title: Transcriptional regulation of germinal center B and plasma cell fates by dynamical control of IRF4

status: Public on May 04 2013

city: Chicago

first: Mark

last: Maienschein-Cline

state: IL

address: 900 S Ashland Ave, Rm 1058, M/C 937

department: Research Resources Center

institute: University of Illinois at Chicago

summary:
The transcription factor IRF4 regulates immunoglobulin class switch recombination and plasma cell differentiation. Its differing concentrations appear to regulate mutually antagonistic programs of B and plasma cell gene expression. We show IRF4 to be also required for generation of germinal center (GC) B cells. Its transient expression in vivo induced the expression of key GC genes including Bcl6 and Aicda. In contrast, sustained and higher concentrations of IRF4 promoted the generation of plasma cells while antagonizing the GC fate. IRF4 cobound with the transcription factors PU.1 or BATF to Ets or AP-1 composite motifs, associated with genes involved in B cell activation and the GC response. At higher concentrations, IRF4 binding shifted to interferon sequence response motifs; these enriched for genes involved in plasma cell differentiation. Our results support a model of "kinetic control" in which signaling-induced dynamics of IRF4 in activated B cells control their cell-fate outcomes.

Regrettably three of the FASTQ raw sequence files in our study were corrupted during storage. FASTQ data from our experimental and control groups are available for download via GEO SRA; however, two groups are missing select raw sequence files. These include one PU.1 Day 3 group file (Sample GSM1133499) and two of four input files used to generate a concatenated “super” input file (Sample GSM1133490); the raw data provided for input consists of the two input files recovered. Importantly, FASTA sequences for both of these datasets are available as supplementary data through GEO, and we can make available upon request (rsciamma@uchicago.edu) all files in our study in the ELAND-extended alignment format. Please note that GEO no longer supports this format.

overall_design:
ChIP was performed by using anti-IRF4, -PU.1, and control IgG antibodies (Santa Cruz Biotech. and Abcam) (Sciammas et al., 2006). For massively parallel sequencing, 200 μg of chromatin fragments were immunoprecipitated by using anti-IRF-4 and anti-PU.1 antibodies, and DNA libraries were prepared. DNA was sequenced by using the Illumina GA2, data was processed with the Solexa pipeline package, and sequences were aligned to the mouse genome (mm9) by using ELAND software.

For transcriptome analysis, total RNA was prepared from triplicate cell samples by using Trizol and processed for hybridization to Affymetrix mouse 430A chips by using standard procedures. For qRT-PCR analysis of sorted SWHEL B cells, total RNA was prepared by sorting 5,000 cells directly into RLT buffer from the RNeasy Micro Kit (QIAGEN).

More details are provided in the supplementary information (web link) and in the manuscript.

first: K

last: Ochiai

last: Simonetti

first: J

last: Chen

first: R

last: Rosenthal

last: Brink

first: A

last: Chong

first: U

last: Klein

last: Dinner

first: H

last: Singh

last: Sciammas


title: Transcriptional regulation of germinal center B and plasma cell fates by dynamical control of IRF4 (ChIP-seq)

country: USA

type: Genome binding/occupancy profiling by high throughput sequencing

summary:
Temporal analysis of Irf4 and PU.1 genome binding during B cell activation and differentiation in vitro using antigen (NP-Ficoll) CD40L and IL-2/4/5 cytokines (see Molecular Systems Biology 7:495 for details of cellular system). The results provide insight in the target genes and binding specificity of IRF4 and PU.1 during coordination of different programs of B cell differentiation.

Regrettably three of the FASTQ raw sequence files in our study were corrupted during storage. FASTQ data from our experimental and control groups are available for download via GEO SRA; however, two groups are missing select raw sequence files. These include one PU.1 Day 3 group file (Sample GSM1133499) and two of four input files used to generate a concatenated “super” input file (Sample GSM1133490); the raw data provided for input consists of the two input files recovered. Importantly, FASTA sequences for both of these datasets are available as supplementary data through GEO, and we can make available upon request (rsciamma@uchicago.edu) all files in our study in the ELAND-extended alignment format. Please note that GEO no longer supports this format.

overall_design: Resting mature peripheral primary B cells were enriched from the spleens of B1-8i (anti-NP gene targeted) mice. We sought to compare the genome-binding landscape of Irf4 and PU.1 prior to differentiation yet after B cell activation (Day 1) and after B cell differentiation (Day 3) of activated B cells into plasma cells (see Molecular Systems Biology 7:495 for description of cellular system). To this end, we used ChIP-seq (using the Illumina GA2 system) to obtain millions of unbiased, genome-wide, binding events. Sequences were mapped to the reference genome (mm9) and enrichment was calculated, relative to an Input sample, using QuEST algorithms.

first: Roger

first: Kyoko

first: Harinder

first: Aaron


title: Genome-scale methylome profiling of human pheochromocytomas and paragangliomas (Illumina methylation beadchips) and mouse chromaffin cells (RRBS)

status: Public on May 06 2013

city: Paris

first: Eric

last: Letouzé

address: 15 rue de l'école de médecine

country: France

institute: Ligue Contre Le Cancer

type: Methylation profiling by high throughput sequencing

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: A high dimensional deep sequencing study of non-small cell lung adenocarcinoma in never-smoker Korean females [Seq]

status: Public on May 10 2013

city: Daejeon

first: Sanghyuk

last: Lee

address: 125 Gwahak-ro

department: KOBIC

country: South Korea

institute: KRIBB

type: Expression profiling by high throughput sequencing

type: Non-coding RNA profiling by high throughput sequencing

summary: One of the most fertile applications of next generation sequencing will be in the field of cancer genomics. Here, we report a high-throughput multi-dimensional sequencing study of primary non-small cell lung adenocarcinoma tumors and adjacent normal tissues of 6 never-smoker Korean female patients. Our data encompass results from exome-seq, RNA-seq, small RNA-seq, and MeDIP-seq. We identified and validated novel genetic aberrations including 47 somatic mutations and 20 fusion transcripts. We also characterized gene expression profiles which we sought to integrate with genomic aberrations and epigenetic regulations into functional networks. Importantly, among others the gene network module governing G2/M cell check point emerged as the primary source of disturbance in these patients. In addition, our study strongly suggests that microRNAs make key regulatory inputs into this gene network module. Our study offers a paradigm for integrative genomics analysis and proposes potential target pathways for the control of non-small cell lung adenocarcinoma.

overall_design: Study of primary non-small cell lung adenocarcinoma tumors and normal tissues of 6 patients.

first: Jhingook

last: Kim

first: Jaesang

first: Jong-Eun


title: Genome-wide maps of CENP-A nucleosomes in three neocentromere-containing cell lines

city: Philadelphia

first: Ben

last: Black

address: 422 Curie Blvd.

institute: University of Pennsylvania

summary: We used native ChIP-seq of CENP-A-containing particles from normal centromeres on alpha-satellite DNA and three naturally-occurring neocentromeres to test the proposed models for the major form of the fundamental repeating unit of centromeric chromatin.  We found that the predominant form of the CENP-A particle at the centromere is an octameric nucleosome with loose terminal DNA.  Additionally, we found CENP-A nucleosomes are strongly phased on the 171 bp alpha-satellite monomers of normal centromeres, and also display strong positioning and neocentromeres.

overall_design: Comparison of CENP-A and bulk nucleosome DNA lengths and positions in three different human neocentromere-containing cell lines


title: Identification and characterization of microRNAs in pepper from fruit sample (3rd stage) by high-throughput sequencing

status: Public on May 03 2013

city: SEOUL

first: Chanseok

last: Shin

address: Gwanak-gu Daehak-dong

institute: Seoul National University

summary: High-throughput sequencing was employed to identify microRNAs in pepper from fruit sample (3rd stage)

overall_design: 1 sample examined

first: DongGyu

last: Hwang

first: JuneHyun

last: Park

first: JaeYun

last: Lim


title: A receptor tyrosine kinase network comprised of FGFRs, EGFR, ERBB2, and MET drives growth and survival of HNSCC cell lines.

city: Cleveland

first: Jihye

state: Ohio

address: 9500 Euclid Ave.

department: Quantitative Health Sciences

institute: Cleveland Clinic

summary: We have previously shown that some gefitinib insensitive head and neck squamous cell carcinoma (HNSCC) cell lines exhibit dominant autocrine fibroblast growth factor receptor (FGFR) signaling. Herein, we deployed a whole genome loss-of-function screen to identify genes whose knockdown potentiated the inhibitory effect of the FGFR inhibitor, AZ12908010, in HNSCC cell lines. Three HNSCC cell lines expressing a genome-wide shRNA library were treated with AZ8010 and the abundance of shRNA sequences was assessed by deep sequencing. Synthetic lethal hits were validated through use of specific inhibitors and independent shRNAs. We found that multiple alternate receptors provided protection from FGFR inhibition, including the receptor tyrosine kinases (RTKs), epidermal growth factor receptor 2 (ERBB2) and hepatocyte growth factor receptor (MET).  We showed that specific knockdown of either ERBB2 or MET in combination with FGFR inhibition led to increased inhibition of growth relative to FGFR tyrosine kinase inhibitor (TKI) treatment alone. These results were confirmed using specific small molecule inhibitors of either ERBB family members or MET. Moreover, the combination of FGFR, MET and ERBB family inhibitors showed the largest inhibition of growth as compared to the double combinations. These results reveal a role for alternate RTKs in maintaining pro-growth and survival signaling in HNSCC cells in the setting of FGFR inhibition. Thus, improved therapies for HNSCC patients could involve rationally designed combinations of TKIs targeting FGFR, ERBB family members and MET.

overall_design: Using a genome-wide shRNA library in combination with deep sequencing, we screened for gene targets that were synthetic lethal with the FGFR inhibitor, AZ12908010 in HNSCC cells. Three HNSCC cell lines were screened in triplicate and the abundance of shRNA sequences in drug treated cells was compared to control treated cells.

first: Katherine

middle: R

last: Singleton

first: Trista

middle: K

last: Hinz

first: Lindsay

middle: A

last: Marek

first: Matias

last: Casas-Selves

first: Clark

last: Hatheway

first: Aik

middle: C

last: Tan

first: James

last: DeGregori

first: Lynn

middle: E

last: Heasley


title: Zinc deficiency impacts CO2 assimilation and disrupts copper homeostasis in Chlamydomonas reinhardtii

city: Los Angeles

first: Sabeeha

last: Merchant

state: California

address: 607 Charles E. Young Drive East

department: Department of Chemistry and Biochemistry

institute: University of California Los Angeles

summary: Zinc is an essential nutrient because of its role in catalysis and in stabilizing protein structure, but excess zinc can also be deleterious. Four nutritional zinc states have been identified in the alga Chlamydomonas reinhardtii: zinc toxic, zinc replete, zinc deficient and zinc limited. Growth is inhibited in zinc-limited and zinc toxic cells relative to zinc-replete cells, while zinc-deficiency is visually asymptomatic but distinguished by the accumulation of transcripts encoding ZIP family transporters. To identify targets of zinc deficiency and mechanisms of zinc acclimation, we used RNA-seq to probe zinc nutrition responsive changes in gene expression. We identified a subset of genes encoding zinc-handling components, including ZIP family transporters and candidate zinc chaperones. In addition, we noted an impact on two other regulatory pathways, the carbon concentrating mechanism (CCM) and the nutritional copper regulon. Targets of transcription factor Ccm1 and various CAH genes are up-regulated in zinc-deficiency, as a likely consequence of reduced carbonic anhydrase activity, which is validated by mass spectrometry and immunoblot analysis of Cah1, Cah3 and Cah4. Chlamydomonas is therefore not able to grow photoautotrophically in air in zinc limiting conditions, but supplementation with 1% CO2 restores growth to wild-type rates, suggesting that the inability to maintain CCM is a major consequence of zinc limitation.  Surprisingly, we noted also that the Crr1 regulon, which responds to Cu limitation, is also turned on in zinc deficiency, and in fact, Crr1 is required for growth in zinc-limiting conditions. Zinc deficient cells are functionally copper deficient, as evidenced by reduced plastocyanin abundance, even though they hyperaccumulate copper up to 50-fold over normal levels. We suggest that zinc-deficient cells sequester Cu in a bio-unavailable form, perhaps to prevent mis-metallation of critical zinc sites.

