoshi

last: Higo


title: Genome-wide distribution of AdpA, a global regulator for secondary metabolism and morphological differentiation in Streptomyces [solid]

overall_design: Time course analysis of genome-wide distribution of AdpA on solid culture


title: Genome-wide distribution of AdpA, a global regulator for secondary metabolism and morphological differentiation in Streptomyces, revealed the extent and complexity of the AdpA regulatory network.


title: Genome-Wide Analysis of Acute Myeloid Leukemia Reveals Leukemia-Specific Methylome and Subtype-Specific Hypomethylation of Repeats

status: Public on Apr 03 2012

city: Cantanhede

first: Jacek

middle: Dariusz

last: Marzec

address: Biocant Park

country: Portugal

institute: CoLAB AccelBio

type: Methylation profiling by high throughput sequencing


title: Transcription factor Ebf1 regulates differentiation stage-specific signaling, proliferative expansion and survival of B cells [H3K4me2 and H3K4me3]

city: Chicago

first: Sebastian

last: Pott

state: Il

address: 920 58th Street

institute: University of Chicago

summary: Transcription factor Ebf1 is an important determinant of early B lymphopoiesis.  To gain insight into differentiation stage-specific functions of Ebf1, we conditionally inactivated Ebf1.  We found that Ebf1 is required for proliferation, survival and signaling of pro-B cells and peripheral B cell subsets.  The proliferation defect of Ebf1-deficient pro-B cells, including the impaired expression of IL-7Ra and several cell cycle regulators, is overcome by transformation with v-Abl.  The survival defect of transformed Ebf1fl/fl pro-B cells can be rescued by the forced expression of the Ebf1 targets c-Myb or Bcl-xL.  In mature B cells, Ebf1 deficiency interferes with the BAFF-R and BCR-dependent Akt signaling pathways, as well as with germinal center formation and class switch recombination.  Genome-wide analyses of Ebf1 binding and Ebf1-mediated gene expression in mature B cells and comparison with reported data sets in pro-B cells provide insight into the basis for lineage- and stage-specific functions of Ebf1.

overall_design: Localistaion of histone modification (H3K4me2 and H3K4me3)  in splenic B cells in mice by ChIP-seq

first: Ildiko

last: Györy

first: Rudolf

last: Grosschedl


title: High-throughput Illumina Hiseq2000 seuqencing of small RNAs from Hevea brasiliensis PB260 and PB217 clones

city: shenzhen

first: Lei

last: Gao

state: guangdong

address: Nanhai Ave 3688

department: College of Life Sciences

institute: shenzhen university

summary: MicroRNAs (miRNAs) are non-coding, short, single-stranded RNAs with essential roles in gene regulation in various organisms including higher plants.  In contrast to the vast information on miRNAs from many economically important plants, almost nothing has been reported on the identification or analysis of miRNAs from rubber tree (Hevea brasiliensis L.), the most important natural rubber-producing crop.  To identify miRNAs and their target genes in rubber tree, high throughput sequencing combined with a computational approach was performed. Four small RNA libraries were constructed for deep sequencing from mature and young leaves of two rubber tree clones, PB 260 and PB 217, which provide high and low latex yield, respectively. 237 miRNAs belonging to 37 known miRNA families were identified, and northern hybridization validated miRNA expression and revealed developmental stage-dependent and clone-specific expression for some miRNAs. We took advantage of the newly released rubber tree genome assembly as well as the genomic databases from leafy spurge and cassava, two species related to rubber tree, and predicted 15 novel miRNAs.

overall_design: 4 samples examined: PB260 mature leaves, PB260 young leaves, PB217 mature leaves, and PB217 young leaves.

first: Jarunya

last: Narangajavana

first: Xuemei

last: Chen

first: Manassawe

last: Lertpanyasampatha


title: Transcription factor Ebf1 regulates differentiation stage-specific signaling, proliferative expansion and survival of B cells [EBF1]

overall_design: EBF1 binding in splenic B cells in mice


title: SINGLET OXYGEN RESISTANT 1 links reactive electrophile signaling to singlet oxygen acclimation in Chlamydomonas reinhardtii

city: Dübendorf

first: Beat

last: Fischer

address: Ueberlandstr. 133

department: Department of Environmental Toxicology

country: Switzerland

institute: Eawag

summary: To identify components involved in the signal transduction and activation of the singlet oxygen-mediated response, a mutant selection was performed. This led to the isolation of the singlet oxygen resistant 1 (sor1) mutant, which is more tolerant to singlet oxygen-producing chemicals and shows a constitutively higher expression of GPXH and GSTS1. Map-based cloning revealed that the SOR1 gene encodes a novel bZIP transcription factor, which seems to control its own expression as well as that of a large number of oxidative stress response and detoxification genes. In the promoter region of many of these genes a highly conserved 8-bp palindromic sequence element was found to be enriched. This element was shown to be essential for GSTS1 overexpression in sor1 and for induction by increased levels of lipophilic reactive electrophile species (RES) suggesting that it functions as an electrophile response element (ERE). RES can be formed after singlet oxygen-induced lipid peroxidation, indicating that a RES-stimulated and SOR1-mediated response of detoxification genes is part of the singlet oxygen-induced acclimation process in C. reinhardtii.

overall_design: The sor1 mutant isolated in a screen for singlet oxygen resistant mutants and the corresponding wild-type strain 4A+ were grown in a 12 h light dark cycle for several days before total RNA was extracted 6 h after the light came on.

first: Heidi

last: Ledford

first: Setsuko

last: Wakao

first: Shih-Yau

last: Huang

first: David

last: Casero

first: Matteo

last: Pellegrini

first: Sabeeha

last: Merchant

first: Andreas

last: Koller

first: Rik

middle: I

last: Eggen

first: Krishna

last: Niyogi


title: AMPKα Modulation in Cancer Progression: Multilayer Integrative Analysis of the Whole Transcriptome in Asian Gastric Cancer

first: Han

last: Liang

address: 1400 Pressler Street

department: Bioinformatics and Computational Biology

institute: University of Texas MD Anderson Cancer Center

summary: Gastric cancer is the most common cancer in Asia and most developing countries. To identify the molecular underpinnings of gastric cancer in the Asian population, we applied an RNA-sequencing approach to gastric tumor and noncancerous specimens to quantitatively characterize the entire transcriptome of gastric cancer (including mRNAs and microRNAs). A multi-layer analysis was then developed to identify multiple types of transcriptional aberrations associated with different stages of gastric cancer, including differentially expressed mRNAs, recurrent somatic mutations and key differentially expressed microRNAs. Through this approach, we identified the central metabolic regulator AMPK-α as a potential functional target in Asian gastric cancer. Further, we experimentally demonstrated the translational relevance of this gene as a potential therapeutic target for early-stage gastric cancer in Asian patients. Together, our findings not only provide a valuable information resource for identifying and elucidating the molecular mechanisms of Asian gastric cancer, but also represent a general integrative framework to develop more effective therapeutic targets.