overall_design:
Zn-limited wild-type cells were generated by transfer of cells from the first round of growth in medium with no supplemental Zn into TAP medium supplemented or not with 2.5 µM Zn-EDTA
The control samples for this study are represented in GSE25622

first: Davin

last: Malasarn

first: Janette

last: Kropat

first: Scott

middle: I

last: Hsieh

first: Giovanni

last: Finazzi

first: David

last: Casero

first: M

middle: D

last: Page

first: Joseph

last: Loo

first: Matteo

last: Pellegrini

first: Francis-André

last: Wollman

middle: S


title: Genome-wide mapping of Mash1 from mouse spinal cord

city: Singapore

first: Wenjie

last: SUN

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

country: Singapore

institute: Genome Institute of Singapore

summary: We performed ChIP-seq on a complex developing tissue (the spinal cord) at mouse embryonic day 12.5. Genome-wide binding sites of Mash1 were generated.

overall_design: ChIP-seq of Mash1/Ascl1 in a heterogeneous tissue.

first: Diogo

last: Castro

first: Daniela

last: Drechsel

first: François

last: Guillemot


title: Methanopyrus kandleri small RNA analysis

city: Marburg

first: Lennart

last: Randau

address: Karl-von-Frisch Strasse 10

country: Germany

institute: MPI for terrestrial microbiology

summary: We report the profiling of small RNAs from Methanopyrus kandleri by high throughput sequencing. Over 83 million Illumina Hi Seq2000 reads were obtained for six independent RNA libraries. The reads were mapped to the M. kandleri AV19 genome (Genbank: NC_003551, 1694969 bp). The small RNome of M. kandleri was analyzed.

overall_design: Analysis of small RNome from six Methanopyrus kandleri RNA samples

first: Randau

last: Lennart


title: Comparing proteomics and RISC immunoprecipitations to identify targets of Epstein-Barr viral miRNAs [RISC-IP-seq]

status: Public on May 01 2013

city: Madison

first: Bill

last: Sugden

state: WI

address: 1111 Highland Ave

institute: University of Wisconsin-Madison

summary: Epstein-Barr virus is a gamma-herpes virus that is causally associated with several lymphomas and carcinomas. This virus encodes at least 25 pre-miRNAs, which are expressed in infected cells to yield more than 50 detected mature miRNAs. miRNAs are small, non-coding RNAs that inhibit gene expression by promoting the inhibition of translation or of degradation of mRNAs. Currently, the function of these viral miRNAs and the contribution they provide to EBV's life-cycle remain largely unknown, due to difficulties in identifying cellular and viral genes regulated by these miRNAs. We have compared and contrasted two methods to identify targets of viral miRNAs in order to identify the advantages and limitations of each method to aid in uncovering the functions of EBV's miRNAs.

overall_design: Examination of RISC (RNA Induced Silencing Complexes) associated transcripts under 2 conditions in BJAB cells

first: Malika

last: Kuzembayeva

first: Ya-Fang

last: Chiu


title: Global spatial organization of γ proteobacterial nucleoids is determined by specific long-range interactions (E. coli)

status: Public on May 02 2013

city: Auckland

first: Justin

middle: M.

last: O'Sullivan

address: 85 Park Road, Grafton

department: Liggins Institute

country: New Zealand

institute: University of Auckland

summary:
GCC was used to determine the structure of E. coli grown in LB or treated with SHX.

The bacterial genome is highly condensed into a nucleoid structure. Here we present global analyses of the genome spatial organization for two γ-proteobacteria: Escherichia coli and Pseudomonas aeruginosa by Genome Conformation Capture. Long distance interactions occurred within the E. coli and P. aeruginosa nucleoids with frequencies that were affected by growth condition and gene dosage. Spatial clustering of genes that are either up or down-regulated depended on the environmental signals, indicating a non-random functional organization of the nucleoid. The largest changes in gene expression upon amino acid starvation occurred in genes that participate in long-range interactions. These genes remained highly spatially clustered when transcript levels decreased. Environment specific interactions were related to DNA motifs but did not correlate with binding sites for nucleoid associated proteins. Overall we identify spatial organization as a significant factor in bacterial gene regulation and suggest that the prokaryotic operon is not simply a linear entity.

overall_design:
4 samples grown in LB; 4 samples treated with SHX.

For Genome Conformation Capture (GCC) analyses, E. coli strains were recovered from -80°C on LB agar (2%) plates for 24 hours. LB medium (3ml) starter cultures were inoculated and grown (37°C, 220rpm, 16h). The Optical Density (OD600) of cultures was measured and used to inoculate LB test cultures to an OD600 of ~0.02. The test cultures were grown (37°C, 220rpm) until the OD600 reached ~0.25 and then harvested. For the serine hydroximate (SHX) treated samples the cultures were treated with SHX (500 µg/ml, 30 min) before harvesting.

E. coli chromatin was prepared according to Rodley et al. (2009)with the following modifications. A total of 5*109 cells were cross-linked with formaldehyde (1% final v/v, 20min, RT) and then quenched with glycine (125mM final, 10min). Cells were collected by centrifugation (4000rpm, 15min, 4°C), washed twice (1% PBS, 1% TritonX-100, 5ml/50ml culture) and pelleted (4000rpm, 15min, 4°C). Cell pellets were suspended in 800μl of B1 lysis buffer (10mM Tris pH 8.0, 50mM NaCl, 10mM EDTA, 20% (w/v) sucrose, 1mg/ml lysozyme) and incubated (37°C, 30min). 800μl of B2 lysis buffer (200mM Tris pH 8.0, 600mM NaCl, 4% TritonX-100, 1 protease inhibitor tablet (Roche) per 10ml of buffer added just before use) was gently added, mixed by inversion 3-4 times and incubated (37°C, 10min). The cell lysate was centrifuged (21,500g, 20min, 4°C) and the supernatant decanted. The chromatin was washed once with 1ml of chromatin digestion buffer (10mM Tris-HCl pH 8.0, 5mM MgCl2, 0.1% TritonX-100) by inverting the tube 3-4 times and centrifuged (21,500g, 20min, 4°C). The supernatant was decanted and the chromatin pellet was suspended in 500μl chromatin digestion buffer. Chromatin samples were aliquoted into 10 sets of 5*108 cells. Samples were digested with HhaI (100U, New England Biolabs). Following digestion a ligation control was added and the samples were ligated with T4 DNA ligase (20U, Invitrogen). Following ligation, cross-links were removed in the presence of proteinase K (0.45U, Fermentas). RNA was removed and pUC19 plasmid (27.4pg/2ml) was added as a sequencing control prior to three extractions with 1:1 Phenol:Chloroform. DNA was column purified (Zymo, DNA clean and concentratorTM-5 kit) according to the manufacturer’s instructions and eluted in milliQ H2O before combining for sequencing. 3μg of purified DNA was sent for pared-end sequencing at the ATC sequencing facility (Rockville, MD, USA) on an Illumina Hi-Seq.

External ligation controls were produced by PCR amplification of short regions from the Lambda phage genome and the pRS426. Primers (Table S4 were designed to include an HhaI site at one end of the final product. PCR products were purified using a PCR purification kit (Qiagen), digested with HhaI (4U, 37°C, 2h) and purified again. Purified, digested PCR products were introduced into the GCC samples at a 1:1 ratio with the number of genomes prior to the ligation step during GCC preparation. The pRS426 fragment was introduced into the exponential phase (LB grown) samples and resulted in 220 separate ligation events with HhaI restriction fragments on the genome. The Lambda phage fragment was introduced into the SHX treated samples and resulted in 2 ligation events with HhaI fragments on the genome.

Network assembly was performed using the Topography suite v1.19. GCC networks were constructed from 100bp (E. coli) paired end Illumina Genome Analyser sequence reads. Topography uses the SOAP algorithm to position PE tags and single ends which contain a HhaI (E. coli) restriction site onto the E. coli (NC_000913) reference genomes, respectively. Each reference genome also contained the pUC19 (SYNPUC19CV) sequence. For the E. coli alignment the sequences of the pRS426 plasmid and Lambda phage ligation controls were also included in the file to which the sequences were aligned. No mismatches or unassigned bases (N) were allowed during positioning.

Except where indicated, bioinformatics and statistical analyses were performed on interactions in which the sequence reads were able to be mapped uniquely onto the reference genome and were above the FDR cut-off value (5). All bioinformatics analysis was perfumed using in house Perl scripts.

first: C

last: Cagliero

first: J

middle: M


title: Genome-scale methylome analysis of the desert locust, Schistocerca gregaria

city: Heidelberg

first: Guenter

last: Raddatz

address: Im Neuenheimer Feld 580

institute: German Center for Cancer Research

summary: DNA methylation is a widely conserved epigenetic modification that is established and maintained by the cooperative activity of DNA methyltransferases. While the complement of DNA methyltransferase genes can vary substantially between animal species, whole-genome methylation analyses have suggested that major features of animal methylomes are widely conserved. We have now used genome-scale bisulfite sequencing to analyze the methylome of the desert locust, Schistocerca gregaria, which represents an economically important pest with a high degree of phenotypic plasticity. Interestingly, in this system, DNA methylation appears to be both established and maintained by Dnmt1 methyltransferases, which distinguishes locusts from most other known organisms. Our results indicate that the S. gregaria methylome shares preferential methylation of CpG dinucleotides and exons with other animal methylomes. In contrast to other invertebrates, however, overall methylation levels were substantially higher and a significant fraction of transposons was methylated. Additionally, genes were densely methylated in a pronounced bimodal pattern, suggesting a role for DNA methylation in the regulation of locust gene expression. Altogether, our results uncover a unique pattern of genome methylation in locusts and also suggest that animal methylomes may be more diverse than previously thought.

overall_design: Whole exome methylation analysis of S. gregaria. Two samples were analyzed, one sample containing DNA from brain, one sample containing DNA from MTG.  To date, there exists no sequenced genome of Schistocerca gregaria; thus,  we could only map the data against an EST database (Locust2 EST project) representing the coding part of the genome.

first: F

last: Lyko

first: L

last: Schoofs


title: Holo-TFIID controls the magnitude of a transcription burst and fine-tuning of transcription.