overall_design: Using Life Technologies SOLiD™ sequencing platform, we performed transcriptome-wide profiling of gastric cancer samples from 30 anonymous, unrelated Asians of both sexes. Included were six noncancerous gastric tissue samples and 24 gastric tumor samples that represented stages I through IV of tumor development. From the WT-seq protocol we generated a WT-seq dataset of 2.1 billion 50-nt short reads from the 30 samples; Applying the second small RNA-seq protocol to 19 gastric tumor samples (5 of the original 24 yielded insufficient sample amounts) and 6 noncancerous gastric tissue samples resulted in a small RNA-seq dataset.

first: Yon

last: Kim

first: Xiuping

last: Liu

first: Ju-Seog

last: Lee

first: Jae

middle: Y

last: Cho

first: Jae-Ho

last: Cheong

first: Hoguen

first: Min

last: Li

first: Thomas

middle: J

last: Downey

first: Matthew

last: Dyer

first: Yongming

last: Sun

first: Jingtao

first: Ellen

middle: M

last: Beasley

first: Hyun

middle: C

last: Chung

first: Sung

last: Noh

first: John

middle: N

last: Weinstein

first: Chang-Gong

first: Garth

last: Powis


title: PAPD5, a noncanonical poly(A) polymerase with an unusual RNA-binding motif [PAR-CLIP]

city: Palo Alto

first: Biter

last: Bilen

address: 3801 Miranda Ave.

institute: Stanford University

summary: PAPD5 is one of the seven members of non-canonical poly(A) polymerases in human cells. There are previous reports about polyadenylation dependent degradation of pre-ribosomal RNAs and uridylation dependent degradation of histone mRNAs in vivo. In this study, we observed polyadenylation but not polyuridylation activity of PAPD5 with in vitro assays. We aimed to get genome-wide targets of PAPD5 and used PAR-CLIP and deep sequencing for this purpose.

overall_design: Recombinant version of PAPD5 is expressed in HEK293 human cell lines and its genome wide targets are obtained with PAR-CLIP and deep sequencing as two replicate experiments. The short reads in the deep sequencing libraries of PAPD5 replicates and an unrelated protein to polymerization from a previous study, IGF2BP1, are aligned to the hg18 human genome assembly. The biological variance of the read counts in overlapping 100-nucleotide-long-windows is estimated between the PAPD5 replicates and further used in the differential expression estimations between the 100-nucleotide windows in PAPD5 replicates and IGF2BP1. The top differentially expressed windows in PAPD5 and IGF2BP1 are further annotated using gene and repeat tracks from UCSC.

first: Christiane

last: Rammelt

first: Mihaela

last: Zavolan

first: Walter

last: Keller


title: Transcriptional Amplification in Tumor Cells with Elevated c-Myc

first: Richard

last: Young

summary: Excessive expression of c-Myc occurs frequently in human cancers, where high levels are associated with tumor aggression and poor clinical outcome, but the effect of high levels of c-Myc on global gene regulation is poorly understood.  We report here that in tumor cells expressing high levels of c-Myc, the transcription factor binds to E-box sequences in the core promoters of most actively transcribed genes and, unexpectedly, the enhancers of these active genes.  The predominant effect of increasing c-Myc levels at both proximal and distal promoter elements is to produce higher levels of transcription at existing active genes by promoting RNA polymerase II elongation, as opposed to stimulating transcription of novel target genes. Our results argue that c-Myc overexpression drives increased transcription of growth-promoting genes, and does so by amplifying the levels of transcripts associated with the entire gene expression program of the cancer cell. Thus, excess c-Myc functions to elicit the transcriptional amplification of existing active genes through the invasion of enhancers across the cancer cell genome, thereby reducing the rate-limiting constraints required for continuous tumor growth and proliferation.

overall_design: ChIP-Seq of multiple factors and histone modifications in a variety of human tumor cell lines

first: Charles

last: Lin

first: Peter

middle: B

last: Rahl

first: Jakob

last: Loven

first: Tong


title: Transcription factor Ebf1 regulates differentiation stage-specific signaling, proliferative expansion and survival of B cells

first: Sebastian

last: Pott

department: Human Genetics

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

overall_design:
Refer to individual Series
See related Series GSE36061.


title: DNA-Seq of DrosDel deficiency lines and w1118 lines

city: Bethesda

first: Brian

last: Oliver

state: MD

address: 50 South Drive

department: LBG

institute: NIDDK, NIH

type: Genome variation profiling by high throughput sequencing

summary: DNASeq reads from pools of female or male DrosDel and w1118 (parental strain for the Df/+ flies). Samples are named in this dataset according to the following sample naming scheme: tissue_genotype shorthand_sex_DNASeq_biological replicate #.

overall_design: We sequenced DNA from pools of female or male DrosDel and w1118 (parental strain for the Df/+ flies).

last: Malone

first: Dong-Yeon

last: Cho

first: Nicolas

middle: R

last: Mattiuzzo

first: Carlo

last: Artieri

first: Lichun

last: Jiang

first: Ryan

last: Dale

first: Harold

middle: E

last: Smith

first: Jennifer

last: McDaniel

first: Sarah

last: Munro

first: Marc

last: Salit

first: Justen

last: Andrews

first: Teresa

last: Przytycka


title: RNA-Seq on SOLiD platform of DrosDel deficiency and w1118 flies

summary: RNA-Seq reads of DrosDel deficiency (Df/+) and parental strain diploid flies along with spike-in controls was performed on the SOLiD platform. Samples are named in this dataset according to the following sample naming scheme: tissue_genotype shorthand_sex_biological replicate #_platform_technical replicate.

overall_design: We sequenced mRNA from pools of female or male DrosDel and w1118 (parental strain for the Df/+ flies).  The same sample was sequenced across two flow cells on the SOLiD platform.

first: John

middle: G

first: Tersa


title: Genome-wide study of DNA methylation in AML by MeDIP-seq

status: Public on Apr 02 2012

first: Jacek

last: Marzec

summary: Methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq) has the potential to identify changes in DNA methylation important in cancer development. In order to understand the role of epigenetic modulation in the development of acute myeloid leukemia (AML) we have applied MeDIP-seq to the DNA of 12 AML patients and 4 normal bone marrows. This analysis revealed leukemia-associated differentially methylated regions that included gene promoters, gene bodies, CpG islands and CpG island shores. Two genes (SPHKAP and DPP6) with significantly methylated promoters were of interest and further analysis of their expression showed them to be repressed in AML. We also demonstrated considerable cytogenetic subtype specificity in the methylomes affecting different genomic features. Significantly distinct patterns of hypomethylation of certain interspersed repeat elements were associated with cytogenetic subtypes. The methylation patterns of members of the SINE family tightly clustered all leukemic patients with an enrichment of Alu repeats with a high CpG density (P < 0.0001). We were able to demonstrate significant inverse correlation between intragenic interspersed repeat sequence methylation and gene expression with SINEs showing the strongest inverse correlation (R2 = 0.7). We conclude that the alterations in DNA methylation that accompany the development of AML affect not only the promoters, but also the non-promoter genomic features, with significant demethylation of certain interspersed repeat DNA elements being associated with AML cytogenetic subtypes. MeDIP-seq data were validated using bisulfite pyrosequencing and the Infinium array.