city: Waltham

first: Michael

last: Marr

state: Massachusetts

address: 415 South St

department: Biology

institute: Brandeis University

summary: TFIID is a central player in activated transcription initiation. Recent evidence suggests that the role and composition of TFIID is more diverse than previously understood. To investigate the effects of changing the composition of TFIID in a simple system we depleted TAF1 from Drosophila cells and determined the consequences on metal induced transcription at an inducible gene, Metallothionein B (MtnB). We observe a marked increase in the levels of both the mature message and pre-mRNA in TAF1 depleted cells. Under conditions of continued metal exposure, we show that TAF1 depletion increases the magnitude of the initial transcription burst, but has no effect on the timing of that burst. We also show that TAF1 depletion causes delay in the shut-off of transcription upon removal of the stimulus. Thus TAFs are involved in both establishing an upper limit of transcription during induction and efficiently turning the gene off once the inducer is removed. Using genomewide nascent-seq we identify hundreds of genes that are controlled in a similar manner indicating that the findings at this inducible gene are likely generalizable to a large set of promoters. There is a long-standing appreciation for the importance of the spatial and temporal control of transcription. Here we uncover an important third dimension of control, the magnitude of the response. Our results show that the magnitude of the transcriptional response to the same signaling event, even at the same promoter, can vary greatly depending on the composition of the TFIID complex in the cell.

overall_design: Nascent RNA was sequenced from replicate samples of Drosophila S2 cells treated with double-stranded RNA directed against E. coli LacI (Control) or against Drosophlia TAF1 (experimental). Reads per kilo-base per million (RPKM) was determined for each gene and the control and experimental samples were compared to determine the genes that were affected by the depletion of TAF1.

first: Sharon

first: Katie

last: Pennington

first: Gung-wei

last: Chirn


title: Buccals are likely to be a more informative surrogate tissue than blood for epigenome-wide association studies [Bisulfite-Seq]

city: London

first: Robert

last: Lowe

address: 4 Newark Street

department: Blizard Institute

country: United Kingdom

institute: QMUL

summary: There is increasing evidence that interindividual epigenetic variation is an etiological factor in common human diseases. Such epigenetic variation could be genetic or non-genetic in origin, and epigenome-wide association studies (EWASs) are underway for a wide variety of diseases/phenotypes. However, performing an EWAS is associated with a range of issues not typically encountered in genome-wide association studies (GWASs), such as the tissue to be analyzed. In many EWASs, it is not possible to analyze the target tissue in large numbers of live humans, and consequently surrogate tissues are employed, most commonly blood. But there is as yet no evidence demonstrating that blood is more informative than buccal cells, the other easily accessible tissue. To assess the potential of buccal cells for use in EWASs, we performed a comprehensive analysis of a buccal cell methylome using whole-genome bisulfite sequencing. Strikingly, a buccal vs. blood comparison reveals >6X as many hypomethylated regions in buccal. These tissue-specific differentially methylated regions (tDMRs) are strongly enriched for DNaseI hotspots. Almost 75% of these tDMRs are not captured by commonly used DNA methylome profiling platforms such as Reduced Representational Bisulfite Sequencing and the Illumina Infinium HumanMethylation450 BeadChip, and they also display distinct genomic properties. Buccal hypo-tDMRs show a statistically significant enrichment near SNPs associated to disease identified through GWASs. Finally, we find that, compared with blood, buccal hypo-tDMRs show significantly greater overlap with hypomethylated regions in other tissues. We propose that for non-blood based diseases/phenotypes, buccal will be a more informative tissue for EWASs.

overall_design: Buccal Profile generated from 14 Buccal Individuals


title: Comparison of the nucleotide-resolution, genome-wide, 5'-end maps of the transcriptomes of Escherichia coli K12 strain BW25113 and Streptomyces coelicolor A3(2) strain M145

city: Leeds

first: David

last: Romero A.

state: West Yorkshire

address: Woodhouse lane

institute: University of Leeds

summary: The differential RNA-seq data contained within this entry is complemented by global RNA-seq.

sample_organism:
Streptomyces coelicolor A3(2)
Escherichia coli BW25113

overall_design: S. coelicolor and E. coli reference strains were grown in batch culture with shaking. The E. coli sample was extracted during mid-exponential growth, while the S. coelicolor sample was extracted at the start of pigmented antibiotic production. Each of the two samples was incubated with or without TAP (tobacco acid pyrophosphatase) before library construction. Thus, 4 libraries were analysed. TAP treatment allows the cloning and sequencing of 5' ends that were originally triphosphorylated.

last: Romero

first: Yu-fei

last: Lin

first: Kenneth

middle: J

last: McDowall


title: Integrative genomic analysis of CREB defines a critical role for transcription factor networks in mediating the fed/fasted switch in liver

city: RTP

first: Logan

last: Everett

state: North Carolina

address: 109 T.W. Alexander Dr

department: Office of Research and Development

institute: U.S. Environmental Protection Agency


title: Trancriptional landscape of Aspergillus niger at breaking of conidial dormancy revealed by RNA-sequencing

city: Nottingham

last: Archer

address: University Park

department: School of Biology

institute: Nottingham University

summary: Genome-wide analysis was performed to assess the transcriptional landscape of germinating A. niger conidia for first hour using next generation RNA-sequencing. The transcriptome of dormant conidia was shown to be highly differentiated from that of germinating conidia. The breaking of dormancy was associated with increased transcript levels of genes involved in the biosynthesis of proteins, RNA turnover and respiratory metabolism. Increased transcript levels of genes involved in metabolism of nitrate and proline at the onset of germination implies their use as sources of nitrogen. The transcriptome of dormant conidia contained a significant component of antisense transcripts that changed during germination. Dormant conidia contained transcripts of genes involved in fermentation, gluconeogenesis and the glyoxylate cycle. The presence of such transcripts in dormant conidia may indicate the generation of energy from non-carbohydrate substrates during starvation-induced conidiation or for maintenance purposes during dormancy. The immediate onset of metabolism of internal storage compounds after the onset of germination, and the presence of transcripts of relevant genes, suggest that conidia are primed for the onset of germination. For some genes, antisense transcription is regulated in the transition from resting conidia to fully active germinants.

overall_design: Duplicate samples from each time point: dormant conidia T0 (DA 21 + DA 22) and conidia germinated for one hour T1 (DA 23 + DA 24)

first: Michaela

last: Novodvorska

first: Kimran

last: Hayer

first: Steven

middle: T

last: Pullan

first: Raymond

last: Wilson

first: Martin

last: Blythe

first: Hein

last: Stam

first: Malcolm

last: Stratford

middle: B


title: High-throughput sequencing of methylated cytosine enriched by modification-dependent restriction endonuclease MspJI

city: Shenzhen

first: Desheng

last: Gong

address: No.7 PengFei road

country: China

institute: Agricultural Genomes Institute at Shenzhen

summary: In this study, we combine MspJI digestion and electrophoretic band selection with next generation high-throughput sequencing technology to detect 5-methylcytosines in Arabidopsis genome. By developing a bioinformatics workflow to attribute the CNNR sites recognized by MspJI to the reference genome, we fulfilled the systematic assessment of this method. According to the assessment, here we provide the method for generating a detailed map of plant methylome that could be feasible, reliable and economical in methylation investigation.

overall_design:
Extracting the MspJI digested fragments, constructing sequencing library according to the Illumina protocol and sequencing with Illumina HiSeq2000. Repeatability and reproducibility studies were performed between two samples from the same individual. Specificity and sensitivity of the method was examined by comparing our data with WGBS data downloaded from GEO (GSE15922: GSM399600).

**The WGBS seq data ('aerial_tissues_BS_seq_CNNR.gff') was generated from the GSM399600 Sample supplementary files ('aerial_tissues_BS_seq_alignment_batch-*.gff.gz') and was utilized to do comparative study with our MspJI-seq data  including sensitivity and specificity analysis.

first: Xiaojun

last: Huang

first: Hanlin

last: Lu


title: Single-cell DNA methylation analysis of epimutations in mouse liver cells.

city: Cologne

first: Kemal

last: Akman

address: Carl-von-Linné-Weg 10

department: Plant Breeding Research

institute: Max Planck Institute Cologne

summary: We report a single-cell DNA methylation analysis using BS-Seq, which reveals enrichment of demethylating epimutations in gene bodies and repeat regions.

overall_design: Comparison of single cells vs. a reference cell pool from Mouse liver tissue using an Illumina HiSeq machine.

first: Silvia

last: Gravina


title: Integrative genomic analysis of CREB defines a critical role for transcription factor networks in mediating the fed/fasted switch in liver [ChIP-seq]

summary: Metabolic homeostasis in mammals critically depends on the regulation of fasting-induced genes by CREB in the liver. Previous genome-wide analysis has shown that only a small percentage of CREB target genes are induced in response to fasting-associated signaling pathways. The precise molecular mechanisms by which CREB specifically targets these genes in response to alternating hormonal cues remain to be elucidated. We performed chromatin immunoprecipitation coupled to high-throughput sequencing of CREB in livers from both fasted and re-fed mice. In order to quantitatively compare the extent of CREB-DNA interactions genome-wide between these two physiological conditions we developed a novel, robust analysis method, termed the ‘single sample independence’ (SSI) test that greatly reduced the number of false positive peaks. We found that CREB remains constitutively bound to its target genes in the liver regardless of the metabolic state.  Integration of the CREB cistrome with expression microarrays of fasted and re-fed mouse livers and ChIP-seq data for additional transcription factors revealed that the gene expression switches between the fasted and fed states are associated with co-localization of additional transcription factors at CREB sites. Our results support a model in which CREB is constitutively bound to thousands of potential target genes and combinatorial interactions between DNA-binding factors are necessary to achieve the specific transcriptional response of the liver to fasting. Furthermore, our genome-wide analysis identifies thousands of novel CREB target genes in liver, including a previously unknown role for CREB in regulating ER stress genes in response to nutrient influx.

overall_design: CREB ChIP-seq was performed on mouse liver from fasted and re-fed mice, using 5 separate biological replicates for each condition. GR and C/EBP(beta) ChIP-seq were performed as single replicates on ad-lib fed mice.

first: John

last: Le Lay

first: Sabina

last: Lukovac

first: Diana

last: Bernstein

last: Steger

first: Mitchell

last: Lazar

first: Klaus

middle: H

last: Kaestner


title: Transcriptome sequencing of Cleome hassleriana for different tissues

first: Zhijun

last: Zheng

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

institute: BGI

summary: We have sequenced the whole genome of Cleome hassleriana by NGS [SRA accession number SRA058749] and build a reference sequence for this genome. In order to improve quality of gene models, a mix transcriptome sample extracted from the bud, leaf, petiole, stems and flowers of Cleome.

overall_design: A mixed RNA pool extracted from the tissues of bud, leaf, petiole, stems and flower is sequenced for Cleome hassleriana.

first: Shifeng

last: Cheng

first: Zhiwu

last: Quan


title: HL-60 treated with TPA

status: Public on May 05 2013

city: Nanjing

first: zhu

last: dihan

address: Hankou 22

institute: Nanjing university

summary: Comparing the difference between TPA stimulated HL-60 and control

overall_design: HL-60 stimulated by TPA for 12h


title: Imprinted expression of genes and small RNA is triggered by localized demethylation of the maternal genome in rice endosperm

city: Berkeley

first: Toshiro

last: Nishimura

state: CA

address: 211 Koshland Hall

department: Plant and Microbial Biology

institute: University of California at Berkeley

summary: We report that rice endosperm shows a specific hypomethylation of DNA in the maternal genome, preferring regions of high DNA accessibility. Maternally expressed imprinted genes are enriched for hypomethylation at putative promoter regions and transcriptional termini, and paternally expressed genes at promoters and gene bodies, mirroring our recent results in A. thaliana. However, unlike in A. thaliana, rice endosperm sRNA populations are dominated by specific strong sRNA-producing loci, and imprinted 24-nt sRNAs are expressed from both parental genomes and correlate with hypomethylation. Overlaps between imprinted sRNA loci and imprinted genes expressed from opposite alleles suggest that sRNAs may regulate genomic imprinting. Whereas sRNAs in seedling tissues primarily originate from small Class II (cut and paste) transposable elements, those in endosperm are much more uniformly derived, including sequences from other TE classes, as well as genic and intergenic regions. Our data indicate that the endosperm exhibits a unique pattern of sRNA expression and suggest that demethylation of maternal endosperm DNA is conserved in flowering plants.

overall_design: Examination of DNA methylation and small RNA expression in the seeds of two cultivars from the japonica subspecies of Oryza sativa L.

first: Jessica

last: Rodrigues

first: Randy

last: Ruan

first: Pamela

last: Ronald

first: Robert

last: Fischer

first: Daniel

last: Zilberman


title: Small RNA analysis of ADAR1-knock down HeLa cells by RNAi

city: Philadelphia

first: Kazuko

last: Nishikura

state: Pennsylvania

address: 3601 Spruce Street

department: Gene Expression & Regulation

institute: The Wistar Institute

summary: Small RNA expression was analysed in total RNA of HeLa cells treated with siRNA toward Luciferase (negative cotrol) or ADAR1.

overall_design: Small RNA: 2 samples examined: HeLa cell with siLuc (negative cotrol), with siADAR1

first: Masayuki

last: Sakurai

first: Ravi

last: Gupta

first: Ramana

middle: V

last: Davuluri


title: Distribution of Drosophila insulator proteins DREF in Drosophila Kc167 cells

city: Atlanta

first: Gurudatta

last: Baraka Vishwanathan

state: GA

address: 1510 clifton road ne

department: Biology Department

institute: Emory University

summary: Here we map the localization of DREF Drosophila replication related element binding factor in Drosophila Kc cells.

overall_design: Examination of genomic occupancy for DREF, control samples used for Drosophila Kc were previously described (Wood, Van Bortle et al., 2011 GSE30740 and GSE32584). The GSM number for the input for Total Chip_seq data is GSM762849.

first: Victor

last: Corces

last: Vishwanathan


title: Next-generation sequencing and microarray-based interrogation of microRNAs from formalin-fixed, paraffin-embedded tissue: Assessment of cross-platform concordance.

city: Boston

first: Katherine

last: Hill

state: MA

address: 330 Brookline Avenue, DA-679

department: Medicine

institute: Beth Israel Deaconess Medical Center

summary: We examine the concordance between miRNA-seq and cDNA-mediated annealing, selection, extension, and ligation (DASL) miRNA assays.

overall_design:
We profiled two technically replicated FFPE specimens on both DASL and RNA-seq.

This submission represents the RNA-seq component of study.

The DASL samples used in the study are GSM876557, GSM876558, GSM876564, and GSM876565.

first: Andrew

last: Kelly

first: Dimitrios

last: Spentzos

first: Mick

last: Correll

first: Lan

last: Hu


title: Genome-wide reprogramming of the chromatin landscape underlies endocrine therapy resistance in breast cancer

city: Lebanon

first: xiaoyang

last: zhang

address: 1 Medical Center Driver

institute: Dartmouth Medical School

summary: The estrogen receptor alpha (ERa) drives the growth of two-thirds of all breast cancers. Endocrine therapy impinges on estrogen-induced ERa activation to block tumor growth. However, half of ERa-positive breast cancers are tolerant or acquire endocrine therapy resistance. Here we demonstrate that breast cancer cells undergo genome-wide reprogramming of their chromatin landscape, defined by epigenomic maps and chromatin openness, as they acquire resistance to endocrine therapy. This reveals a role for the Notch pathway while excluding classical ERa signaling. In agreement, blocking Notch signaling, using gamma-secretase inhibitors, or targeting its downstream gene PBX1 abrogates growth of endocrine therapy-resistant breast cancer cells. Moreover Notch signaling through PBX1 directs a transcriptional program predictive of tumor outcome and endocrine therapy response.

overall_design: Comparing histone modifications (H3K4me2 and H3K36me3), chromatin openness (FAIRE) and PBX1 binding between endocrine therapy sensitive MCF7 and resistant MCF7-LTED cells.

first: Xiaoyang

last: Zhang


title: Combined miRNA and mRNA Sequencing Identifies Protective Transcriptional Signature of Enhanced PI3Kalpha Signaling in Cardiac Hypertrophy

city: Taipei

first: Kai-Chien

last: Yang

address: No.1, Sec. 1, Jen-Ai Rd, 1150R

department: Graduate Institute and Department of Pharmacology

country: Taiwan

institute: National Taiwan University

summary: A molecular and bioinformatic pipeline permitting comprehensive analysis and quantification of myocardial miRNA and mRNA expression with next-generation sequencing was developed and the impact of enhanced PI3Kalpha signaling on the myocardial transcriptome signature of pressure overload-induced pathological hypertrophy was explored.

overall_design: miRNA and mRNA-Seq were carried out in four groups of mouse LV samples: WT sham, WT+TAC, caPI3Kalpha sham, caPI3Kalpha+TAC

first: Jeanne

last: Nerbonne


title: Small RNA analysis of wildtype Mouse embryo and Adar1 null mouse embryo at E11.0 and E11.5 together with mRNA-seq results of E11.5

summary: Adar1 is an essential gene for mouse embryonic development. Adar1 null mouse embryos dies around E11.5 because of massive apoptosis.

overall_design: Small RNA: 4 samples examined: wild type E11.0, ADAR1 null E11.0, wild type E11.5, ADAR1 null E11.5, mRNA-seq: wild type E11.5, ADAR1 null E11.5.

first: Hiromitsu

last: Ota

first: Louis

last: Valente

first: Bjorn-Erik

last: Wulff

first: Kentaro

last: Ariyoshi

first: Hisashi

last: Iizasa


title: Murine polyploid trophoblast giant cells from day E9.5 implantation sites

status: Public on Apr 30 2013

city: Cambridge

first: Jessica

last: Von Stetina

address: 9 Cambridge Center

institute: Whitehead Institute

type: Genome variation profiling by genome tiling array

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

overall_design: Refer to individual Series


first: luca

last: magnani

state: NH

address: NCCC one medical drive

department: Genetics

institute: Dartmouth College


title: microRNA (miRNA) expression profiles of murine polyploid trophoblast giant cells (TGCs) from day 9.5 implantation sites.

summary: We employed miRNA-seq to profile all miRNAs from a pure population of hand-dissected polyploid TGCs from embryonic day 9.5. These data set of polyploid-specific TGCs microRNAs will provide insights into TGCs differentiation and endoreplication.

overall_design: TGCs were micro-dissected from day E9.5 nine implantation sites from C57BL/J6 mice. The portion of the TGCs in direct contact with the spongiotrophoblast layer and the labyrinth layer were manually removed to avoid collecting any polyploid cells from the former or multi-nucleated syncytiotrophoblast cells from the latter.

first: Noa

last: Sher

first: George

middle: W

last: Bell

first: Shinobu

last: Matsuura

first: Katya

last: Ravid

first: Terry

middle: L

last: Orr-Weaver


title: High Resolution Mapping of H1 Linker Histone Variants in Embryonic Stem Cells

status: Public on Apr 29 2013

first: Yuhong

last: Fan

state: Georgia

address: IBB 2313, 315 Ferst Drive

department: School of Biology and the Petit Institute for Bioengineering and Bioscience

institute: Georgia Institute of Technology

summary: H1 linker histones facilitate higher order chromatin folding and are essential for mammalian development.  To achieve high resolution mapping of H1 variants H1d and H1c in embryonic stem cells (ESCs), we have established a knock-in system and shown that the N-terminally tagged H1 proteins are functionally interchangeable to their endogenous counterparts in vivo.  H1d and H1c are depleted from GC- and gene-rich regions and active promoters, inversely correlated with H3K4me3, but positively correlated with H3K9me3 and associated with characteristic sequence features.  Surprisingly, both H1d and H1c are significantly enriched at major satellites, which display increased nucleosome spacing compared with bulk chromatin. While also depleted at active promoters and enriched at major satellites, overexpressed H10 displays differential binding patterns in specific repetitive sequences compared with H1d and H1c.  Depletion of H1c, H1d and H1e causes pericentric chromocenter clustering and de-repression of major satellites.  These results integrate the localization of an understudied type of chromatin proteins, namely the H1 variants, into the epigenome map of mouse ESCs, and we identify significant changes at pericentric heterochromatin upon depletion of this epigenetic mark.

overall_design: Mapping linker histone H1 variants H1d, H1c and H10 as well as 3 core histone modifications in mouse ESC genome.

first: Kaixiang

last: Cao

first: Nathalie

last: Lailler

first: Xiao

last: Dong

first: Eric

last: Bouhassira


title: RNA expression profiles of murine polyploid trophoblast giant cells (TGCs) from day 9.5 implantation sites.

summary: We employed RNA-seq to transcriptionally profile a pure population of hand-dissected polyploid TGCs from embryonic day 9.5. These data provide a set of polyploid-specific TGCs transcripts that will aid in the understanding of TGCs differentiation and endoreplication.

overall_design: TGCs were micro-dissected from day E9.5 nine implantation sites from C57BL/6J mice. The portion of the TGCs in direct contact with the spongiotrophoblast layer and the labyrinth layer were manually removed to avoid collecting any polyploid cells from the former or multi-nucleated syncytiotrophoblast cells from the latter.