overall_design: Examination of DNA methylation of 12 AML patients versus normal bone marrow from 4 healthy donors

first: Marwa

last: Saied

first: Sabah

last: Khalid

first: Paul

first: Thomas

last: Down

first: Vardhman

middle: K

last: Rakyan

first: Gael

last: Molloy

first: Manoj

last: Raghavan

first: Silvana

last: Debernardi

first: Bryan

middle: D

last: Young


title: Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga Nannochloropsis oceanica CCMP1779

city: East Lansing

first: Guangxi

last: Wu

state: MI

address: S308 Plant Bio Bld

institute: Michigan State University

summary: Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high value lipid products. First success in applying reverse genetics makes Nannochloropsis species attractive models to investigate the cell and molecular biology and biochemistry of this fascinating organism group. (Principle findings) Here we present the assembly of the 28.7 Mb genome of Nannochloropsis oceanica CCMP1779. RNA sequencing data from N-replete and N-depleted growth conditions support a total of 11,973 genes, which in addition to automatic annotation were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism,  114 predicted transcription factors and 109 transcriptional regulators were annotated. In addition, we provide protocols for the transformation of the sequenced strain. (Significance) The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols provides a blueprint for future detailed gene functional analysis and phylogenetic comparison of Nannochloropsis species by a growing academic community focused on this genus.

overall_design: one sample each of nitrogen-replete and nitrogen-depleted conditions

first: Christoph

last: Benning

first: Astrid

last: Vieler


title: Novel roles for Klf1 in regulating the erythroid transcriptome revealed by mRNA-seq

status: Public on Apr 01 2012

city: St Lucia

first: Michael

middle: Robert

last: Tallack

state: QLD

address: Services Road

institute: Institute for Molecular Bioscience, The University of Queensland

summary: Klf1 (formerly known as Eklf) regulates the development of erythroid cells from bi-potent progenitor cells via the transcriptional activation of a diverse set of genes. Mice lacking Klf1 die in utero prior to E15 from severe anemia due to the inadequate expression of genes controlling hemoglobin production, cell membrane and cytoskeletal integrity, and the cell cycle and proliferation. We have recently described the full repertoire of Klf1 binding sites in vivo by performing Klf1 ChIP-seq in primary erythroid tissue (E14.5 fetal liver). Here we describe the Klf1-dependent erythroid transcriptome by comparing mRNA-seq from Klf1+/+ and Klf1-/- erythroid tissue. This has revealed novel target genes not previously obtainable by traditional microarray technology and provided novel insights into the function of Klf1 as a transcriptional activator such as interactions with Gata1, Scl/Tal1 and p300. We also describe a set of erythroid specific promoters not previously identified that drive high level expression of otherwise ubiquitously expressed genes in erythroid cells. Additionally, our study has identified for the first time two novel lnc-RNAs that are dynamically expressed during erythroid differentiation as well as a role for Klf1 in directing apoptotic gene expression to drive the terminal stages of erythroid maturation.

overall_design:
Examination of mRNA expression in 3 Klf1-/- and 3 Klf1+/+ fetal livers

This submission represents mRNA-Seq component of study.

first: Andrew

last: Perkins


title: The anti-Shine-Dalgarno sequence drives translational pausing and codon choice in bacteria

city: San Francisco

first: Gene-Wei

last: Li

address: 1700 4th Street, BH404

institute: UCSF

summary: Protein synthesis by ribosomes takes place on a linear substrate but at variable speeds. Transient pausing of ribosomes can impact a variety of co-translational processes, including protein targeting and folding. These pauses are influenced by the sequence of the mRNA. Thus redundancy in the genetic code allows the same protein to be translated at different rates. However, our knowledge of both the position and the mechanism of translational pausing in vivo is highly limited. Here we present a genome-wide analysis of translational pausing in bacteria using ribosome profiling-deep sequencing of ribosome-protected mRNA fragments. This approach enables high-resolution measurement of ribosome density profiles along most transcripts at unperturbed, endogenous expression levels. Unexpectedly, we found that codons decoded by rare tRNAs do not lead to slow translation under nutrient-rich conditions. Instead, Shine-Dalgarno-(SD) like features within coding sequences cause pervasive translational pausing. Using an orthogonal ribosome possessing an altered anti-SD sequence, we demonstrated that pausing is due to hybridization between mRNA and the 16S rRNA of the translating ribosome. In protein coding sequences, internal SD sequences are disfavoured, which leads to biased usage, avoiding codons and codon pairs that resemble canonical SD sites. Our results indicate that internal SD-like sequences are a major determinant of translation rates and a global driving force for the coding of bacterial genomes.

sample_organism: Bacillus subtilis subsp. subtilis str. 168

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: Identification of translation pause sites in vivo using ribosome profiling

first: Eugene

last: Oh

first: Jonathan

middle: S

last: Weissman


title: Drosophila DrosDel deficiency and diploid control flies

first: Brian

last: Oliver

overall_design: Refer to individual Series

first: John

middle: H

last: Malone

first: Dong-Yeon

last: Cho

first: Nicolas

last: Mattiuzzo

first: Carlo

last: Artieri

first: Lichun

last: Jiang

first: Ryan

last: Dale

first: Harold

last: Smith

first: Jennifer

last: McDaniel

first: Sarah

last: Munro

first: Marc

last: Salit

first: Justen

last: Andrews

first: Teresa

last: Przytycka


title: RNA-Seq on Illumina platform of Drosophila DrosDel deficiency lines

summary: RNA-Seq reads of DrosDel deficiency (Df/+) and parental strain diploid flies along with spike-in controls was performed on the Illumina platform. Samples are named in this dataset according to the following sample naming scheme: tissue_genotype shorthand_sex_biological replicate #_platform.

overall_design: We sequenced mRNA from pools of female or male DrosDel and w1118 (parental strain for the Df/+ flies).


title: RNA-sequencing of TGF-ß1-driven gene expression in human kidney cell line

city: Dublin

first: Melissa

last: Morine

address: Belfield

country: Ireland

institute: University College Dublin

summary: TGF-beta1 is the major cytokine driver of fibrotic scarring observed in diabetic nephropathy and other fibrosis-related diseases. RNA-sequencing offers the potential for more sensitive assessment of the TGF-ß1-driven transcriptome.