middle: R


title: Ldb1-nucleated transcription complexes function as primary mediators of erythroid gene activation

first: Raja

last: Jothi

state: NC

address: 111 TW Alexander Drive; A314

department: National Institute of Environmental Health Sciences

institute: National Institutes of Health

summary:
We used ChIP-Seq to map Ldb1, Scl and Gata1 binding sites in mouse total bone marrow cells. Together with functional studies comparing gene expression in Murine Erythroleukemia (MEL) cells expressing Ldb1 shRNA or control shRNA  and bioinformatics analysis, we systematically determined the transcriptional program controlled by Ldb1 complexes in erythropoiesis.
This represents the ChIP-Seq component of the study only

overall_design: To evaluate the role of Ldb1complexes in erythroid gene activation

first: LiQi

last: Li

first: Johannes

last: Freudenberg

first: Kairong

last: Cui

first: Ryan

last: Dale

first: Sang-Hyun

last: Song

first: Ann

last: Dean

first: Keji

last: Zhao

first: Paul

last: Love


title: Epstein-Barr virus maintains lymphomas via its miRNAs

city: Madison

first: Bill

last: Sugden

address: 1111 Highland Ave

institute: University of Wisconsin-Madison

summary: Epstein‐Barr virus (EBV) has evolved exquisite controls over its host cells, human B lymphocytes, not only directing these cells during latency to proliferate and thereby expand the pool of infected cells, but also to survive and thereby persist for the lifetime of the infected individual.  Although these activities ensure the virus is successful, they also make the virus oncogenic, particularly when infected people are immunosuppressed. Here we show, strikingly, that one set of EBV’s miRNAs both sustain BL (Burkitt’s lymphoma) cells in the absence of other viral oncogenes and promote the transformation of primary B lymphocytes.  Burkitt’s Lymphoma cells were engineered to lose EBV and found to die by apoptosis and could be rescued by constitutively expressing viral miRNAs in them.  Two of these EBV miRNAs were found to target Caspase 3 to inhibit apoptosis at physiological concentrations.

overall_design: Examination of RISC associated transcripts under 4 conditions in Sav S1-1 cells

first: David

last: Vereide

first: Eri

last: Seto

first: Ya-Fang

last: Chiu

first: Mitchell

last: Hayes

first: Takanobu

last: Tagawa

first: Adam

last: Grundhoff

first: Wolfgang

last: Hammerschmidt


title: A master cistromic circuit governing hepatic fibrogenesis [ChIP-seq]

city: La Jolla

first: Ruth

last: Yu

address: 10010 N Torrey Pines Rd

department: Gene Expression Lab

institute: Salk Institute

summary: Liver fibrosis is a reversible wound-healing response to liver injury and hepatic stellate cells (HSCs) are central cellular players that mediate hepatic fibrogenesis. However, the molecular mechanisms that govern this process remain unclear. Here, we reveal a novel cistromic circuit in HSCs comprising the vitamin D receptor (VDR) and SMAD transcription factors that restrains the intensity of hepatic fibrogenesis. Ligand-activated VDR suppresses TGFβ1-induced pro-fibrotic gene expression in HSCs. Administration of a vitamin D analogue, calcipotriol, diminishes the fibrotic response in a mouse model of liver fibrosis, while VDR knockout mice spontaneous develop extensive hepatic fibrosis by age 6 months. Using ChIP-Seq, we find that the anti-fibrotic properties of VDR are due to crosstalk with SMAD, mediated by their co-occupancy of DNA-binding sites on pro-fibrotic genes. Specifically, SMAD binding potentiates local chromatin accessibility to enhance VDR recruitment at the same cis-regulatory elements, which reciprocally antagonizes the interaction between SMAD3 and chromatin and limits the assembly of transcriptional activation complexes at fibrotic genes, a process that is enhanced by the presence of VDR agonists. These results not only establish this coordinated VDR/SMAD cistromic circuit as a master regulator of hepatic fibrogenesis, but also support VDR as a potential drug target to ameliorate liver fibrosis.

overall_design: Identification of VDR, SMAD3 and H3 binding sites in human stellate LX2 cells that were pre-treated with calcipotriol (100nM) for 16 hrs (where calcipotriol treatment is indicated) followed by incubation of calcipotriol (100nM) or TGFβ1 (1ng/ml) for another 4 hours (where indicated).

first: Ning

last: Ding

first: Ronald

last: Evans


title: Quantitative Mammalian Transcriptomics using Designed Primer-based Amplification

city: la jolla

first: vipul

last: bhargava

address: 9500 gilman drive

institute: ucsd

summary:
We describe a novel quantitative cDNA expression profiling strategy, involving amplification of the majority of mouse transcriptome using a defined set of 44 heptamer primers. The amplification protocol allows for efficient amplification from as low as 50pg of mRNA and did not alter the expression of the transcripts even with 200 fold dilution of the minimum requirement of the starting material (10ng of mRNA) for standard RNA-seq protocols.
We implemented our methodology on embryological lineage segregation, achieved by graded activation of Activin A/TGFβ signaling in mouse embryonic stem cells (mESCs). The fold changes in transcript expression were in excellent agreement with quantitative RT-PCR and we observed a dynamic range spanning more than five orders of magnitude in RNA concentration with a reliable estimation of low abundant transcripts. Our transcriptome data identified key lineage markers, while the high sensitivity showed that novel lineage specific transcripts anticipate the differentiation of specific cell types.
We compared our strategy with Std. RNA-seq (Mortazavi et al. 2008) and SMART-seq (Ramsköld et al. 2012). We also showed potential of our methodology to suppress the representation of highly expressing ribosomal transcripts.

overall_design: Sequencing was performed on day 4 differentiating mouse ESCs treated for two days with 3 different dosages of Activin A (3ng/mL, 15ng/mL and 100ng/mL). The cells were also treated with SB-431542. Serial dilutions of mRNA derived Activin A(3ng/mL) samples were used to detemine the minimum amount of mRNA required to construct relaible sequencing library. SMARTseq libraries were prepared for both Activin A(3ng/mL) and Activin A(100ng/mL) samples. Three Different primer sets were designed to suppress the representaiton of Ribosomal transcripts.

first: Vipul

last: Bhargava

first: Shankar

last: Subramaniam


title: Gene Expression Analysis of Zebrafish Melanocytes, Iridophores, and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin

status: Public on Apr 26 2013

city: St. Louis

first: Charles

last: Higdon

state: Missouri

address: 4444 Forest Park Blvd.

institute: Washington University

summary: In order to facilitate understanding of pigment cell biology, we developed a method to concomitantly purify melanocytes, iridophores, and retinal pigmented epithelium from zebrafish, and analyzed their transcriptomes.  Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co-enrichment in melanocytes and retinal pigmented epithelium, as well as in melanocytes and iridophores.  We found 214 genes co-enriched in melanocytes and retinal pigmented epithelium, indicating the shared functions of melanin-producing cells.  We found 62 genes significantly co-enriched in melanocytes and iridophores, illustrative of their shared developmental origins from the neural crest.  This is also the first analysis of the iridophore transcriptome.  Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine-based reflective pigment.  We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate-limiting substrate for purine synthesis, phosphoribosyl pyrophosphate, thus constituting a guanine cycle.  The purification procedure and expression analysis described here, along with the accompanying transcriptome-wide expression data, provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types, and will be a useful resource for further studies of pigment cell biology.

overall_design: mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing

first: Rob

last: Mitra

first: Stephen

last: Johnson


title: Transcriptomic sequencing of the murine testis during the first wave of spermatogenesis

city: Jokioinen

first: Anu

last: Sironen

address: Myllytie 1

department: Biotechnology and Food Research

country: Finland

institute: Agrifood Research Finland

summary: Our experimental design includes samples at different time points during the first wave of spermatogenesis. Each time point corresponds to specific cell content in the testis. First time point is post natal day (PND) 7, when only somatic cells and spermatogonia are present in the testis. Thereafter additional cell types are present in selected samples in chronological order: PND 14 contains early spermatocytes, PND 17 late spermatocytes, PND 21 round spermatids and finally PND 28 elongating spermatids. Our results highlight differentially expressed genes and gene isoforms, which are important for correct sperm development and male fertility. In addition, functional analysis suggests a highly controlled gene expression pattern during the first wave of spermatogenesis.

overall_design: Transcriptome sequencing of the mouse testes at PND 7, 14, 17, 21 and 28 for analysis of differences in gene expression pattern during the first wave of spermatogenesis. In total 10 samples were analysed, replicates for each time point.

first: Asta

last: Laiho


title: A Master Cistromic Circuit Governing Hepatic Fibrogenesis

sample_organism:
Homo sapiens
Rattus norvegicus

platform_organism:
Homo sapiens
Rattus norvegicus


title: The interactome of Pou5f1 enhancer in mouse embryonic stem cells

status: Public on Apr 28 2013

city: Los Angeles

first: Fan

last: Gao

address: 1501 San Pablo St. ZNI 245

department: ZNI

institute: USC

summary: We report the application of enyzme-based 4C-Seq technique for exploring Pou5f1 enhancer interactome in mouse ES cells.

overall_design: We explored the interactome of Pou5f1 upstream enhancer in mouse ES cells by using an enzyme digestion based 4C-Seq protocol. The interactome is involved in gene active regulation.

first: Zong

last: Wei

first: Kai

last: Wang

first: Wange

last: Lu


title: The histone demethylase Jarid1b ensures faithful mouse development by protecting developmental genes from aberrant H3K4me3 [ChIP-Seq]

city: Copenhagen

first: Sandra

middle: Ursula

last: Schmitz

address: Ole Maaløes Vej 5

country: Denmark

institute: Biotech Research and Innovation Centre

summary:
Embryonic development is tightly regulated by transcription factors and chromatin-associated proteins. H3K4me3 is associated with active transcription and H3K27me3 with gene repression, while the combination of both keeps genes required for development in a plastic state. Here we show that deletion of the H3K4me2/3 histone demethylase Jarid1b (Kdm5b/Plu1) results in major neonatal lethality due to respiratory failure. Jarid1b knockout embryos have several neural defects including disorganized cranial nerves, defects in eye development and increased incidences of exencephaly. Moreover, in line with an overlap of Jarid1b and Polycomb targets genes, Jarid1b knockout embryos display homeotic skeletal transformations typical for Polycomb mutants. Genome-wide analysis demonstrated that normally inactive genes encoding developmental regulators acquire aberrant H3K4me3 in early Jarid1b knockout embryos. H3K4me3 accumulates as embryonic development proceeds, leading to increased expression of neural master regulators in knockouts. Taken together, these results suggest that Jarid1b contributes to mouse development by protecting developmental genes from inappropriate acquisition of active histone modifications.
* Lack of Jarid1b leads to major neonatal lethality and defects in neural systems
* Jarid1b mutants display homeotic skeletal transformations
* H3K4me3 is increased at inactive transcriptional regulators in Jarid1b-/- embryos
* Chromatin changes are accompanied by elevated levels of key neural transcription factors

overall_design: Determining H3K4me3 and H3K27me3 in early (E8.5) mouse embryos

first: Mareike

last: Albert

middle: U


title: ADAR1 forms a complex with Dicer to promote microRNA processing and RNA-induced gene silencing

status: Public on Apr 25 2013

first: Kazuko

last: Nishikura


title: De novo piRNA cluster formation in the Drosophila germline triggered by transgenes containing a transcribed transposon fragment

city: Moscow

first: Sergei

last: Ryazansky

address: Kurchatov sq., 2

department: Department of Molecular Genetics of Cell

country: Russia

institute: Institute of Molecular Genetics

summary: PIWI interacting RNAs (piRNAs) provide defense against transposable element (TE) expansion in the germline of metazoans. piRNAs are processed from the transcripts encoded by specialized heterochromatic clusters enriched in damaged copies of transposons. How these regions are recognized as a source of piRNAs is still elusive. The aim of this study is to determine how transgenes that contain a fragment of the LINE-like I transposon lead to an acquired TE resistance in Drosophila. We show that such transgenes, being inserted in unique euchromatic regions which normally do not produce small RNAs, become de novo bidirectional piRNA clusters that silence I-element activity in the germline. Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequences — not only from the transposon fragment. Chromatin immunoprecipitation analysis shows that in ovaries the trimethylated histone 3 lysine 9 (H3K9me3) mark associates with transgenes producing piRNAs. We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs.

overall_design: The fractions of small RNAs (19-29 nt) from ovaries of wild type and 11 transgenic lines of Drosophila melanogaster were sequenced using Illumina HiSeq 2000.

first: Ivan

last: Olovnikov

first: Sergey

last: Shpiz

first: Alla

last: Kalmykova


title: Minotaur is critical for primary piRNA biogenesis [smallRNA-Seq]

status: Public on Apr 27 2013

city: Cold Spring Harbor

first: Vasily

last: Vagin

state: NY

address: 1 Bungtown Rd

institute: Cold Spring Harbor

summary: Piwi proteins and their associated small RNAs are essential for fertility in animals.  This is due, in part, to their roles in guarding germ cell genomes against the activity of mobile genetic elements.  piRNA populations direct Piwi proteins to silence transposon targets and as such form a molecular code that discriminates transposons from endogenous genes.  Information ultimately carried by piRNAs is encoded within genomic loci, termed piRNA clusters.  These give rise to long, single-stranded, primary transcripts that are processed into piRNAs.  Despite the biological importance of this pathway, neither the characteristics that define a locus as a source of piRNAs nor the mechanisms that catalyze primary piRNA biogenesis are well understood. We searched an EMS-mutant collection annotated for fertility phenotypes for genes involved in the piRNA pathway. Twenty-seven homozygous-sterile strains showed transposon-silencing defects.  One of these, which strongly impacted primary piRNA biogenesis, harbored a causal mutation in CG5508, a member of the Drosophila glycerol-3-phosphate O-acetyltransferase (GPAT) family. These enzymes catalyze the first acylation step on the path to the production of phosphatidic acid (PA).  Though this pointed strongly to a function for phospholipid signaling in the piRNA pathway, a mutant form of CG5508, which lacks the GPAT active site, still functions in piRNA biogenesis.  We have named this new biogenesis factor Minotaur.

overall_design: Examination of small RNA profile in heterozygous and homozygous CG5508 mutant ovaries

first: Yang

last: Yu

first: Anna

last: Jankowska

first: Yicheng

last: Luo

first: Kaja

last: Wasik

first: Colin

last: Malone

first: Emily

last: Harrison

first: Adam

last: Rosebrock

first: Barbara

last: Wakimoto

first: Delphine

last: Fagegaltier

first: Felix

last: Muerdter

first: Gregory

last: Hannon


title: Minotaur is critical for primary piRNA biogenesis [RNA-Seq]

overall_design: Examination of transcriptom profile in heterozygous and homozygous CG5508 mutant ovaries


title: Human PA-1 cells treated with scrambled or specific ASOs

city: Shanghai

first: Ling-Ling

last: Chen

address: 320 Yue-Yang Road

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

summary: We describe the discovery of sno-lncRNAs, a class of nuclear-enriched intron-derived long noncoding RNAs (lncRNAs) that are processed on both ends by the snoRNA machinery. During exonucleolytic trimming, the sequences between the snoRNAs are not degraded, leading to the accumulation of lncRNAs flanked by snoRNA sequences but lacking 5' caps and 3' poly(A) tails. Such RNAs are widely expressed in cells and tissues and can be produced by either box C/D or box H/ACA snoRNAs. Importantly, the genomic region encoding one abundant class of sno-lncRNAs (15q11-q13) is specifically deleted in Prader-Willi Syndrome (PWS). The PWS region sno-lncRNAs do not colocalize with nucleoli or Cajal bodies, but rather accumulate near their sites of synthesis. These sno-lncRNAs associate strongly with Fox family splicing regulators and alter patterns of splicing. These results thus implicate a previously unannotated class of lncRNAs in the molecular pathogenesis of PWS.

overall_design: We have used deep sequencing to explore the gene expression from poly(A)+ RNAs in embryonal carcinoma (EC) line PA-1 cells treated with scrambled or specific antisense oligodeoxynucleotides (ASOs).

first: Ling

first: Qing

middle: F

last: Yin

first: Li

last: Yang

last: Zhang

first: Jian

last: Xiang

first: Yue

last: Wu

first: Gordon

middle: G

last: Carmichael


title: Minotaur is critical for primary piRNA biogenesis

first: Vasily

last: Vagin

first: Yang

last: Yu

first: Anna

last: Jankowska


title: Bactrocera dorsalis Gene expression

status: Public on Apr 24 2013

city: Hilo

first: Brian

last: Hall

state: Hawai'i

address: 64 NOWELO ST

institute: USDA PBARC

summary: Comprehensive RNA sequencing was performed on a laboratory colony of B. dorsalis with a focus on attempting to capture as many genes in the sequencing from throughout the entire developmental life history. De novo assembly and analysis of the resulting sequence

overall_design: One sample each for the egg, larvae, pupae, adult male, adult female and mated female life stages was sequenced.

first: Scott

last: Geib


title: RNA-sequencing project for zebrafish embryo and larva development

first: Hongxing

address: 3888 Chenhua Rd, Songjiang District

institute: Shanghai Chenshan Botanical Garden

summary: Zebrafish is an important model system for the study of vertebrate embryonic development and adaptive immunese response. Recent years have seen great advancement in the understanding of the regulatory mechanisms during zebrafish embryogenesis and immune processes, yet large gaps still remain in the functional pathways critical for each developmental stage, especially for the late embryonic development. We sequenced the polyA-extracted mRNA from 9 stages covering 7 major developmental periods of zebrafish. Whole genome gene expression pattern were analyzed to reveal unknown pathways or factors with implicated roles during each stage of vertebrate development.

overall_design: Analysis of total mRNA by highthroughput sequencing in 9 stages covering 7 periods during the embryonic and larval development of zebrafish

first: Jihua

last: Yao

first: Hong

last: Ma


title: High-throughput single cell gene expression analysis in primary blood stem and progenitor cells identifies new links between key transcriptional regulators

first: Rebecca

middle: Louise

last: Hannah

address: Addenbrookes Hospital, Hills Road

department: Haematology

institute: University of Cambridge

summary: Cellular decision-making is mediated by a complex interplay of external stimuli with the intracellular environment, in particular transcription factor regulatory networks.  Here we have determined the expression of a network of 18 key haematopoietic transcription factors (TFs) in 597 single primary blood stem and progenitor cells isolated from mouse bone marrow.  We demonstrate that different stem/progenitor populations are characterised by distinctive TF expression states, and through comprehensive bioinformatic analysis reveal positively and negatively correlated TF pairings, including previously unrecognised relationships between Gata2, Gfi1 and Gfi1b.  Validation using transcriptional and transgenic assays confirmed direct regulatory interactions consistent with a novel regulatory triad in immature blood stem cells, where Gata2 may function to modulate cross-inhibition between Gfi1 and Gfi1b.  Single cell expression profiling therefore identifies network states and allows reconstruction of network hierarchies involved in controlling stem cell fate choices, and provides a blueprint for studying both normal development and human disease.

overall_design: Examination of Gata2 and Gfi1 binding patterns in murine mast cells

first: Vicki

last: Moignard

first: Iain

last: Macaulay

first: Gemma

last: Swiers

first: Florian

last: Buettner

first: Judith

last: Schuette

first: Fernando

last: Calero-Nieto

first: Sarah

last: Kinston

first: Anagha

last: Joshi

first: Fabian

last: Theis

first: Sten

middle: E

last: Jacobsen

first: Marella

last: de Bruijn

first: Berthold

last: Gottgens


title: Genome-wide control of RNA polymerase II activity by cohesin

city: St Louis

first: Cheri

middle: A

last: Schaaf

state: MO

address: 1100 South Grand Ave

department: Biochemistry & Molecular Biology

institute: Saint Louis University School of Medicine

type: Genome binding/occupancy profiling by genome tiling array

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

overall_design: Refer to individual Series


title: Genome-wide control of RNA polymerase II activity by cohesin (sequencing)

summary: Cohesin is a well-known mediator of sister chromatid cohesion, but it also influences gene expression and development. These non-canonical roles of cohesin are not well understood, but are vital: gene expression and development are altered by modest changes in cohesin function that do not disrupt chromatid cohesion. To clarify cohesin’s roles in transcription, we measured how cohesin controls RNA polymerase II (Pol II) activity by genome-wide chromatin immunoprecipitation and precision global run-on sequencing. On average, cohesin-binding genes have more transcriptionally active Pol II and promoter-proximal Pol II pausing than non-binding genes, and are more efficient,  producing higher steady state levels of mRNA per transcribing Pol II complex. Cohesin depletion frequently increases pausing at cohesin-binding genes, indicating that cohesin  often facilitates transition of paused Pol II to elongation. In many cases this likely reflects a role for cohesin in transcriptional enhancer function. Strikingly, more than 95% of predicted extragenic enhancers bind cohesin, and cohesin depletion can reduce their association with Pol II, indicating that cohesin facilitates enhancer-promoter contact. Cohesin directly promotes transcription of the myc gene, and cohesin depletion reduces Pol II activity at most Myc target genes. The multiple transcriptional roles of cohesin revealed by these studies likely underlie the growth and developmental deficits caused by minor changes in cohesin activity.

overall_design: The PRO-seq method was used to measure transcriptionally engaged Pol II genome-wide in two replicates each of mock RNAi-treated, Nipped-B RNAi-treated, and Rad21 RNAi-treated ML-DmBG3-c2 cells.

first: Hojoong

last: Kwak

first: Ziva

last: Misulovin

first: Amanda

last: Koenig

first: John

last: Lis

first: Dale

last: Dorsett


title: RNA-seq analysis of Mycobacterium avium TMC724 vs. M. avium 104 non-coding and coding transcriptome

status: Public on Apr 23 2013

city: Berlin

first: Dmitriy

last: Ignatov

address: Charitéplatz 1

institute: Max Planck Unit for the Science of Pathogens

summary: Deep sequencing was implemented to study the transcriptional landscape of Mycobacterium avium TMC724. High-resolution transcriptome analysis identified the transcription start points for 652 genes. One third of these coincided with the start codons and therefore belong to leaderless transcripts, whereas the rest of the transcripts had 5' UTRs with the mean length of 83 nt. In addition, the 5' UTRs of 6 genes contained SAM-IV and Ykok types of riboswitches. 87 antisense RNAs and 9 intergenic small RNAs were mapped. Four of the revealed intergenic small RNAs, including igMAV_1034-1035 expressed at a very high level, have no homologs in M. tuberculosis, whilst M. avium lacks several intergenic sRNAs present in M. tuberculosis. Among those, MTS479 and MTS1338 are of special interest due to their possible implication in pathogenesis. Elucidation of differences in the repertoire of intergenic sRNAs between the two mycobacterial species may improve our understanding of mycobacterial diseases pathogenesis.