overall_design: There were two treatment groups: vehicle, 48 hr TGFb1. Each treatment was carried out in triplicate. Upon quality control assessment, one TGFß1 treated sample was excluded from further analyses, leaving 3 unstimulated and 2 TGFß1 samples.

first: Finian

last: Martin


title: Integrative deep sequencing of the mouse lung transcriptome reveals differential expression of diverse classes of small RNAs in response to respiratory virus infection.

status: Public on Mar 31 2012

city: Seattle

first: Michael

last: Katze

state: WA

address: Rosen Building 960 Republican St.

department: Microbiology

institute: University of Washington

summary:
We previously reported widespread differential expression of long non-protein-coding RNAs (ncRNAs) in response to virus infection. Here, we expanded the study through small RNA transcriptome sequencing analysis of the host response to both severe acute respiratory syndrome coronavirus (SARS-CoV) and influenza virus infections across four founder mouse strains of the Collaborative Cross, a recombinant inbred mouse resource for mapping complex traits. We observed differential expression of over 200 small RNAs of diverse classes during infection. A majority of identified microRNAs (miRNAs) showed divergent changes in expression across mouse strains with respect to SARS-CoV and influenza virus infections and responded differently to a highly pathogenic reconstructed 1918 virus compared to a minimally pathogenic seasonal influenza virus isolate. Novel insights into miRNA expression changes, including the association with pathogenic outcomes and large differences between in vivo and in vitro experimental systems, were further elucidated by a survey of selected miRNAs across diverse virus infections. The small RNAs identified also included many non-miRNA small RNAs, such as small nucleolar RNAs (snoRNAs), in addition to nonannotated small RNAs. An integrative sequencing analysis of both small RNAs and long transcripts from the same samples showed that the results revealing differential expression of miRNAs during infection were largely due to transcriptional regulation and that the predicted miRNA-mRNA network could modulate global host responses to virus infection in a combinatorial fashion. These findings represent the first integrated sequencing analysis of the response of host small RNAs to virus infection and show that small RNAs are an integrated component of complex networks involved in regulating the host response to infection.
IMPORTANCE: Most studies examining the host transcriptional response to infection focus only on protein-coding genes. However, mammalian genomes transcribe many short and long non-protein-coding RNAs (ncRNAs). With the advent of deepsequencing technologies, systematic transcriptome analysis of the host response, including analysis of ncRNAs of different sizes, is now possible. Using this approach, we recently discovered widespread differential expression of host long (>200 nucleotide[nt]) ncRNAs in response to virus infection. Here, the samples described in the previous report were again used, but we sequenced another fraction of the transcriptome to study very short (about 20 to 30 nt) ncRNAs. We demonstrated that virus infection also altered expression of many short ncRNAs of diverse classes. Putting the results of the two studies together, we show that small RNAs may also play an important role in regulating the host response to virus infection.

overall_design: The small RNA transcriptome deep sequencing analysis was performed on lung samples from our previously published study (Unique signatures of long noncoding RNA expression in response to virus infection and altered innate immune signaling , X Peng, MBio. 2010 Oct 26;1(5). pii: e00206-10.). We infected four of the eight founder mouse strains used in generating the Collaborative Cross, a recombinant inbred mouse resource for mapping complex traits (41). These strains included 129S1/SvImJ (129/S1), WSB/EiJ (WSB), PWK/PhJ (PWK), and CAST/EiJ (CAST) mice. Ten-week-old mice were intranasally infected with phosphate-buffered saline (PBS) alone or with 1X10^5 PFU of mouse adapted severe acute respiratory syndrome coronavirus (SARS-CoV; rMA15), or 500 PFU of influenza A virus strain A/Pr/8/34 (H1N1; PR8). To match the previous whole-transcriptome analysis, we performed small RNA transcriptome sequencing analysis on the same eight samples from mice with SARS-CoV infections, including one SARS-CoV rMA15-infected mouse and one matched mock-infected mouse from each of the four strains at 2 days postinfection (dpi). In addition, we sequenced the small RNA transcriptome for 12 samples obtained from influenza virus infected mice, including two PR8-infected mice and one matched mockinfected mouse from each of the four strains at 2 dpi.

first: Xinxia

last: Peng

first: Lisa

last: Gralinski

first: Martin

middle: T

last: Ferris

first: Matthew

last: Frieman

last: Thomas

first: Sean

last: Proll

first: Marcus

last: Korth

last: Tisoncik

first: Mark

last: Heise

first: Shujun

last: Luo

first: Gary

middle: P

last: Schroth

first: Terrence

last: Tumpey

first: Chengjun

last: Li

first: Yoshihiro

last: Kawaoka

first: Ralph

last: Baric


title: Gene expression profiles of tumor-associated macrophages (TAMs) overexpressing miR-511-3p

city: Oslo

first: Camilla

last: Stormo

address: Kirkeveien 166

department: Department of Medical Genetics

country: Norway

institute: Oslo University Hospital

summary: Hematopoietic stem/progenitor cells (HS/PCs) were transduced with lentiviral vectors overexpressing OFP and either miR-511-3p or a control, mutated miRNA sequence (miR-511-3p-mut). The transduced HS/PCs were then transplanted in recipient C57BL/6 mice. Tumors (Lewis lung carcinomas, LLC) were injected s.c. 4 weeks after HS/PC transplant. Lentiviral vector-transduced (OFP+), tumor-associated macrophages (TAMs) were isolated 4 weeks after LLC injection by fluorescence-activated cell sorting. Gene expression profiles of TAMs overexpressing either miR-511-3p or miR-511-3p-mut were obtained from 3 independent biological samples/each. Gene expression profiles of miRNA-overexpressing TAMs were then compared with the gene expression profiles of wild-type TAMs isolated from LLCs grown in nontransplanted C57BL/6 mice; the latter TAMs were subfractioned into MRC1+CD11c(low) or CD11c+MRC1(low) subsets before RNA isolation and analysis. Comparison of gene expression profiles of TAMs revealed that miR-511-3p overexpression tunes down the expression of multiple genes that are classically upregulated in protumoral MRC1+CD11c(low) TAMs.

overall_design: TAMs were isolated from Lewis lung carcinomas grown s.c. in C57BL/6 mice.

first: Mario

middle: L

last: Squadrito

first: Michele

last: De Palma

first: Robert

last: Lyle

first: Gregor

last: Gilfillan


title: Small RNAs in the Physcomitrella dcl4 mutant

city: University Park

first: Michael

last: Axtell

state: PA

address: 280 Mueller Lab

department: Biology

institute: Pennsylvania State University

summary: To study the function of Physcomitrella DCL4, a knockout plant line was created.  Small RNAs from ~10-day old protonemata were obtained and sequenced using a SOLiD 2 instrument.

overall_design: A single library each was made from dcl4 mutant protonemata and wild-type protonemata.  The dcl4 library was sequenced in four separate technical replicates.  The wild-type sample was subject to a single sequencing run.


title: Transcriptome sequencing of Foxtail millet Setaria italica for different tissues

city: Shenzhen

first: Zhijun

last: Zheng

address: #11 Building 2/F│Beishan Industrial Zone│Yantian District

institute: BGI

summary: Transcriptome sequencing of Foxtail millet Setaria italica (Zhang-gu) for different tissues.

overall_design: Four RNA pools were created corresponding to four different tissues: root, leaf, stem, spica (tassel) at developmental stage, then each pool was sequenced.

first: Xin

last: Liu

first: Shifeng

last: Cheng


title: Whole-genome landscape of histone H2AX and γ-H2AX

city: Singapore

first: Jung Kyoon

last: Choi

address: 60 Biopolis Street

country: Singapore

institute: Genome Institute of Singapore

summary: Phosphorylation of the histone variant H2AX forms γ-H2AX that marks DNA double-strand break (DSB).  Here we generated the sequencing-based maps of H2AX and γ-H2AX positioning in resting and proliferating cells before and after ionizing irradiation.  Genome-wide locations of possible endogenous and exogenous DSBs were identified based on γ-H2AX distribution in dividing cancer cells without irradiation and that in resting cells upon irradiation, respectively.  γ-H2AX-enriched regions of endogenous origin in replicating cells included telomeres and active transcription start sites, apparently reflecting replication- and transcription-mediated stress during rapid cell division.  Surprisingly, H2AX itself, prior to phosphorylation, was specifically located at these endogenous hotspots.  This phenomenon was only observed in dividing cancer cells but not in resting cells.  Endogenous H2AX was concentrated on the transcription start site of actively transcribed genes but was irrelevant to pausing of RNA polymerase II (pol II), which precisely coincided with γ-H2AX of endogenous origin.  γ-H2AX enrichment upon irradiation also coincided with actively transcribed regions, but unlike endogenous γ-H2AX, it extended into the gene body and was not specifically concentrated on the pausing site of pol II.  Subtelomeres were not responsive to external DNA damage.  Our findings provide insight into DNA repair programs of cancer and may have implications for cancer therapy.

overall_design: Profiles of H2AX and gamma-H2AX in normal resting and cancer T cells with and without ionizing irradiation.

first: Jung-Kyoon


title: A Bcl6-Smrt/Ncor repression program controls atherosclerosis

status: Public on Mar 30 2012

city: La Jolla

first: Ruth

last: Yu

state: CA

address: 10010 N Torrey Pines Rd

department: Gene Expression Lab

institute: Salk Institute


title: Identification of allele-specific alternative mRNA processing via transcriptome sequencing

city: Los Angeles

first: Gang

address: 611 Charles E. Young Drive Rm. 660

institute: UCLA

summary: Establishing the functional roles of genetic variants remains a significant challenge in the post-genomic era. Here, we present a method, allele-specific alternative mRNA processing (ASARP), to identify genetically influenced mRNA processing events using transcriptome sequencing (RNA-Seq) data. The method examines RNA-Seq data at both single nucleotide and whole-gene/isoform levels to identify allele-specific expression (ASE) and existence of allele-specific regulation of mRNA processing. We applied the methods to data obtained from the human glioblastoma cell line U87MG and primary breast cancer tissues and found that 26–45% of all genes with sufficient read coverage demonstrated ASE, with significant overlap between the two cell types. Our methods predicted potential mechanisms underlying ASE due to regulations affecting either whole-gene-level expression or alternative mRNA processing, including alternative splicing, alternative polyadenylation and alternative transcriptional initiation. Allele-specific alternative splicing and alternative polyadenylation may explain ASE in hundreds of genes in each cell type. Reporter studies following these predictions identified the causal single nucleotide variants (SNVs) for several allele-specific alternative splicing events. Finally, many genes identified in our study were also reported as disease/phenotype-associated genes in genome-wide association studies. Future applications of our approach may provide ample insights for a better understanding of the genetic basis of gene regulation underlying phenotypic diversity and disease mechanisms.

overall_design: Examine allele-specific gene expression and alternative RNA processing in U87MG cell line

first: Jae-Hoon

last: Bahn

first: Xinshu

last: Xiao


title: Uncovering small RNA-mediated responses to cold stress in a wheat thermosensitive genic male sterile line by deep sequencing

status: Public on Mar 29 2012

city: Wuhan

first: Zhonghui

last: Tang

address: Huazhong Agricultural University

institute: Huazhong Agricultural University

summary: The male sterility of thermosensitive genic male sterile (TGMS) lines of wheat (Triticum aestivum) is strictly controlled by temperature. The early phase of anther development is especially susceptible to cold stress. MicroRNAs (miRNA) play an important role in plant development and in responses to environmental stress. In this study, deep sequencing of small RNA (smRNA) libraries obtained from spike tissues of the TGMS line under cold and control conditions identified a total of 81 unique miRNA sequences from 30 families, and trans-acting small interfering RNAs (tasiRNAs) derived from two TAS3 genes. We identified 26 targets of 16 miRNA families and three targets of tasiRNAs. Comparing smRNA sequencing datasets and TaqMan qPCR results, we identified six miRNAs and one tasiRNA (tasiRNA-ARF) as cold stress-responsive smRNAs in spike tissues of the TGMS line. We also determined the expression profiles of target genes that encode transcription factors in response to cold stress. Interestingly, expressions of cold-stress responsive smRNAs integrated in the auxin-signaling pathway and their target genes were largely anticorrelated. We investigated tissue-specific expression of smRNAs using a tissue microarray approach. Our data indicated that miR167 and tasiRNA-ARF play roles in regulating the auxin-signaling pathway, and possibly in the developmental response to cold stress. These data provide evidence that smRNA regulatory pathways are linked with male sterility in the TGMS line during cold stress.