overall_design: Transcriptional profile of Mycobacterium avium TMC724, grown at 37°C in Dubos broth until mid-logarithmic growth phase

first: Sofia

last: Malakho

first: Konstantin

last: Majorov

first: Timofey

last: Skvortsov

first: Alexander

last: Apt

first: Tatyana

last: Azhikina


title: Endogenous mammalian histone H3.3 exhibits chromatin-related functions during development

city: Davis

first: Henriette

last: O'Geen

address: 1 Shields Avenue

institute: UC Davis

summary:
Background.
The histone variant H3.3 plays key roles in regulating chromatin states and transcription. However, the role of endogenous H3.3 in mammalian cells and during development has been less thoroughly investigated. To address this gap, we report the production and phenotypic analysis of mice and cells with targeted disruption of the H3.3-encoding gene, H3f3b.
Results.
H3f3b KO mice exhibit a semi-lethal phenotype traceable at least in part to defective cell division and chromosome segregation. H3f3b KO cells have widespread ectopic CENP-A protein localization suggesting one possible mechanism for defective chromosome segregation. KO cells have abnormal karyotypes and cell cycle profiles as well. The transcriptome and euchromatin-related epigenome were moderately affected by loss of H3f3b in MEFs with ontology most notably pointing to changes in chromatin regulatory and histone coding genes. Reduced numbers of H3f3b KO mice survive to maturity and almost all survivors from both sexes are infertile.
Conclusions.
Taken together, our studies suggest that endogenous mammalian histone H3.3 has important roles in regulating chromatin and chromosome functions that in turn are important for cell division, genome integrity, and development.

overall_design: Examination of H3K9Ac and H3K4me3 in wild-type and H3.3 null MEFs

first: Kelly

last: Bush

first: Benjamin

last: Yuen

first: Bonnie

last: Barrilleaux

last: Riggs

first: Rebecca

last: Cotterman

first: Paul

middle: S

last: Knoepfler


title: Deep sequencing of endogenous mRNA from Caenorhabditis elegans in the presence and absence of arsenite

city: Jena

first: JenAge

last: Project

address: Beutenbergstr. 11

institute: Leibniz Institute for Age Research - Fritz Lipmann Institute

summary:
Background: Arsenite is one of the most toxic chemical substances known and is assumed to exert detrimental effects on viability even at lowest concentrations. By contrast and unlike higher concentrations, we here find that exposure to low-dose arsenite promotes growth of cultured mammalian cells.
In the nematode C. elegans, low-dose arsenite promotes resistance against thermal and chemical stressors, and extends lifespan of this metazoan, whereas higher concentrations reduce longevity.
While arsenite causes a transient increase in reactive oxygen species (ROS) levels in C. elegans, co-exposure to ROS scavengers prevents the lifespan-extending capabilities of arsenite, indicating that transiently increased ROS levels act as transducers of arsenite effects on lifespan, a process known as mitohormesis.
The RNA-seq data comprises 2 biological replicates for worms exposed to 100nM Arsenite 48h after L4 and 2 biological replicates of the same age as controls

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

overall_design: 4 samples: 2 mRNA profiles of C.elegans 48h after L4 exposed to Arsenite; 2 mRNA profiles of C.elegans 48h after L4 as controls (H20). The N2 wild type (var. Bristol) strain was used.

first: Sebastian

last: Schmeisser

first: Kathrin

first: Sandra

last: Weimer

first: Marco

last: Groth

first: Steffen

last: Priebe

first: Eugen

last: Fazius

first: Doreen

last: Kuhlow

first: Denis

last: Pick

first: Jürgen

last: Einax

first: Reinhard

last: Guthke

first: Matthias

last: Platzer

first: Kim

last: Zarse

first: Michael

last: Ristow


title: Identification of drought stress-responsive transcription factor in ramie (Boehmeria nivea L．Gaud)

city: Changsha

first: Touming

last: Liu

address: NO.348, western road of xianjia lake

department: Institute of Bast Fiber Crops

institute: Chinese Academy of Agricultural Sciences

summary: Drought is a main environment stress which severely inhibits the stem growth of ramie and lead to a decrease of the fiber yield. The mechanisms of ramie responding to drought stress are poorly understood. Using Illumina sequencing, approximately 4.8 million (M) 21-nt cDNA tags were sequenced in the cDNA library derived from the drought-stressed ramie (DS), and about 4.7 M were sequenced in the cDNA library constructed from the control ramie under well water condition (CO). The tags generated from two libraries were aligned with ramie transcriptome to annotate their function and a total of 23,912 and 22,826 ramie genes were matched by these tags of DS and CO library, respectively. Comparison of gene expression level between CO and DS ramie based on the differences of tag frequencies appearing in two libraries revealed that 1101 and 505 genes were respectively up- and down- regulated under drought stress. Pathway enrichment analysis identified a set of significantly enriched pathways of DEGs. A series of candidate genes and pathways that may contribute to drought tolerance in ramie will be helpful for further improving ramie drought tolerance ability.

overall_design: 3' tag-based DGE libraries were generated to exam the differentially expressed gene between drought-stressed and well-watered ramie


title: Processing-Independent CRISPR RNAs Limit Natural Transformation in Neisseria meningitidis

city: Cologne

first: Konrad

middle: U.

last: Förstner

state: North Rhine-Westphalia

address: Gleueler Str. 60

department: Information Services

institute: ZB MED - Information Centre for Life Sciences

summary: In bacteria and archaea, CRISPR loci confer adaptive, sequence-based immunity against viruses and plasmids. CRISPR interference is specified by CRISPR RNAs (crRNAs) that are transcribed and processed from CRISPR spacers and repeats. Pre-crRNA processing is essential for CRISPR interference in all systems studied thus far. Here we examine crRNA biogenesis and CRISPR interference in naturally competent Neisseria spp., including the human pathogen N. meningitidis. Our studies reveal a unique crRNA maturation pathway in which crRNA transcription is driven by promoters that are embedded within each repeat, yielding crRNA 5’ ends are not formed by processing. Although crRNA 3’ end formation occurs through RNase III cleavage of a pre-crRNA/tracrRNA duplex, as in other Type II CRISPR systems, this processing event is dispensable for interference. The meningococcal pathway is the most streamlined CRISPR/cas system characterized to date. Endogenous CRISPR spacers frequently target genomic sequences of other Neisseria strains and so limit natural transformation, which is the primary source of genetic variation that contributes to immune evasion, antibiotic resistance, and virulence in N. meningitidis.

overall_design: dRNA-seq approach for RNA samples from cultures of N. lactamica 020-06, harvested at mid-log. Two cDNA libraries from total RNA were prepared to distinguish between transcripts with either primary orprocessed 5’ ends: one library is generated from untreated RNA, whereas the other is treated with terminator exonuclease (TEX),

first: Yan

last: Zhang

first: Nadja

last: Heidrich

first: Biju

last: Joseph

first: Carl

last: Gunderson

first: H

last: Seifert

first: Christoph

last: Schoen

first: Jörg

last: Vogel

first: Erik

last: Sontheimer


title: The interactome of POU5F1 upstream enhancer in mouse embryonic stem cells revealed by 4C-Seq

first: Fan

last: Gao

state: California

summary: We report the application of 4C-Seq technique for exploring POU5F1 enhancer interactome in mouse embryonic stem cells. A statistical model was built to identify enriched interacting regions from raw 4C data. The biological replicate data were compared to identify reproducible interacting regions. The interacting sites in the reproducible regions are enriched with active histone marks as well as transcription factors Oct4, Klf4, Esrrb, Tcfcp2i1 and Zfx that are critical for reprogramming and pluripotency.

overall_design: Generation of Illumina HiSeq2000 sequencing data using 4C-Seq protocol

first: Zong

last: Wei

first: Kai

last: Wang

first: Wange

last: Lu


title: RNA-sequencing technology identifies specific genes expressed in the ICM of mouse embryos

city: Lyon

first: Irene

last: Aksoy

address: 18 avenue du Doyen Lepine

institute: Stem Cell and Brain Research Institute

summary: RNA-seq data from whole mouse embryos at E3.75 (stage where the three cell types: TE, PrE and EPI are well resolved) and from dissected ICMs in order to identify genes expressed specifically in the ICM

overall_design: We used E3.75 whole mouse embryos at the blastocyst stage and immunosurgically dissected ICMs for RNA extraction and sequencing

first: Lawrence

last: W Stanton


title: Genome-wide maps of Sox/Oct partnerships pluripotent and lineage-committed cells

summary: We show here by using genome-wide ChIP-sequencing that lineage segregation involves multiple Sox/Oct partnership. In undifferentiated ES cells Oct4 interacts with Sox2 and both TFs bind on the 'canonical' motif, whereas in cells commited to PrE lineage Oct4 switches from Sox2 to Sox17 interaction and this complex bind to a unique "compressed" motif.

overall_design: ChIP-sequencing has been done for Sox2, Sox17 and Oct4 in the pluripotent context or PrE context

first: Larry

last: Stanton


title: Inactive or moderately active human promoters are enriched for inter-individual epialleles [Seq]

status: Public on Apr 22 2013

city: Cambridge

first: Thomas

last: Down

address: Tennis Court Road

department: WT/CRUK Gurdon Institute

summary: We first demonstrate that non-genetically determined inter-individual differentially methylated regions (iiDMRs) can be temporally stable for at least two years. Then, we show that iiDMRS are associated with concomitant changes in chromatin state as measured by inter-individual differences in the levels of the histone variant H2A.Z. However, the correlation of promoter iiDMRs with gene expression is negligible and this correlation is not improved even by integrating H2A.Z information. We find that most promoter epialleles, whether genetically or non-genetically determined, are associated with low levels of transcriptional activity, depleted for house keeping genes, and either depleted for H3K4me3/enriched for H3K27me3, or lacking both these marks in human embryonic stem cells. These findings validate in an independent cohort. Interestingly, the key features of iiDMRs are reminiscent of those previously observed for promoters that undergo hyper-methylation in various cancers, in vitro cell culture, and human chronological ageing.

overall_design: H2A.z ChIP-seq, RNA-seq, and DNA methylation data (submitted separately) were collected for five normal individuals.   T21/T22 and T31/T32 are monozygotic twins.

first: C

last: Gemma

first: S

last: Ramagopalan

first: T

last: Beyan

first: M

middle: I

last: Hawa

last: Holland

first: P

middle: J

last: Hurd

first: G

last: Giovannoni

first: R

middle: D

last: Leslie

middle: C

last: Ebers

first: V

middle: K

last: Rakyan


title: Transcriptomic profile of Mycobacteriophage Giles

city: Pittsburgh

first: Rebekah

middle: M

last: Dedrick

address: 4249 Fifth Ave

department: Biological Sciences

institute: University of Pittsburgh

summary: The gene expression during early (30min) and late (2.5hrs) infection of Mycobacteriophage Giles was analyzed, as well as in a Giles lysogen.

overall_design: Total RNA was isolated, depleted of rRNA and prepped for sequencing (Illumina) during early and late infection of Giles and a lysogen.

first: Graham

last: Hatfull


title: Analysis of Drosophila salivary glands and Kc cells with depleted levels of linker histone H1 (Illumina smRNA-Seq]

city: Bronx

first: Sandeep

middle: Nagaraj

last: Wontakal

address: 1300 Morris Park Ave Chanin 402

department: Cell Biology

institute: Albert Einstein College of Medicine

summary: Salivary glands or larval ovaries were isolated from transgenic flies expressing RNAi targeting Nautilus (control) or linker histone H1 using a Tub-Gal4 driver.