overall_design: Examination of 7 small RNA libraries in spike tissues during cold and control condition

first: ZHONGHUI

last: TANG

first: CHANGPING

last: ZHAO

first: YONGLIAN

last: ZHENG


title: TCF7 is a key regulator of the switch of self-renewal and differentiation in a multipotential hematopoietic cell line

city: PALO ALTO

first: Jiaqian

last: Wu

address: 1501 California Avenue

summary: A critical problem in biology is understanding how cells choose between self-renewal and differentiation. To generate a comprehensive view of the mechanisms controlling early hematopoietic precursor self-renewal and differentiation, we used systems-based approaches and murine EML multipotential hematopoietic precursor cells as a primary model.  EML cells give rise to a mixture of self-renewing Lin-SCA+CD34+ cells and partially differentiated non-renewing Lin-SCA-CD34- cells in a cell autonomous fashion. We identified and validated the HMG box protein TCF7 as a key regulator in this self-renewal/differentiation switch, and it operates in the absence of canonical Wnt signaling. We found that TCF7 is the most downregulated transcription factor when CD34+ cells switch into CD34- cells using RNA-Seq. We subsequently identified the target genes bound by TCF7 using ChIP-Seq. We show that TCF7 binds to Runx1 (Aml1) promoter region, and RUNX1 and TCF7 co-regulate. Gene Set Enrichment Analysis suggests that TCF7 primarily acts as a positive regulator of genes preferentially expressed in CD34+ cells. Consistent with this possibility, knocking-down TCF7 represses many up-regulated genes in Lin-CD34+ cells. Finally a network of up-regulated transcription factors of CD34+ cells which defines the self-renewing state was constructed. These studies in EML cells demonstrate fundamental cell-intrinsic properties of the switch between self-renewal and differentiation, and yield valuable insights for manipulating HSCs and other differentiating systems.

overall_design: Examining the transcription factor binding targets of TCF7 and RUNX1.

first: Jia

middle: Q

first: Montrell

last: Seay

first: Manoj

last: Hariharan

last: Snyder

first: Sherman

last: Weissman


title: Genome-wide location analysis of SMRT and NCoR in wild-type and Bcl6 knockout macrophages

summary: Using ChIP-seq, we reveal the SMRT and NCoR co-repressor cistromes, which each consist of over 30,000 half-shared binding sites.  Moreover, we identify Bcl6-bound sub-cistromes for each co-repressor, which are strongly concentrated on NF-κB-driven inflammatory and tissue remodeling genes.  These results reveal a critical role for Bcl6 and its corepressors SMRT and NCoR in the prevention of atherosclerosis and chronic inflammation.

overall_design: Identification of SMRT and NCoR binding sites in wild-type and Bcl6 knockout primary bone-marrow derived macrophages

first: Grant

last: Barish

first: Ronald

last: Evans


title: DEEP SEQUENCING OF MODELS OF BREAST DUCTAL CARCINOMA IN SITU REVEALS ALDH5A1 AS A NOVEL POTENTIAL THERAPEUTIC TARGET

city: Detroit

first: RAYMOND

last: MATTINGLY

address: 540 East Canfield

department: Pharmacology

institute: Wayne State University

summary: We attempted to identify alterations in gene expression that occur during the progression from normal breast to ductal carcinoma in situ (DCIS) with the aim to elucidate significant genes and pathways underlying the premalignant transformation.  To determine the expression changes that are common to multiple DCIS models (MCF10.DCIS, SUM102 and SUM225) and normal mammary epithelial cells (MCF10A), we grew the cells in three dimensional overlay culture with reconstituted basement membrane and used the extracted RNA for 76 cycles of deep sequencing (mRNA-Seq) using Illumina Genome Analyzer GAIIx.  Analysis of mRNA-Seq results showed 295 consistently differentially expressed transcripts in DCIS models as compared to MCF10A.  These differentially expressed genes are associated with a number of signaling pathways such as integrin, fibroblast growth factor and TGFß signaling.  Many differentially expressed transcripts in DCIS were found to be involved in cell-cell signaling, cell-cell adhesion and cell proliferation.  We further investigated ALDH5A1 gene that encodes for the enzyme, aldehyde dehydrogenase 5A1, which is involved in glutamate metabolism. Further, inhibition of ALDH5A1 with different pharmacological drugs resulted in significant inhibition of cell growth and proliferation in the DCIS models.

overall_design: Four cell lines examined: normal mammary epithelial cell line (one sample) and three ductal carcinoma in situ cell lines (three samples). Each sample has two duplicates

first: Hitchintan

last: Kaur

first: Shihong

last: Mao

first: Quanwen

last: Li

first: Mansoureh

last: Sameni

first: Stephen

middle: A

last: Krawetz

first: Bonnie

middle: F

last: Sloane

first: Raymond

last: Mattingly


title: Virulence regulator EspR of Mycobacterium tuberculosis is a nucleoid-associated protein

city: Lausanne

first: Benjamin

last: Blasco

address: Station 19

department: SV-GHI

institute: EPFL

summary: The principal virulence determinant of Mycobacterium tuberculosis (Mtb), the ESX-1 protein secretion system, is positively controlled at the transcriptional level by EspR. Depletion of EspR reportedly affects a small number of genes, both positively or negatively, including a key ESX-1 component, the espACD operon.  EspR is also thought to be an ESX-1 substrate.  Using EspR-specific antibodies in ChIP-Seq experiments (chromatin immunoprecipitation followed by ultra-high throughput DNA sequencing) we show that EspR binds to at least 165 loci on the Mtb genome. Included in the EspR regulon are genes encoding not only EspA, but also EspR itself, the ESX-2 and ESX-5 systems, a host of diverse cell wall functions, such as production of the complex lipid PDIM (phenolthiocerol dimycocerosate) and the PE/PPE cell-surface proteins.  EspR binding sites are not restricted to promoter regions and can be clustered.  This suggests that rather than functioning as a classical regulatory protein EspR acts globally as a nucleoid-associated protein capable of long-range interactions consistent with a recently established structural model.  EspR expression was shown to be growth phase-dependent, peaking in the stationary phase.  Overexpression in Mtb strain H37Rv revealed that EspR influences target gene expression both positively or negatively leading to growth arrest.  At no stage was EspR secreted into the culture filtrate.  Thus, rather than serving as a specific activator of a virulence locus, EspR is a novel nucleoid-associated protein, with both architectural and regulatory roles, that impacts cell wall functions and pathogenesis through multiple genes.

overall_design: ChIP-Seq of EspR in Mtb H37Rv at mid-log phase of growth. Two independent experiments were performed. Input DNA (No IP) was used as a control.

first: Jeffrey

middle: M

last: Chen

first: Ruben

last: Hartkoorn

first: Claudia

last: Sala

first: Swapna

last: Uplekar

first: Jacques

last: Rougemont

first: Florence

last: Pojer

first: Stewart

last: Cole


title: Histone Phosphoacetylation ChIP-seq of Kc167 cells from Drosophila

city: Atlanta

first: Wendy

last: Kellner

state: GA

address: 1510 Clifton Road

institute: Emory University

summary: ChIP-seq was performed using Drosophila Kc167 cells using antibodies against H3K4me3 to identify active promoters and H3K4me1 to identify active enhancers. H3K27ac ChIPseq was performed to identify active promoters and enhancers. Once enhancers and promoters were identified, JIL-1 and histone phosphorylation, H3K9acS10ph and H3K27acS28ph, ChIP-seq was performed to look at binding trends. JIL-1 and phosphoacetlation is found at low levels at inactive enhancers and shows increase at active enhancers and promoters.

overall_design: Here we examine histone phosphorylation by JIL-1 and acetylation of H3K27ac by CBP at transcriptionally active vs. inactive promoters and enhancers. ChIP-seq is performed in Kc167 Drosophila cells using antibodies against JIL-1, H3K27acS28ph, H3K9acS10ph, H3K4me3, H3K4me1, and H3K27ac.

first: Victor

last: Corces

first: Edward

last: Ramos

first: Kevin

last: Van Bortle

first: Naomi

last: Takenaka


title: Combinatorial role of two paralogous splicing factors, hnRNP L and L-like, in multiple-exon regulation of CD45 alternative splicing

city: Giessen

first: Lee-Hsueh

last: Hung

state: Hessen

address: Heinrich-Buff-Ring 58

country: Germany

institute: University of Giessen

summary: The goal of this study was to investigate the role of hnRNP L-like in alternative pre-mRNA splicing in human B-cells through an RNA-Seq approach.

overall_design: RNA-Seq was performed in DG75 cell line with over expression of hnRNP L-like or GFP as control.

first: Albrecht

last: Bindereif


title: DCL4-Dependent Clusters of PhasiRNAs in Rice.

city: Beijing

first: xiaofeng

last: cao

address: West Lincui Road, Chaoyang District

department: State Key Laboratory of Plant Genomics

institute: Institute of Genetics and Developmental Biology

summary: The osdcl4-1 mutant exhibits much severer developmental defects than dcl4 in Arabidopsis, suggesting that Os DCL4 may process broader substrates in rice. By deep sequencing of small RNAs from different tissues of wild types and osdcl4-1, we revealed that 21-nucleotide siRNAs were largely dependent on Os DCL4. Besides several tasiRNA loci reported in Arabidopsis and rice, over one thousand 21-nucleotide and several dozen 24-nucleotide phased siRNA (phasiRNA) clusters were identified in panicles but not in seedlings and grains. Further analyses identified two conserved 22-nucleotide motifs among the cleavage sites of the 21- and 24- phasiRNA loci, and the cleavage sites of over 90% of 21- and 24-nucleotide phasing clusters were confirmed by PARE/degradome analysis from 93-11 panicles. MiR2118 and miR2275, expressed specifically in panicles, were predicted to trigger cleavages at 21- and 24-nucleotide phasiRNA clusters, respectively. The triggers of phasiRNAs are more dependent on Os DCL4 than Os DCL1. Furthermore, the processing of 21-nucleotide phasiRNAs was largely Os DCL4-dependent, whereas the processing of 24-nucleotide phasiRNAs was slightly affected by Os DCL4, but not by Os DCL3a and Os DCL1. Our results revealed distinct roles of Os DCL4 in a novel 21- and 24-nucleotide phasiRNA biogenesis pathway in rice.

overall_design: Six small RNA libraries were constructed from seedlings and panicles of 93-11 (a wild-type Indica rice variety) and osdcl4-1, as well as those from Nipponbare panicles and seedlings (a wild-type Japonica rice variety).

first: Pingchuan

last: Li

first: Xianwei

last: Song

first: Jixian

last: Zhai

first: Ming

last: Zhou

first: Lijia

last: Ma

first: Bin

last: Liu

first: Dong-Hoon

last: Jeong

first: Mayumi

last: Nakano

first: Shouyun

last: Cao

first: Chunyan

first: Chengcai

last: Chu

first: Xiu-jie

last: Wang

first: Pamela

middle: J

last: Green

first: Blake

middle: C

last: Meyers

first: Xiaofeng


title: Transcriptome mapping of blaCMY-2 positive IncA/C plasmid pAR060302

status: Public on Mar 28 2012

city: Saint Paul

first: Timothy

middle: J.

last: Johnson

state: MN

address: 1971 Commonwealth Ave

department: Veterinary Biosciences

institute: University of Minnesota

summary: The multidrug resistance-encoding plasmids belonging to the IncA/C incompatibility group have recently emerged among Escherichia coli and Salmonella enterica in the United States.  These plasmids have a unique genetic structure compared to other enterobacterial plasmid types, a broad host range, and propensity to acquire large numbers of antimicrobial resistance genes via their accessory regions.  Using E. coli strain DH5α harboring the prototype IncA/C plasmid pAR060302, we sought to define the baseline transcriptome of IncA/C plasmids under laboratory growth and in the face of selective pressure.  The effects of ampicillin, florfenicol or streptomycin exposure were compared to cells left untreated at logarithmic phase using Illumina sequencing (RNA-Seq).  Under growth in Luria-Bertani broth lacking antibiotics, much of the backbone of pAR060302 was transcriptionally inactive, including its putative transfer regions.  A few plasmid backbone genes of interest were highly transcribed, including genes of a putative toxin-antitoxin system and an H-NS-like transcriptional regulator.  In contrast, numerous genes within the accessory regions of pAR060302 were highly transcribed, including the resistance genes floR, blaCMY-2, aadA, and aacA.  Antibiotic treatment with ampicillin or streptomycin resulted in no genes being differentially expressed compared to controls lacking antibiotics, suggesting that many of the resistance-associated genes are not differentially expressed due to exposure to these antibiotics.  In contrast, florfenicol treatment resulted in the up-regulation of floR and numerous chromosomally encoded genes.  Overall, the transcriptome mapping of pAR060302 suggests that it mitigates the fitness costs of carrying resistance-associated genes through global regulation with its transcriptional regulators.

overall_design: Bacterial strains and growth conditions.  E. coli strain DH5α harboring pAR060302 was grown in 10 mL DifcoTM Luria-Bertani (LB) broth aliquots at 37º C with shaking until an OD600 of 0.5.  A total of 8 cultures were independently grown representing two biological replicates per condition tested.  Six of the cultures were amended, 2 cultures per antibiotic, with ampicillin (50 µg/mL final concentration), florfenicol (30 µg/mL final concentration), or streptomycin (50 µg/mL final concentration) and allowed to incubate at 37º C with shaking for an additional 30 min.  Two cultures were not amended with any antibiotic.  Cells were pelleted and RNA was purified using a commercially available RNA extraction kit (Qiagen).  RNA preparations were then subjected to a DNase treatment to eliminate DNA contamination from the sample (Qiagen).  A treatment was also included to deplete ribosomal RNA using a commercially available kit (MicrobExpress, Ambion).  The two biological replicates for each growth condition were pooled for sequencing.  Ilumina sequencing for transcriptome mapping.  cDNA libraries were generated with an insert size of 100 bp  and sequenced with 76-base cycles of single-end reads using a Genome Analyzer II (Illumina) platform according to manufacturer’s protocols at the Biomedical Genomics Center (University of Minnesota, Minneapolis, Minnesota, USA).  Approximately 160,000 plasmid-mapped reads each were obtained for the ampicillin and streptomycin treated samples, and 260,000 plasmid-mapped reads each for the control and florfenicol treatment samples.  Genome-mapped read counts were as follows:  control, ~6.4 million reads, florfenicol treatment, ~5.7 million reads, ampicillin treatment, ~1.7 million reads, and streptomycin treatment, ~5.2 million reads.   We only used those reads uniquely mapped on plasmid or chromosomal DNA for global normalization and further analysis.  RNAseq data analysis.  cDNA reads were trimmed so that the quality at each base position was above 30 (~15-20 bp) and then mapped either to the E. coli K-12 MG1655 published genome sequence (Genbank accession no. NC_000913) or to the pAR060302 published sequence (Genbank accession no. NC_092692) using BOWTIE.  The E. coli strain DH5α has an incomplete annotation and for this reason the E. coli K-12 annotation was used, representing an estimation of differentially expressed genes due to exposure of antimicrobials. The reads mapped per kilobase of gene per million (RPKM) reads was calculated using either the E. coli chromosome or the pAR060302 annotation and was used for global normalization.  The per kilobase cDNA length normalized the effect of different length of cDNAs such that the sequence reads have a equal chance to map on the long cDNA regions and the short cDNA regions.  After RPKM normalization, each sample is comparable to each other.  An R package, DEGseq, was used to identify differentially expressed genes between the control and each antibiotic treatment condition.  A cutoff of q-value < 0.05 and a fold change of > 3 were used to measure statistical significance.