overall_design: ~200 larvae were used to isolate salivary glands or ovaries, independently.  Total RNA was isolated using Trizol reagent following manufacturer's guidelines. Then 5 µg of total RNA was separated on a polyacrylamide gel, and 18-29 nt small RNAs were isolated for cloning.

first: Xingwu

last: Lu

first: Arthur

last: Skoultchi


title: The histone demethylase Jarid1b ensures faithful mouse development by protecting developmental genes from aberrant H3K4me3

first: Sandra

last: Schmitz

address: Ole Maaløes Vej 5


title: Functional characterisation of SVP by a Chromatin Immunoprecipitation sequencing approach

city: Milan

first: Giulio

last: Pavesi

address: Via Celoria 26

department: Biosciences

country: Italy

institute: Università di Milano

summary: SVP is a key MADS-box transcription factor for Arabidopsis development since it acts both during vegetative and reproductive phases where it plays different roles probably by interacting with different partners to regulate specific sets of target genes. In fact, whereas SVP functions as a repressor of floral transition during the vegetative phase, it works as floral meristem gene during reproductive phase. We studied the behavior of SVP during two distinct developmental phases: the vegetative and reproductive phase. The aim of these studies is to identify subsets of genes that are directly bound by SVP by means of ChIP sequencing (Illumina Solexa Sequencing) approach during the two distinct phases of development.

overall_design: Arabidopsis thaliana seedlings and inflorescences were selected at successive stages of early development for chromatin extraction and subsequent immunoprecipitation using GFP antibody. The identification of genome wide binding sites of SVP using the ChIP-SEQ approach were performed in the vegetative phase using pSVP::SVP-GFP svp-41 and wild-type seedlings grown for 2 weeks in Short Day (SD) conditions (8 h light/16 h dark); for the reproductive phase we used wild-type and pSVP::SVP-GFP svp-41 inflorescences grown for 2 weeks in SD conditions and then moved in (LD) conditions (16 h light/16 h dark). The inflorescences were collected at 2 weeks after bolting.

first: Veronica

last: Gregis

first: Alice

last: Sessa

first: Fernando

last: Lalaguna

first: Rosalinda

last: Guerra

first: Julieta

last: Mateos

first: Federico

last: Zambelli

first: Gianmarco

last: Prazzoli

first: Lucia

last: Colombo

first: Martin

last: Kater


title: Role of EGR2 transcription factor in T cell anergy

status: Public on Apr 21 2013

city: Chicago

first: Yan

last: Zheng

state: IL

institute: University of Chicago


title: Genome-wide maps of EGR2 transcription factor binding sites in T cell anergy

summary: We report the application of sequencing technology for mapping the EGR2 transcriptional program in T cell anergy

overall_design: Examination of Egr2 genomic binding sites in T cells following overnight treatment with immobilized anti-CD3 mAb (1ug/ml)

first: Yuanuan

last: Zha

first: Robbert

last: Spaapen

first: Rebecca

last: Mathew

first: Kenneth

last: Barr

first: Albert

last: Bendelac

first: Thomas

last: Gajewski


title: Identification of direct targets and modified bases of RNA cytosine methyltransferases

city: SLC

first: Vahid

last: Khoddami

state: UT

address: 4350, 2000 Circle of Hope, HCI

department: Oncological Sciences

institute: Huntsman Cancer Institute-University of Utah

summary: We report development of a mechanism-based technique, Aza-IP, that utilizes the stable covalent linkage formed in vivo between an RNA methyltransferase (m5C-RMT) and 5-azacytidine (5-aza-C) incorporated within the target RNA to enable specific target enrichment, coupled with high-throughput sequencing to provide target identification. We apply Aza-IP to two enzyme types, DNMT2 and NSUN2, the latter with important roles in fertility, intellectual ability, stem cells and cancer. For both, Aza-IP provided >200-fold enrichment of their known human tRNA targets. For NSUN2, we reveal many novel tRNA and non-coding RNA targets, all linked to NSUN2 nucleolar localization, greatly increasing the potential repertoire underlying loss-of-function pathologies.  Strikingly, we observe a C>G transversion ‘signature’ specifically at the target cytosine, which enables the identification of the exact target cytosine within the RNA in the same experiment.

overall_design: The general design of Aza-IP involves nine steps: 1) Expression of an epitope-tagged m5C-RMT derivative (or use of an antibody capable of immuno-precipitating the RNA-bound enzyme), 2) cell growth in the presence of 5-aza-C, which incorporates at low/moderate levels into nascent RNA, 3) cell lysis, 4) immuno-precipitation of the subject m5C-RMT, a portion of which is covalently attached to target RNAs bearing 5-aza-C, 5) stringent washing to remove RNA contaminants, 6) RNA fragmentation, release and purification, 7) ligation of adaptor oligos to the RNA, and the creation of a cDNA library in a manner that enables strand-specific assignments, 8) cDNA sequencing, and 9), mapping and examination of sequence reads to define RNA targets and site of cross-linking/catalysis.

first: Bradley

middle: R

last: Cairns


title: Global Analysis of Yeast mRNPs

status: Public on Apr 18 2013

city: Boulder

first: Sarah

middle: Fitzgerald

last: Mitchell

state: CO

address: 3415 Colorado Ave

institute: University of Colorado

summary: Proteins regulate gene expression by controlling mRNA biogenesis, localization, translation and decay. Identifying the composition, diversity and function of mRNPs (mRNA protein complexes) is essential to understanding these processes. In a global survey of S. cerevisiae mRNA binding proteins we identified 120 proteins that cross-link to mRNA, including 66 new mRNA binding proteins. These include kinases, RNA modification enzymes, metabolic enzymes, and tRNA and rRNA metabolism factors. These proteins show dynamic subcellular localization during stress, including assembly into stress granules and P-bodies (Processing-bodies). CLIP (cross-linking and immunoprecipitation) analyses of the P-body components Pat1, Lsm1, Dhh1 and Sbp1 identified sites of interaction on specific mRNAs revealing positional binding preferences and co-assembly preferences.  Taken together, this work defines the major yeast mRNP proteins, reveals widespread changes in their subcellular location during stress, and begins to define assembly rules for P-body mRNPs.

overall_design: CLIP-seq analysis of Dhh1, Lsm1, Pat1 and Sbp1

first: Roy

last: Parker


title: Analysis of Drosophila salivary glands and Kc cells with depleted levels of linker histone H1

first: Sandeep

last: Wontakal

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

overall_design: Refer to individual Series

first: Xingwu

last: Lu

first: Arthur

last: Skoultchi


title: High resolution transcriptome-wide RNA cytosine methylome of Mouse Embryonic Fibroblasts

summary: We report high resolution transcriptome-wide RNA cytosine methylome of Mouse Embryonic Fibroblasts (MEFs) revealed by an optimized new RNA bisulfite sequencing approach.  Comparison of RNA methylation profile from wild-type and Dnmt2 -/- MEFs shows that only C38 in three tRNAs (tRNA-Asp, Gly and Val) is the target of Dnmt2 in MEFs at normal conditions.

overall_design: Harvested MEFs, from 13.5 isogenic (wt or Dnmt2-/-) embryos, were subjected to total RNA isolation and DNase treatment. Small RNA fraction was separated using mirVana kit (Ambion). Large RNA fraction was prepared by ribosomal RNA depletion using RiboMinus™ Transcriptome Isolation Kit (Invitrogen) followed by RNA fragmentation using RNA Fragmentation Reagent (Ambion). Each one of the fractions (small or large) from each one of the samples (wt or Dnmt2-/-) were split into two: one for direct RNA sequencing and one for RNA bisulfite sequencing. For bisulfite treatment, small and large RNA fractions of each sample (wt or Dnmt2-/-) were separately subjected to bisulfite treatment. Total of 8 samples (bisulfite treated and untreated of small and large RNA fractions of wt or Dnmt2-/-) were separately subjected to library preparation with Illumina’s directional mRNA-Seq sample preparation protocol followed by 101 cycle single-end high-throughput sequencing using Illumina’s HiSeq 2000 sequencing system.


title: Epigenome analysis of DNA modification 5-hydroxymethylcytosine in normal, abnormal, and globozoospermia sperm genomes

city: Beijing

first: Bao-Fa

last: Sun

address: Da-Tun Road

institute: Beijing Institute of Genomics (BIG) of Chinese Academy of Sciences (CAS)

summary: Genome wide of 5-hydroxymethylcytosine profiling of normal and abnormal, and globozoospermia sperm genomes.Two semen samples were collected from a healthy man and a globozoospermia patient who had consulted a physician at ShengJing Hospital of China Medical University. Other semen samples obtained from two volunteers who were good health generally. 5-hmC enriched genomic DNA libraries were generated following the Illumina protocol for “Preparing Samples for CHIP sequencing of DNA”. 100bp single end sequencing on Illumina Hiseq2000 to get 5-hmC-enriched DNA fragment sequence was performed.

overall_design: Examination of 5hmC levels in normal, abnormal, and globozoospermia  sperm genomes

first: Yun-Gui

last: Yang

first: Xiu-Xia

last: Wang

first: You-Qi

last: Liao


title: Transcriptome analysis of the livers from mice that had been fed water-soluble extract of Pacific Krill

city: Kitakami-shi

first: Hidetoshi

last: Yamada

state: Iwate

country: Japan

institute: Iwate Biotechnology Reserch Center

summary:
Background: We previously reported that intake of a water-soluble extract of Pacific Krill (WEPAK) by mice decreases triglyceride accumulation in liver and suppresses weight gain induced by a high-fat diet.  However, the mechanisms mediating these phenomena were not investigated or revealed in our previous study.

Methods and Findings: Here, we investigated the effect of WEPAK in mouse liver using transcriptome analysis and discovered that WEPAK induced the expression of genes categorized into the gene ontology (GO) term lipid metabolic process.  Furthermore, two regulators of fatty acid β-oxidation, AMPK and PPARδ, were induced in the liver and muscle of mice that had taken in WEPAK.

Conclusions: These results indicate that WEPAK increased the expression of genes related to fatty acid β-oxidation via activation of AMPK and PPARδ.  WEPAK may have the effect of improving lipid metabolism and, therefore, may be beneficial in the prevention of obesity.

overall_design: Examination of 4 different feeding condtion (4mice/group)


title: RIP-Seq analysis of Trypanosoma brucei ELAV-like protein Tb927.8.6650

city: Montreal

first: Hamed

last: Najafabadi

state: QC

address: 740 Dr. Penfield Avenue, Room 7202

department: Human Genetics

country: Canada

institute: McGill University

summary: A tagged ectopic version of the ELAV-like protein Tb927.8.6650 of T. brucei was expressed in stable cell lines and pulled down. Co-purifying transcripts were analyzed by sequencing to identify RNAs associated with Tb927.8.6650.

overall_design: Stable procyclic form cell lines expressing tetracycline-inducible TAP-tagged Tb927.8.6650 were created. Cells were harvested 48h after tet-induction, followed by tandem affinity purification of Tb927.8.6650, extraction of co-purified RNA, and sequencing.

first: Zhiquan

first: Reza

last: Salavati