first: Kevin

last: Lang

first: Jessica

last: Danzeisen

first: Wayne

last: Xu


title: Depletion of RUNX1/ETO in t(8;21) AML cells leads to genome-wide changes in chromatin structure and transcription factor binding (ChIP-seq)

city: Birmingham

first: Salam

middle: Adli

last: Assi

address: IBR

department: Institute for Cancer and Genomic Sciences

institute: University of Birmingham

summary: The t(8;21) translocation fuses the DNA binding domain of the hematopoietic master regulator RUNX1 to the ETO protein. The resultant RUNX1/ETO fusion protein is a leukemia-initiating transcription factor that interferes with RUNX1 function. The result of this interference is a block in differentiation and, finally, the development of acute myeloid leukemia (AML). To obtain insights into RUNX1/ETO-dependant alterations of the epigenetic landscape we measured genome-wide RUNX1- and RUNX1/ETO bound regions in t(8;21) cells and assessed to what extent the effects of RUNX1/ETO on the epigenome depend on its continued expression in established leukemic cells. To this end we determined dynamic alterations of histone acetylation, RNA Polymerase II binding and RUNX1 occupancy in the presence or absence of RUNX1/ETO using a knockdown approach. Combined global assessments of chromatin accessibility and kinetic gene expression data show that RUNX1/ETO controls the expression of important regulators of hematopoietic differentiation and self-renewal. We show that selective removal of RUNX1/ETO leads to a widespread reversal of epigenetic reprogramming and a genome-wide re-distribution of RUNX1 binding, resulting in the inhibition of leukemic proliferation and self-renewal and the induction of differentiation. This demonstrates that RUNX1/ETO represents a pivotal therapeutic target in AML.

overall_design: RUNX1 siMM and RUNX1 siRE ChIP-Seq samples and a paired-end ChIP-Seq samples from patients with t(8;21) AML are used in this study; there are two paired-end ChIP-Seq runs and control per patient

first: Anetta

last: Ptasinska

first: Deepak

last: Mannari

first: Sally

middle: R

last: James

first: Dan

last: Williamson

first: Jenny

last: Dunne

first: Maarten

last: Hoogenkamp

first: Wu

last: Mengchu

first: Matthew

last: Care

first: Hesta

last: McNeill

first: Pierre

last: Cauchy

last: Cullen

first: Reuben

last: Tooze

last: Tenen

first: Bryan

last: Young

first: Peter

middle: N

last: Cockerill

first: David

last: Westhead

first: Olaf

last: Heidenreich

first: Constanze

last: Bonifer


title: Sindbis virus induces the production of a novel class of endogenous siRNAs in Aedes aegypti mosquitoes

city: Blacksburg

first: Zach

middle: N.

last: Adelman

state: VA

address: 305 Fralin Life Science Institute

department: Entomology

institute: Virginia Tech

summary: We examined the biogenesis of mRNA-derived endogenous short-interfering RNAs (endo-siRNAs) in the disease vector mosquito, Aedes aegypti. Under standard conditions, mRNA-derived endo-siRNAs were produced from the bidirectional transcription of tail-tail overlapping gene pairs. Upon infection with the alphavirus, Sindbis virus (SINV), another class of mRNA-derived endo-siRNAs was observed. Genes producing SINV-induced endo-siRNAs were not enriched for overlapping partners or nearby genes, but were enriched for transcripts with long 3'UTRs. Endo-siRNAs from this class derived uniformly from the entire length of the target transcript, and were found to regulate the transcript levels of the genes from which they were derived. Strand-specific qPCR experiments demonstrated that antisense strands of targeted mRNA genes were produced to exonic, but not intronic regions. Finally, small RNAs mapped to both sense and antisense strands of exon-exon junctions, suggesting double-stranded RNA precursors to SINV-induced endo-siRNAs may be synthesized from mature mRNA templates. These results suggest additional complexity in small RNA pathways and gene regulation in the presence of an infecting virus in disease vector mosquitoes.

overall_design: Examination of endo-siRNA production in Aedes aegypti mosquitoes

first: Michelle

last: Anderson

first: Mingming

last: Liu

first: Liqing

last: Zhang

first: Kevin

last: Myles


title: Role of DNMT3B in the regulation of early neural and neural crest specifiers

city: Davis

first: Janine

last: LaSalle

address: Medical Microbiology and Immunology

institute: UC Davis

summary: The de novo DNA methyltransferase DNMT3B functions in establishing DNA methylation patterns during development. We performed RNAi knockdown of DNMT3B in human embryonic stem cells (ESCs) in order to investigate the mechanistic contribution of DNMT3B on DNA methylation and early neuronal differentiation.  Genome-wide analyses of DNA methylation by MethylC-seq identified novel regions of hypomethylation in the DNMT3B knockdowns along the X chromosome as well as pericentromeric regions, rather than changes to specific dysregulated gene promoters.  While DNMT3B was not required for early neuroepithelium specification, DNMT3B deficient neuroepithelium exhibited accelerated maturation with earlier expression of mature neuronal markers (such as NEUROD1) and early neuronal regional specifiers (such as neural crest) relative to normal ESCs. Our results suggest that DNMT3B mediates large-scale methylation patterns in human ESCs and that DNMT3B deficiency alters the timing of neuronal maturational differentiation in human neuronal cultures.

overall_design: Examined DNA methylation in human embryonic stem cells, both with and without DNMT3B knockdown

first: Diane

middle: I

last: Schroeder